Synthesis, properties, and hepatic metabolism of strongly fluorescent fluorodipyrrinones

Article abstract of DOI:10.1016/j.tet.2006.04.046

From non-fluorescent 8-H fluorophenyldipyrrinones, highly fluorescent (φ{symbol}_F 0.4-0.6) analogs have been synthesized by reaction with 1,1′-carbonyldiimidazole to bridge the dipyrrinone nitrogens and form an N,N′-carbonyldipyrrinone (3H,5H-d

Full text of DOI:10.1016/j.tet.2006.04.046

Tetrahedron 62 (2006) 7043–7055
Synthesis, properties, and hepatic metabolism of strongly
fluorescent fluorodipyrrinones
Stefan E. Boiadjiev,a Zachary R. Woydziak,a Antony F. McDonaghb and David A. Lightnera,
*
aDepartment of Chemistry, University of Nevada, Reno, NV 89557-0020, USA
bDivision of Gastroenterology, Box 0538, University of California, San Francisco, CA 94143-0538, USA
Received 28 February 2006; revised 12 April 2006; accepted 13 April 2006
Available online 22 May 2006
Abstract—From non-fluorescent 8-H fluorophenyldipyrrinones, highly fluorescent (fF 0.4–0.6) analogs have been synthesized by reaction
with 1,10-carbonyldiimidazole to bridge the dipyrrinone nitrogens and form an N,N0-carbonyldipyrrinone (3H,5H-dipyrrolo[1,2-c:20,10-f ]-
pyrimidine-3,5-dione). Amphiphilic, water-soluble 8-sulfonic acid derivatives are then obtained by reaction with concd H2SO4. The resulting
fluorinated and sulfonated N,N0-carbonyl-bridged dipyrrinones, isolated as their sodium salts, are potential cholephilic fluorescence and
19F MRI imaging agents for use in probing liver and biliary metabolism. After intravenous injection in the rat they were excreted rapidly
and largely unchanged in bile. 19F NMR spectroscopy of a pentafluorophenyl-tosylpyrrolinone synthetic precursor exhibited rarely seen
diastereotopicity.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
(A)
Z
C
O
O
H
O
The natural pigment bilirubin (Fig. 1A) is produced contin-
uously in animals during normal catabolism of heme.1 It is
a bichromophoric structure comprised of two Z-dipyrrinones
bearing intramolecularly hydrogen-bonded propionic acid
side-chains.2,3 It is lipophilic and has low solubility in water
at physiologic pH. In humans accumulation of bilirubin is
thwarted by its efficient clearance from the blood by the liver
and elimination in bile as ester glucuronide conjugates of the
propionic acid side-chains.1,4 Formation of the glucuronides
is catalyzed by a specific glucuronosyl transferase enzyme
(UGT1A1)5 and their excretion from the liver is mediated
by a membrane transporter known as MRP2 (multidrug
resistance associated protein 2).6 These two proteins are
important in the metabolism and hepatic elimination of
a large number of xenobiotics in addition to bilirubin.
Genetic deficiencies of either UGT1A1 or MRP2 and a vari-
ety of liver disorders can cause accumulation of bilirubin or
its glucuronides and clinical jaundice.5,6 In the absence of
UGT1A1, elimination of bilirubin is almost totally impaired.
If the concentration of unconjugated bilirubin in the circula-
tion exceeds the binding capacity of serum albumin, move-
ment of the pigment across the blood–brain barrier and
deposition within the brain can cause toxicity.1 In contrast
to bilirubin, xanthobilirubic acid (Fig. 1B),7 a polar but
water-insoluble synthetic dipyrrinone analog for one-half of
N
N
H
H
CH2
O
O
H
H
N
N
H
O
C
Z
Bilirubin
Z
(B)
N
N
H H
CO2H
O
Xanthobilirubic Acid
(C)
R
N
N
O
O
Xanthoglow (R = CH2CH2CO2H)
Sulfoglow (R = SO3Na)
Figure 1. (A) Bilirubin. (B) Xanthobilirubic acid, a dipyrrinone model for
bilirubin. (C) Xanthoglow, a highly fluorescent (fF 0.80, cyclohexane)
N,N0-carbonyl-bridged analog of xanthobilirubic acid and sulfoglow,
a water-soluble analog.
bilirubin, is readily excreted intact in bile without the need
for glucuronidation.8 Thus, bilirubin and its analogs are use-
ful probes for hepatobiliary disfunction and mechanisms of
glucuronidation and biliary excretion.
Keywords: Dipyrroles; Perfluorophenyl; 19F NMR; Fluorescence.
* Corresponding author. Tel.: +1 775 784 4980; fax: +1 775 784 6804;
e-mail: lightner@scs.unr.edu
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.04.046

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S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
Though brightly colored, bilirubin and xanthobilirubic acid
are essentially non-fluorescent at room temperature, e.g.,
with fluorescence quantum yields of fF<10ꢀ3 because of
rapid Z/E isomerization in the excited states.2,3,9
Restricting Z/E isomerization by bridging the two nitro-
gens of a dipyrrinone causes a dramatic increase in fluores-
cence (to fFw1). Recently we found that a carbonyl group is
a convenient bridge, easily inserted by reaction of the dipyr-
rinone with 1,10-carbonyldiimidazole (CDI) in the presence
of strong base (such as DBU) in CH2Cl2, and is very effec-
tive in enhancing fluorescence.10 Thus, xanthobilirubic
acid methyl ester was readily converted to xanthoglow
(Fig. 1C) methyl ester, with fF¼0.80 in cyclohexane at
25 ꢁC for lexc 410 nm and lem 473 nm.10
hyde.12 Commercialpentafluorobenzaldehydewascondensed
with nitroethane in the presence of DBU (Scheme 1), a Henry
reaction that afforded an alcohol that was acetylated and used
without purification in a Barton–Zard reaction with p-tolue-
nemethylisocyanide (TosMIC) to give 15. Replacing nitro-
acetate in Scheme 1 with a nitroalkene from a Knoevenagel
reaction between pentafluorobenzaldehyde and nitroethane
using ammonium acetate13 might also have provided a suit-
able precursor to pyrrole 15; however, this nitroalkene
synthesis was unsuccessful. a-Bromination followed by
treatment with TFA/H2O is a typical way12,14 of transforming
an a-free pyrrole into a pyrrolinone (e.g., 13), and this route
was successfully applied in transforming 16 into 14. The
same approach was found earlier by our group to give unsat-
isfactory results with 15. Therefore, direct oxidation of 15 to
13 using hydrogen peroxidewas examined. This reaction was
optimized over many runs, particularly by changing the con-
tact time from 12to 30 h. Theyield seemed dependent also on
the quality of the peroxide; an aged (lower percent peroxide?)
reagent gave the best results. Chromatographic purification
was necessary to obtain a 69% yield of 13. Quantitative
removal of the tosyl group to yield 10 was achieved using
sodium borohydride in ethanol.14
Seeking a water-soluble xanthoglow analog, we prepared the
first ‘sulfoglow’ (Fig. 1C),10a a xanthoglow analog with the
C(8) propionic acid replaced by sodium sulfonate. When
injected intravenously into rats it was rapidly excreted intact
into bile and urine, which became highly fluorescent.11 These
preliminary studies suggested that it might be possible
to develop highly fluorescent cholephilic (‘bile-loving’)
analogs useful for fluorescence imaging of renal and hepatic
metabolism or for early detection of cholestatic liver disease.
Were they to contain fluorine, such agents might also be use-
ful for 19F-magnetic resonance imaging. To this end we set
out to synthesize cholephilic highly fluorinated sulfoglows
(‘fluoroglows’). From our experience and perspective, a log-
ical target was pentafluoroglow 1. In the course of this study,
we learned, however, that its synthesis was compromised in
the presence of nucleophilic base (ethoxide) in favor of p-
ethoxytetrafluoroglow (2) and regioisomers. We also present
the synthesis of pure 2 here because it provides a key inter-
mediate for synthesizing p-alkoxy analogs of 2 of differing
lipophilicity and hence regulating hepatic versus renal excre-
ability.11 In the following, we describe the syntheses and
spectroscopic properties of new fluorinated, fluorescent di-
pyrrinones 4–6 and the sulfoglow sodium salts 1 and 2,
with pentafluoro- and tetrafluorophenyl groups on the lactam
ring. We also present preliminary studies on the metabolism
and hepatobiliary excretion of 1 and 2. This work is part of a
more comprehensive investigation of cholephilic fluorescent
pharmacophores for understanding transhepatic uptake
and transport and cholestatic disorders, particularly in the
Base-catalyzed condensation of 10 with 3,5-dimethyl-2-for-
myl-1H-pyrrole15 should have led directly to 7, but replace-
ment of aromatic ring fluorine also occurred. Although
substitution of the para-fluorine might have been expected,
we observed a lack of regioselective replacement during
test condensation reactions of pyrrolinone 10. With a 2-
fold excess of aldehyde versus pyrrolinone 10, freshly pre-
pared sodium methoxide or ethoxide, and a reaction time
of 20 h at reflux, the yield of isolated yellow pigment from
methanol was lower than from higher boiling ethanol,
from which w58% of a yellow pigment was obtained after
Ar
Ar
a
c
+
CH3CH2NO2
ArCHO
Ts
O2N
OR
N
H
15: Ar = C6F5
16: Ar = C6H5
Ar = C6F5 or C6H5
R = H
R = Ac
(34%)
(63%)
b, 78%
d
Ar
Ar
newborn.11
Ar
g
4
e
H
H
Target Compounds
O
H
N
N
O
N
H
H
Ts
N
H
H
O
OHC
N
H
R
O
R
O
7: Ar = C6F5
8: Ar = 4-EtOC6F4
9: Ar = C6H5
10: Ar = C6F5
11: Ar = 4-EtOC6F4
12: Ar = C6H5
13: Ar = C6F5
14: Ar = C6H5
(84%)
(74%)
(64%)
(95%)
(71%)
(95%)
(69%)
(86%)
8
8
f
SO3Na
H
N
N
N
N
O
O
h
R
C6F5
p-EtOC6F4
C6H5
1
4
5
6
Ar
Ar
2
SO_3Na+
(39%)
2: Ar = 4-EtOC6F4 (45%)
3: Ar = C6H5
i
H
3
N
N
N
N
O
O
O
O
4: Ar = C6F5
5: Ar = 4-EtOC6F4
6: Ar = C6H5
1: Ar = C6F5
(67%)
(74%)
(93%)
2. Results and discussion
2.1. Syntheses
Scheme 1. Reagents and conditions: (a) DBU; (b) Ac2O, DMAP; (c)
TosMIC, TMG; (d) H2O2 or i: PhNMeþ3 Br3ꢀ, ii: TFA, H2O; (e) NaBH4; (f)
NaOEt; (g) N-methylmorpholine; (h) CDI, DBU; (i) concd H2SO4, then
Na2CO3.
The syntheses of 4 and 5 employed a Barton–Zard pyrrole-
forming reaction sequence explored earlier with benzalde-

S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
7045
chromatography and crystallization. Although the pigment
from each solvent was crystalline and homogeneous by
TLC, 1H NMR spectroscopy revealed onlyw80–85% p-alk-
oxy regioisomeric purity. Recrystallizations, even with
w50% loss, improved the purity of the ethoxy pigment 8
to only w90%. 1H NMR spectroscopy, in particular the
C(5)-methine signal, was indicative of the presence of three
different dipyrrinones, most likely due to ortho- as well as
para-substitution and ortho, para-disubstitution of fluorine
by ethoxy. The 13C NMR spectral lines of the minor isomeric
impurities, heavily split by the fluorines, were very weakly
intense and were thus of little use in analyzing different sub-
stitution patterns. But 19F NMR spectroscopy, with its wide
dispersion range, revealed unequivocally the presence of the
mono ortho-substituted analog of 8, in addition to para.
hydroxide/ethanol) because we suspected that a high
reaction temperature and a nucleophilic base16–18 would
facilitate further substitution of (ortho) fluorines. Thus, con-
densation using a non-nucleophilic base in a non-nucleo-
philic solvent was sought. With a strong non-nucleophilic
base (DBU) in DMF or CH3CN solvent at 85–90 ꢁC,
condensation of 11 with 2-formyl-3,5-dimethylpyrrole
(Scheme 1) led mostly to decomposition products and no
formation of dipyrrinone. Hunig’s base (N,N-diisopropyl-
¨
ethylamine) in CH3CN led to no reaction, and the strong
dibasic amine, 1,4-diazabicyclo[2.2.2]octane (DABCO) in
CH3CN at 90 ꢁC resulted mostly in decomposition products.
However, with N-methylmorpholine in dimethylformamide
at 90 ꢁC, a promising 31% yield of dipyrrinone was
obtained. With a change of solvent to CH3CN and increased
reaction time to 84 h, 11 was converted smoothly into dipyr-
rinone 8 in 74% yield.
The pentafluorophenyl ring is known to be susceptible to
aromatic nucleophilic substitution, and so we considered
pentafluorobenzaldehyde16 reactivity with nucleophiles to
be a model for that of a pentafluorophenyl b-substituent of
a dipyrrinone, where in both cases the carbon attached to
the fluorinated benzene ring is sp2 hybridized. Typical fluo-
rine-replacing nucleophilic aromatic substitution reactions
including the reaction of pentafluorobenzaldehyde with (i)
sodium hydrosulfide in DMF and (ii) sodium thiophenolate
in CH3OH yielded predominantly para-thiophenol and
para-phenylthioether, respectively.17 A different ortho–
para selectivity was found in the reaction of dimethylamine
with pentafluorobenzaldehyde in diethyl ether at room tem-
perature for 1 h: the ortho-amino isomer dominated over the
para by a 3:2 ratio.17 No clear-cut procedure has been
reported for the reaction of pentafluorobenzaldehyde with
sodium methoxide in methanol; however, the para-methoxy
derivative has been isolated in low yield after careful distil-
lation.17 Similarly, methoxide ion replaced predominantly
the para-fluorine in bromopentafluorobenzene after reflux
in methanol for 8 h and isolation in 70% yield by preparative
gas-chromatography.16 Recent papers discussed the regio-
selectivity of nucleophilic replacement (including by ethox-
ide) in di-, tri-, tetra-, and pentafluoropyridines.18 On the
basis of the available literature and the preliminary work de-
scribed above, we deemed it advantageous to find conditions
for regioselective replacement of para-fluorine only.
With the proper conditions for dipyrrinone formation from
a fluorine-containing pyrrolinone having been found, they
were successfully employed on the pentafluorophenyl
substituted starting material (10). Using pure pyrrolinone
10 and 3 equiv of 3,5-dimethylpyrrole-2-aldehyde in pres-
ence of N-methylmorpholine in CH3CN at 95 ꢁC for 84 h,
the desired dipyrrinone 7 bearing a C(3)-pentafluorophenyl
group was isolated in 84% yield (which was even higher
than that obtained with p-ethoxy analog 8). The solubility
of 7 in chloroform (or dichloromethane) is very low, how-
ever, in comparison with 8.
Although the yield at the condensation step was higher for 7
than 8, its conversion to the N,N0-carbonyl bridged fluoro-
phore 4 was not as clean and high-yielding as is usually
observed.10,11 Only 67% of purified tricycle 4 was isolated
from a reaction, which usually gives >90% of product.
The starting material 7 was almost completely consumed,
and a significant amount of rather polar side product was iso-
lated. Its structure was deduced from 1H and 13C NMR spec-
tra to be the analog of 5 with a 1-imidazolyl ring replacing
ethoxy at the para-position. Thus, nucleophilic substitution
of the para-fluorine cannot be completely suppressed even
under the mild conditions used. The adventitious imidazole
nucleophile apparently is released from CDI after the car-
bonyl transfer reaction that forms the bridged dipyrrinone.
Instead of synthesizing the dipyrrinone while simultaneously
and non-selectively replacing fluorine with an ethoxy group,
regioselective para-fluorine substitution was achieved in an
earlier step, under milder conditions. A significant improve-
ment in regioselectivity was achieved when the pyrrolinone
10 was treated at sub-ambient temperature with a freshly pre-
pared solution of sodium ethoxide in ethanol. Optimized
parameters included the exact amount of ethoxide and the
duration of contact, with best results being obtained from
5 equiv of sodium ethoxide and a 1-h reaction time at
w10 ꢁC to give a 71% yield of 11 from 10. In light of recent
literature data, the reaction might be successful even at
a lower temperature. After optimization, similar conditions
were also found to give good regioselectivity for para-substi-
tution relative to ortho in recently published work.18
For comparison of spectroscopic data, the non-fluorinated
parent of 7 was prepared from the known pyrrolinone
12.12 Here, too, condensation with 3,5-dimethylpyrrole-2-
aldehyde was conducted using N-methylmorpholine in
CH3CN at 95 ꢁC for 48 h to give the then unknown C(3)-
phenyl-substituted dipyrrinone (9) in 64% yield. The typical
reaction conditions (KOH/EtOH) gave unacceptable results:
a mixture of two products in low yield. Cyclization of 9,
unlike 7 or 8, smoothly afforded tricyclic 6 in 93% yield.
Insertion of the carbonyl bridge by reacting 8 with CDI and
DBU was sluggish and afforded the N,N0-carbonyl-bridged
dipyrrinone 5, which was isolated in moderate 74% yield.
A similar slow carbonyl insertion and comparable product
yield (67%) were found in the conversion of 7 to 4.
Sulfonation of 5 using concd H2SO4 at 0 ꢁC, followed by
an alkaline quench, gave sodium sulfonate 2, which seemed
to be less soluble in water than its C(3)-n-decyl analog
With p-ethoxypyrrolinone 11 now isolated in pure form, we
examined ways to carry it forward to dipyrrinone 8 while
avoiding strongly nucleophilic ethoxide/ethanol (or sodium

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S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
synthesized earlier.11 After quenching the sulfonation
reaction, the organic material separated as a solid from
both aqueous and organic (chloroform) phases. More com-
plete extraction into chloroform was achieved by addition
of methanol. The crude product was purified by radial chro-
matography to afford a 45% yield of the yellow sulfonate
to rotation19 at coalescence according to: DGz¼RTc
[22.96+ln(Tc/Dn)]. Substituting Tc and Dn gives a barrier
DGz¼13.7 kcal molꢀ1, which indicates only a moderately
hindered atropisomerization process.
Typically, when an AB-spin system is envisioned, methylene
protons in the neighborhood of an element of chirality come
first to mind. However, 19F NMR, with intrinsically high re-
ceptivity of fluorine nuclei, has been used for stereochemical
purposes and for testing basic NMR concepts chronologi-
cally in parallel to 1H NMR. Yet, despite the enormous quan-
tity of 19F NMR spectral data accumulated,20,21 including
material clearly related to stereochemistry and conforma-
tional analysis,19,22 there are few clear-cut examples of AB
fluorine spectra.
1
salt. Its purity was determined by H NMR to be >90%.
Sulfonation of 4 (to 1) was found to be more sluggish, and
under the reaction conditions of 5/2, no product (1) was
found. At 25 ꢁC for 4 h, however, 4 was transformed to 1
in 39% yield. In contrast to these two reactions, attempted
conversion of 6 to 3 resulted in products that were not ex-
tractable, presumably due to multiple sulfonation, of the
phenyl ring and other sites.
2.2. Constitutional structures
The closest literature examples of diastereotopic fluorines in
a CF2 group are in perfluorinated compounds. Variable tem-
perature 19F NMR has been reported for 1-perfluorohexyl-1-
phenylethanol,23 which has one stereogenic center and five
pairs of prochiral fluorines at increasing distance. At
353 K only the two nearest CF2 show diastereotopicity and
at 193 K only the central in the chain CF2 continues to
show a singlet with small satellites as a sign of nonequiva-
lence. The structure of perfluoro 4-ethyl-3,4-dimethyl-
hexan-3-yl carbanion has also been studied by dynamic
19F NMR.24 At 298 K only the two CF2 groups furthest
from the carbanionic center exhibit diastereotopicity
(ABX3 spin system). The CF2 closest to the negative charge
shows anisochronous fluorines below 193 K, and this was in-
terpreted as an indication of frozen rotation around the rele-
vant bonds at the carbanionic center. The only AB fluorine
spin system from an intentionally labeled CF2 was found
in a difluoromethionine incorporated in three different sites
in a recombinant protein.25 The degree of chemical shift
difference is small when the amino acid is at relatively
free surface positions, but the anisochronicity is enhanced
for a methionine incorporated in tightly packed protein
core where there is less conformational freedom. Variable
temperature 19F NMR and 19F{19F} COSY experiments of
free difluoromethionine have been also reported.25
The constitutional structures of 1, 2, 4–16 follow from the
1
method of synthesis and their H and 13C NMR spectra.
Pyrrolinone 12 was reported previously.12 The 19F NMR
spectra of 1, 2, 4, 5, 7, 8, 10, 11, 13, and 15 were also ob-
tained and correlated with the assigned structures. The 19F
NMR spectrum of 13 differed in significant ways from the
other nine. In particular, whereas 10 showed three 19F
NMR signals with second order 19F{19F} couplings
(ꢀ139.51 ppm, ortho; ꢀ152.89 ppm, para; ꢀ161.51 ppm,
meta), 13 exhibited two sharp and two very broad signals
at 298 K (Fig. 2). The unexpected appearance of four chem-
ical shifts at ambient temperature was clarified by lowering
the temperature to 223 K where five chemical shifts were
found, with the two most downfield signals showing fine
structure. From this apparently more complex spectrum,
it became clear that a dynamic process occurs in 13.
Although the same process might occur in the five other pen-
tafluorophenyl substituted compounds (1, 4, 7, 10, and 15), it
was not detected by NMR. What makes 13 unique is the
presence of a stereogenic center at C(4) bearing the tosyl
group. Stereogenic centers are absent in 1, 4, 7, 10, and
15—as well as in 2, 5, 8, and 11 (the p-ethoxy analog of
13 was not prepared). The stereogenic center in 13, com-
bined with the axis of rotation defined by C(3)–C(ipso)
bond (an axis of chirality), renders the ortho- and meta-fluor-
ines diastereotopic. They lie in different electronic environ-
ments in the preferred conformation of 13, and 19F NMR is
sufficiently sensitive to detect different chemical shifts even
at room temperature. Rotation around the C(3)–C(ipso)
bond is rather uninhibited at 298 K, and only the ortho-
fluorines, being closest to the stereogenic center, are aniso-
chronous. At lower temperature, the hindered rotation
extends the diastereotopicity to meta-fluorines as well (at
ꢀ160.57 ppm and ꢀ160.75 ppm). In principle, the phenyl
ortho and meta hydrogens, as well as those from the tosyl
Although difficult to perform at sub-ambient temperatures,
19F{19F} COSY spectrum was acquired on a sample of pyr-
a
rolinone 13 in CDCl3 at 223 K (Fig. 2). The experiment
unequivocally confirmed the assignments made earlier on
the ground of substituent chemical shift increments. At
low temperature the two downfield signals with chemical
shifts at ꢀ136.92 ppm and ꢀ138.71 ppm correlated with
the most shielded fluorine nuclei at ꢀ160.57 ppm and
ꢀ160.75 ppm. With scale expansion it becomes evident
that the signal at ꢀ136.92 ppm correlates with that at
ꢀ160.57 ppm, and the downfield signal at ꢀ138.71 ppm
correlates with that at ꢀ160.75 ppm. Both upfield signals
(ꢀ160.75 ppm and ꢀ160.57 ppm) correlate with the signal
at ꢀ150.28 ppm but the latter did not show any other corre-
lations. This means that the latter signal belongs to the fluo-
rine nucleus attached to the para-position, which does not
show long range (4J) coupling to the ortho-fluorines. Such
coupling (4J¼2.9 Hz), however, was detected at 323 K in
the 1D high resolution 19F NMR spectrum (Fig. 2A,
upper middle trace). From the assignment of the para-
fluorine signal, it follows that the signals at ꢀ160.75 ppm
1
group of 14 are diastereotopic, but the H NMR spectrum
was too complicated to resolve due to overlapping signals.
The coalescence temperature (Tc) of ortho- and ortho0-19F
NMR signals of 13 was 313 K in CDCl3. The first order
rate constant (kc) of the exchange process (i.e., the rotation
1=2
rate) at this temperature is given by: kc ¼ p ꢂ Dn=ð2Þ
where Dn is the signal separation in hertz at stop-exchange
regime.19 From the low temperature experiments
Dn¼841 Hz, which gives a rate kc¼1870 sꢀ1. Dynamic 19
F
NMR also allows for calculation of the activation barrier

S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
7047
(A)
(C)
(B)
F
F
F
F'
F'
H
O
Ts
N
H
13
Figure 2. (A) Expanded partial 19F NMR spectra of tosylpyrrolinone 13 in CDCl3 at 323 K (upper traces), at 298 K (middle traces), and at 223 K (lower traces),
referenced to external C6F6 in CDCl3 at ꢀ162.90 ppm. (B) Stereochemical drawing of 13. (C) Full 19F{19F} COSY spectrum of 13 in CDCl3 at 223 K.
and ꢀ160.57 ppm are from m,m0-fluorines and those at
ꢀ138.71 ppm and ꢀ136.92 ppm from o,o0-fluorines. In
other words, only the most shielded signals (those assigned
to m,m0-fluorines) exhibit a full complement of off-diagonal
peaks, all of which are the result of spin–spin coupling via
three bonds.
substituted dipyrrinone analog.11 The shieldings in 7–9 are
due to the influence of the aromatic p-system attached at
C(3) of the dipyrrinone, because the aromatic ring faces
the C(5)-hydrogen, as supported by molecular mechanics
conformational analysis using PC Model.26 PC Model re-
vealed two isoenergetic minima when the aromatic ring of
7 was allowed to rotate with respect to the lactam, with
dihedral angles C(2)–C(3)–C(ipso)–C(ortho-upper)¼+65ꢁ
and ꢀ65ꢁ.
The structures of 4–6 also followed from the method of syn-
thesis and from 13C NMR spectra that were characteristic
of dipyrrinones (7–9)7,11 and N,N0-carbonyl-bridged di-
pyrrinones (4–6),10,11 with the latter showing a urea-type
carbonyl at w143 ppm. In the 1H NMR, 7–9 exhibited
more shielded C(5)-hydrogens compared to their C(3)-alkyl
counterparts. Thus, C(3)-phenyl 9 showed this proton at
6.06 ppm, the pentafluorophenyl 7 at 5.78 ppm, and its
p-ethoxy analog 8 at 5.82 ppm versus the normally encoun-
tered chemical shift, e.g., 6.13 ppm for the C(3)-n-decyl
2.3. 1H NMR chemical shifts and hydrogen-bonded
dimers
The dipyrrinone NH 1H NMR chemical shifts in CDCl3 re-
veal much about the extensively studied monomer$dimer
equilibrium.27 Comparison of dipyrrinone pyrrole and
lactam NH 1H NMR chemical shifts in 7–9 and in the

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S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
Table 1. Comparison of dipyrrinone 1H NMR NH chemical shiftsa for 7–9 and their C(3)-n-decyl analog in CDCl3 at 25 ꢁC
R
3
H
N
N
H
H
O
Chemical shift
Lactam NH
M
7 (R¼C6F5)
11.97
11.84
11.61
8 (R¼4-EtOC6F4)
11.91
11.76
11.51
9 (R¼C6H5)
11.78
11.53
11.02
R¼(CH2)9CH3
11.30
11.08
10.58
1ꢂ10ꢀ2
1ꢂ10ꢀ3
1ꢂ10ꢀ4
Pyrrole NH
1ꢂ10ꢀ2
1ꢂ10ꢀ3
1ꢂ10ꢀ4
10.51
10.42
10.22
10.51
10.40
10.17
10.56
10.43
10.11
10.44
10.30
9.97
a
d, ppm downfield from Me4Si.
C(3)-decyldipyrrinone synthesized earlier11 was made at the
same sample concentration (Table 1). In all four examples,
evidence of intermolecular hydrogen bonding is evident
from the deshielded NH resonances at the typical chemical
shifts.27 A 10-fold dilution, from 10ꢀ3 to 10ꢀ4 M causes
the expected slight upfield shift, again a shift that correlates
with a monomer$dimer equilibrium. The C(3)-n-decyl
substituted dipyrrinone exhibited somewhat less deshielded
NH chemical shifts than 7–9, suggesting slightly stronger
intramolecular hydrogen bonding in the latter.
simply stronger association. A deshielding edge effect from
the aromatic ring might be felt by the NH, a view supported
by the gradual increase of chemical shift in changing the un-
substituted phenyl to p-ethoxytetrafluorophenyland to penta-
fluorophenyl. Based on data at 10ꢀ3 M concentration, the
electronegative fluorinated rings of 7 and 8 are more effective
in deshielding the lactam NH to 11.84 ppm and 11.76 ppm
than the unsubstituted phenyl ring (11.53 ppm). The calcu-
˚
lated distances, lactam NH to ortho-F, are 4.8–5.0 A (PC
Model).26 It seems probable that both the shielding of
C(5)-H and the deshielding of lactam NH are expressions
of the same magnetic anisotropy of the aryl ring on C(3).
If the aromatic substituents at C(3) increase the donor ability
of the lactam NH (or the acceptor ability of the carbonyl ox-
ygen), then the dimerization constant is expected to increase.
In fact, the lactam NH appears to be more sensitive to C(3)
substituent and concentration than the pyrrole NH. The larg-
est lactam NH deshielding difference (Ddw0.5 ppm) occurs
when changing from C(3)-n-decyl to C(3)-phenyl and not
between, e.g., phenyl and pentafluorophenyl, suggesting
that the relative NH deshielding has an origin other than
2.4. UV–vis absorption and fluorescence emission
All the dipyrrinones and N,N0-carbonyl-bridged dipyrrinones
are yellow compounds forming yellow solutions. The latter
are highly fluorescent. Comparison of their UV–vis data
(Table 2), particularly those in chloroform (Fig. 3), shows
only small perturbations of lmax and 3max due to the C(3)
Table 2. Comparison of UV–vis spectral data of N,N0-carbonyl-bridged dipyrrinones 1–2, 4–6, and 7–9a
Pigment
3max (lmax, nm)
Cyclohexane
C6H6
17,600 (447)
10,700 (282)
CHCl3
CH3OH
(CH3)2SO
H2O
1
2
4
17,700 (437)
12,300 (278)
18,600 (433)
12,900 (275)
18,400 (447)
12,100 (281)
17,700 (433)
22,900 (271)
13,500 (440)
10,800 (283)
13,700 (436)
11,500 (280)
14,400 (432)
11,600 (276)
13,800 (444)
11,300 (281)
13,400 (431)
12,000 (273)
20,400 (451)
20,000 (432)
10,900 (277)
19,200 (449)
19,200 (447)
19,400 (443)
19,300 (445)
sh 19,000 (439)
19,500 (444)
12,300 (279)
19,000 (443)
13,000 (277)
19,200 (441)
12,400 (276)
19,100 (443)
5
6
20,100 (448)
20,200 (428)
13,600 (278)
13,600 (281)
18,200 (433)
14,000 (281)
18,000 (433)
14,200 (279)
17,900 (431)
13,500 (279)
18,200 (433)
19,200 (437)
19,500 (419)
sh 12,900 (281)
13,800 (279)
39,200 (425)
13,600 (278)
38,200 (431)
13,600 (278)
35,800 (429)
7
sh 24,900 (453)
48,000 (427)
6800 (299)
sh 22,000 (456)
42,300 (429)
6200 (299)
6200 (290)
6000 (290)
sh 6100 (287)
35,300 (429)
8
9
a
sh 24,700 (447)
46,100 (423)
7600 (297)
sh 23,000 (452)
42,000 (427)
7000 (298)
37,200 (423)
37,700 (429)
7400 (285)
6900 (286)
7400 (282)
sh 22,600 (439)
43,600 (412)
7500 (290)
sh 22,400 (441)
40,700 (415)
7100 (292)
35,700 (411)
36,900 (417)
34,400 (417)
7200 (282)
6800 (282)
7200 (286)
Concentration range 2.3–3.8ꢂ10ꢀ5 M at 20 ꢁC.

S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
7049
-3
compared to the C(3)-n-decyl substituted dipyrrinone in
chloroform: 3¼33,800 (399 nm) (Fig. 3). A significant (11–
23 nm) bathochromic shift is seen in DMSO, as compared
to C(3)-n-decyl dipyrrinone, 3¼34,600 (406 nm).
ε x 10
30
20
10
0
The influence of the nature of the para-substituent (F vs EtO
vs H) on the absorption wavelength suggests (at least partial)
conjugation between the dipyrrinone p-system and the C(3)-
aryl group p-system. Similar effects are observed in the
N,N0-carbonyl-bridged dipyrrinones (Table 2 and Fig. 3)
and correlate directly with a decreased reactivity of 4 versus
5 in the sulfonation step.
Fluorescence of N,N0-carbonyl-bridged dipyrrinones 4–6
was measured in five solvents, and the intensity of fluores-
cence as fluorescence quantum yields (fF) was quantified
using 9,10-diphenylanthracene standard as described pre-
viously.10c Their excitation, emission wavelengths, and
calculated quantum yields in cyclohexane, benzene, chloro-
form, methanol, and dimethylsulfoxide are given in Table 3.
These data might be compared with similar dipyrrinones de-
scribed earlier,10,28 but the best reference compound is the
dipyrrinone analog with C(8)-free and C(3) substituted
15
10
5
with C(3)-n-decyl, which showed fF¼0.66 and emission
em
max
l
¼ 490 nm in chloroform whereas the 6 analog showed
fF¼0.57 and lemmax ¼ 502 nm. The corresponding p-ethoxy-
tetrafluorophenyl tricycle
5
exhibited fF¼0.59 with
em
em
max
l
¼ 508 nm, and the pentafluorophenyl analog 4 had
fF¼0.57 and maximum emission at
l
¼ 509 nm
max
(Fig. 4). These data show that the quantum yields are of sim-
ilar magnitude whether the C(3) substituent is aryl, fluori-
À
Á
em
max
nated aryl or alkyl, and the emission wavelength
l
0
300
350
400
λ [nm]
450
500
follows the same trend observed in the UV spectra: a signif-
icant bathochromic shift accompanies the replacement of
C(3)-n-decyl with phenyl group, and an additional but
smaller bathochromic shift occurs with fluorine substitution
on the aryl group.
Figure 3. Comparison of UV–vis spectra in chloroform of dipyrrinones
(upper panel) and their N,N0-carbonyl-bridged derivatives (lower panel)
substituted at C(3) with: n-decyl (dotted line), phenyl (dash–dotted line),
p-ethoxytetrafluorophenyl (dashed line), and pentafluorophenyl (solid line).
Sodium sulfonates 1 and 2 exhibited similar high fluores-
cence quantum yields in organic solvents: fFw0.4–0.6.
Even in non-polar benzene, the values are in agreement with
those of 4–6, thus indicating that aggregation or solubility
issues are not influencing the emission. In dimethylsulfoxide
solvent somewhat enhanced fluorescence of 1 and 2 was de-
tected in comparison to 4–6. In aqueous solutions the fluo-
rescence efficiency is diminished to about 0.3 but is still
sufficient for detection in biological samples—and this sol-
vent offers the largest Stokes shift. The fluorescence emis-
sion wavelength from 1 to 2 is not affected significantly by
substituents. The parent dipyrrinone 9 in CHCl3 has
3¼35,700 (411 nm), the tetrafluorinated 8 has 3¼37,200
(423 nm), and the pentafluorinated analog 7 has 3¼39,200
(425 nm); and in DMSO 9 has 3¼34,400 (417 nm), tetra-
fluorinated 8 has 3¼35,300 (429 nm), and the pentafluori-
nated 7 has 3¼35,800 (429 nm)—evidence that the
electron-withdrawing phenyl substituents exert a significant
bathochromic shift (of 12–14 nm) and that non-polar sol-
vents cause a gradual but considerable hyperchromic shift,
Table 3. Fluorescence data for N,N0-carbonyl-bridged dipyrrinonesa of this work and their C(3)-n-decyl analog
Pigment
Cyclohexane
lem
C6H6
CHCl3
CH3OH
(CH3)2SO
lem
fF
fF
lem
fF
lem
fF
lem
fF
1b
—
—
0.58
0.67
0.60
0.78
—
—
480
475
466
463
0.51
0.52
0.58
0.61
0.60
0.74
516
513
508
504
494
472
0.43
0.57
0.57
0.59
0.57
0.66
513
510
509
508
502
490
0.40
0.41
0.39
0.39
0.39
0.36
536
534
541
539
533
523
0.67
0.62
0.56
0.59
0.56
0.67
537
532
533
530
521
501
2c
4
5
6
C(3)-n-decyl
Concentration range 7.7–9.0ꢂ10ꢀ7 M; excitation lex¼413–450.
In H2O, fF¼0.30, lem¼546 nm.
a
b
c
In H2O, fF¼0.29, lem¼542 nm.

7050
S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
the short biliary cannula, onset of the efflux of 1 from the
I
liver must occur within less than 3 min. The smaller less po-
lar peak in the chromatograms is yet to be identified. Its nor-
malized absorption spectrum in the HPLC mobile phase is
almost superimposable on that of 1, suggesting no major
structural modification. Although it could be a metabolite
of 1, Phase I and Phase II metabolic reactions in the liver
generally generate products more polar than the precursor.
The shape of the peak suggests that it might also be a chroma-
tographic artifact caused by colorless co-migrating constitu-
ents of bile. Figure 5b shows a biliary excretion curve for 1,
obtained by plotting HPLC peak areas of 1 in bile normal-
ized to the sample with the maximum peak area. Most pig-
ment was excreted within the first 30 min after injection
and the fraction of the administered dose excreted un-
changed in bile in two animals in 4 h was 0.5. Unchanged
1 and its possible metabolite was also detectable by HPLC
and by fluorescence in urine samples collected intermittently
during the experiment. Thus, 1 is cleared rapidly and prefer-
entially from the circulation by the liver and eliminated
predominantly by biliary excretion, with a smaller fraction
appearing in urine.
450
500
550
λ [nm]
600
650
700
Figure 4. Comparison of fluorescence emission band in chloroform of N,N0-
carbonyl-bridged dipyrrinones substituted at C(3) with: n-decyl (dotted
line), phenyl (dash–dotted line), p-ethoxytetrafluorophenyl (dashed line),
and pentafluorophenyl (solid line). The vertical axis plots relative intensity
(I) of fluorescence emission.
The tetrafluorophenyl analog 2 behaved qualitatively simi-
larly to 1, being excreted rapidly in bile in unchanged
form after intravenous injection, with an apparently smaller
fraction appearing in urine. However, significant amounts of
metabolites of 2, especially less polar metabolites, were not
detected. Determination of the fraction of the dose of 2 ex-
creted in bile was hampered by poor recoveries in the anal-
yses of 2 in the serum injectate. The reason for this is
presently unclear, but may be related to the high affinity of
2 for serum albumin. However, it was clear from the bile
chromatograms that 2 is excreted preferentially via the liver
into bile. Thus, both 1 and 2, like the sulfoglows reported
previously, are cholephilic. They appear to be taken up
rapidly by hepatocytes in vivo in the rat, and then excreted
rapidly across the biliary canalicular membrane into bile.
the presence of the C(8) sulfonate group as compared to the
C(8)-free parents 4–6.
2.5. Hepatic excretion
When 1, dissolved in serum, was injected intravenously as
a bolus in the rat, the endogenous fluorescence of bile rapidly
increased and a prominent peak, followed by a smaller
broader peak, was detected in bile by HPLC within 3 min
of the injection (Fig. 5a). The prominent peak was identified
as unchanged 1, based on its absorption spectrum and reten-
tion time. The highest concentration of 1 in bile was ob-
served in the first sample collected, 3 min after the
intravenous injection. Since bile flows only slowly through
Figure 5. Hepatobiliary excretion of 1 in the homozygous Gunn rat. (a) HPLC chromatograms of bile collected just before (lower trace) and 3 min after (upper
trace) intravenous injection ofw0.25 mg 1 dissolved in 1 mL rat serum. The inset shows a chromatogram of a sample of the serum solution of 1 that was injected.
(b) Biliary excretion curve for 1 generated by plotting HPLC peak areas of unchanged 1 in bile before and after its injection. Values were normalized to the
maximum peak area (at 3 min).

S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
7051
3. Concluding comments
diluted in 10-fold increments to 10 mM. The 1H NMR spec-
tra were acquired using a high sensitivity probe for indirect
detection. The pulse width was set at about the optimum 65ꢁ
flip angle and the total repetition time was kept at 8–10 times
the T1 values of NH signals to allow for complete relaxation.
Artificial high line broadening was used in the processing to
smoothen the low intensity signals, in particular at low con-
centrations. Overall, meticulously identical experimental
conditions with samples at the same concentrations were
used and all spectra were referenced to the residual CHCl3
signal at 7.260 ppm. This precision was necessary to mea-
sure the chemical shifts with 10ꢀ3 ppm confidence.
Two new, highly fluorinated (perfluorophenyl and p-ethoxy-
tetrafluorophenyl substituents) N,N0-carbonyl-bridged di-
pyrrinones were converted to amphiphilic derivatives by
sulfonation. The highly fluorescent sulfoglows (1 and 2) so
obtained are excreted rapidly and principally in unchanged
form in bile in the rat after intravenous infusion. Some excre-
tion in urine also was observed. These compounds may form
the basis for development of 19F MRI and fluorescence
agents for probing liver metabolism and disease.
4. Experimental
4.1.1. 4-Methyl-3-pentafluorophenyl-2-(4-toluenesul-
fonyl)-1H-pyrrole (15).
4.1. General procedures
4.1.1.1. 2-Nitro-1-pentafluorophenylpropanol.
To
a solution of 23.53 g (120 mmol) of perfluorobenzaldehyde
and 11.26 g (150 mmol) of nitroethane in 12 mL of anhyd
CH3CN was slowly added 1.8 mL (12 mmol) of DBU, and
the mixture was stirred for 30 h at room temperature. The
mixture was diluted with 200 mL of CH2Cl2, washed with
3% aq HCl (100 mL), H2O (3ꢂ100 mL), dried over anhyd
MgSO4, filtered, and the solvent was evaporated under vac-
uum (1 h at 40 ꢁC). The crude nitropropanol is a 1:1 mixture
Nuclear magnetic resonance spectra were acquired on
a Varian Unity Plus spectrometer at 11.75 T magnetic field
1
strength operating at an H frequency of 500 MHz, 13C
frequency of 125 MHz, and 19F frequency of 470 MHz in
solutions of CDCl3 (referenced at 7.26 ppm for 1H and
77.00 ppm for 13C) or (CD3)2SO (referenced at 2.49 ppm
1
for H and 39.50 ppm for 13C). All 13C NMR spectra were
broadband 1H decoupled, and J constants indicated for
some signals are from 13C–19F coupling. The 19F NMR
spectra were referenced to external C6F6 in CDCl3 at
ꢀ162.90 ppm. UV–vis spectra were recorded on a Perkin
Elmer Lambda 12 spectrophotometer. All fluorescence spec-
tra were measured on a Jobin Yvon FluoroMax 3 by using
constant spectral parameters: step resolution (increment)
of 1 nm, both excitation and emission slits of 2 nm, and in-
tegration time of 0.5 s. Radial chromatography was carried
out on Merck preparative layer grade silica gel PF254 with
CaSO4 binder, using a Chromatotron (Harrison Research,
Inc., Palo Alto, CA) with 1, 2 or 4 mm thick rotors.
Analytical thin-layer chromatography was carried out on
J.T. Baker silica gel IB-F plates (125 mm layer). Melting
points were determined on a Mel-Temp capillary apparatus
and are uncorrected. Combustion analyses were carried out
by Desert Analytics, Tucson, AZ. High resolution mass-
spectra were obtained from Nebraska Center for Mass
Spectrometry, Lincoln, NE.
of diastereomers: H NMR d: 1.40, 1.76 (2ꢂ1.5H, 2ꢂd,
1
J¼6.9 Hz), 5.03–5.08 (1H, m), 5.43–5.52 (1H, m) ppm;
13C NMR d: 16.1, 19.5, 61.2, 67.3, 85.0, 86.0, 112.4 (m),
1
136.1 (dm, J¼250.7 Hz), 143.4 (dm, 1J¼249.8 Hz), 146.7
1
(dm, J¼250.2 Hz) ppm and was used directly in the next
step.
4.1.1.2. 1-Acetoxy-2-nitro-1-pentafluorophenylpro-
pane. To a solution of the alcohol (26.1 g) from above in
70 mL of anhyd CH2Cl2 and 50 mg of DMAP was added
acetic anhydride (18.1 mL, 193 mmol) during 10 min, and
the mixture was stirred for 16 h. Methanol (25 mL) was
added during 15 min, and after 1 h stirring the mixture was
diluted with 200 mL of CH2Cl2 and then carefully poured
into 150 mL of satd aq NaHCO3. The organic layer was
washed with H2O (3ꢂ100 mL), dried over anhyd MgSO4,
filtered, and the solvent was evaporated under vacuum to
give 29.3 g (w78%) of a 1:1 diastereomeric mixture of nitro-
acetates. 1H NMR d: 1.46, 1.48 (2ꢂ1.5H, 2ꢂd, J¼6.8 Hz),
2.04, 2.21 (2ꢂ1.5H, 2ꢂs), 4.91–4.99 (1H, m), 5.19–5.29
(1H, m) ppm; 13C NMR d: 14.6, 14.7, 20.0, 21.8, 67.4,
67.7, 83.8, 84.0, 108.5 (m), 136.0 (dm, 1J¼250.9 Hz),
143.6 (dm, 1J¼248.7 Hz), 145.9 (dm, 1J¼251.1 Hz),
167.0, 168.1 ppm. This mixture was used directly in the
next step without further purification.
Spectral data were obtained in spectral grade solvents
(Aldrich or Fischer), which were distilled under Ar just prior
to use. Before distillation, CHCl3 was passed through a basic
alumina column (Woelm, Eschwege, Act. 0). Distillation
of (CH3)2SO solvent was carried out under vacuum
(0.5 mmHg) collecting the solvent at 0 ꢁC and thawing it
under Ar. Pyrrole 16,12 tosylpyrrolinone 14 (derived from
16),12 and pyrrolinone 12,12 as well as 3,5-dimethyl-2-
formyl-1H-pyrrole,15 were synthesized according to previ-
ously published methods.
4.1.1.3. Pyrrole ring formation. To a solution of
24.40 g (125 mmol) of 4-toluenesulfonylmethyl isocyanide
(TosMIC)29 and 25 mL (200 mmol) of tetramethylguanidine
in 100 mL of a mixture anhyd THF/isopropyl alcohol¼1:1
(v/v) kept at 0 ꢁC was added over 1 h a solution of the nitro-
acetate from above dissolved in 25 mL of the same solvents.
The mixture was warmed slowly to room temperature and
stirred for 28 h. Then it was diluted with 400 mL of
CH2Cl2, poured into 300 mL of ice-5% aq HCl, and the or-
ganic layer was washed with 5% aq NaHCO3 (100 mL) and
H2O (3ꢂ100 mL). The extract was dried (anhyd MgSO4),
filtered through a silica pad, evaporated, and the residue
was recrystallized from ethyl acetate/hexane to afford
The concentration study (Table 1) of dipyrrinones 7–9 NH
1H NMR chemical shifts was conducted as follows. All so-
lutions were prepared in CDCl3 that had been freshly treated
(freshly distilled in two cases) by passing through Woelm
Activity 0 basic alumina. Stock solutions concentrated to ap-
proximately the upper limit of solubility at 25 ꢁC were pre-
pared (in the case of 9 this limit was 20 mM and for
pentafluorophenyl 7 it was only 10 mM) then consecutively

7052
S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
16.38 g (34% based on perfluorobenzaldehyde) of pyrrole
13C NMR d: 10.2 (t, J¼2.0 Hz), 48.4 (t, J¼2.6 Hz), 108.4
(td, 2J¼18.2 Hz, 4J¼3.9 Hz), 135.7, 137.5, 137.9 (dm,
1J¼253.8 Hz), 141.4 (dm, 1J¼256.7 Hz), 143.9 (dm,
1J¼250.3 Hz), 174.4 ppm; 19F NMR d: ꢀ161.5 (m),
ꢀ152.9 (m, 3J¼20.8 Hz), ꢀ139.5 (m, 3J¼21.7 Hz) ppm.
1
15. It had mp 253–255 ꢁC (dec); H NMR d: 1.87 (3H, s),
2.40 (3H, s), 6.89 (1H, d, J¼1.8 Hz), 7.23 (2H, m,
3J¼8.3 Hz), 7.50 (2H, m, 3J¼8.3 Hz), 9.24 (1H, br s) ppm;
1H NMR ((CD3)2SO) d: 1.79 (3H, s), 2.34 (3H, s), 7.09
3
4
3
(1H, dq, J¼2.2 Hz, J¼0.8 Hz), 7.35 (2H, m, J¼8.4 Hz),
7.55 (2H, m, J¼8.4 Hz), 12.61 (1H, br s) ppm; 13C NMR
Anal. Calcd for C11H6F5NO (263.2): C, 50.20; H, 2.29;
N, 5.31.
3
((CD3)2SO) d: 9.5, 20.9, 108.3 (td, 2J¼19.6 Hz, 4J¼
3.5 Hz), 111.6, 120.6, 123.1, 125.7, 126.4, 129.8, 136.9
(dm, 1J¼249.4 Hz), 138.9, 140.2 (dm, 1J¼251.4 Hz),
143.9, 144.2 (dm, 1J¼248.6 Hz) ppm. 19F NMR d: ꢀ163.4
(m), ꢀ154.8 (m, 3J¼21.0 Hz), ꢀ138.7 (m, 3J¼22.9 Hz)
ppm.
Found: C, 50.01; H, 2.59; N, 5.40.
4.1.4. 3-Methyl-4-(4-ethoxytetrafluorophenyl)-3-pyrro-
lin-2-one (11). To a solution of 789 mg (3.0 mmol) of penta-
fluorophenylpyrrolinone 10 in 10 mL of abs ethanol (cooled
to 10 ꢁC) was added during 1 h a freshly prepared solution
of 15.0 mmol sodium ethoxide (from 345 mg of sodium)
in 15 mL of abs ethanol. The mixture was stirred for an
additional 1 h, before being diluted with 100 mL of CHCl3
and poured into 100 mL of 1% aq HCl. The organic layer
was washed with H2O (3ꢂ50 mL), dried over anhyd
Na2SO4, and filtered. The solvent was evaporated under
vacuum and the residue was purified by radial chromato-
graphy eluting with 0.5–1.5% CH3OH in CH2Cl2 (v/v).
After solvent evaporation, the combined pure fractions
were crystallized from ethyl acetate/hexane to give 617 mg
Anal. Calcd for C18H12F5NO2S (401.3): C, 53.86; H, 3.01;
N, 3.49.
Found: C, 53.71; H, 2.96; N, 3.41.
4.1.2. 3-Methyl-4-pentafluorophenyl-5-(4-toluene-
sulfonyl)-3-pyrrolin-2-one (13). A mixture of 2.41 g
(6 mmol) of pyrrole 15, 240 mL of acetic acid, 120 mL of
CH2Cl2, and 120 mL of 30% H2O2 was heated at reflux
for 24 h. The mixture was cooled, diluted with 200 mL of
H2O, and solid NaCl (w50 g) was added. The product was
extracted with CH2Cl2 (5ꢂ70 mL), and the combined ex-
tracts were washed with 5% aq NaHCO3 (100 mL) and
H2O (2ꢂ100 mL). The solution was dried (anhyd MgSO4),
filtered, and the residue, after evaporation of solvent, was
purified in three portions by radial chromatography eluting
with a gradient of 1–4% CH3OH in CH2Cl2 (v/v). The
pure fractions, after evaporation, were crystallized from
ethyl acetate/hexane to afford 1.73 g (69%) of 13. It had
1
(71%) of pyrrolinone 11. It had mp 128–129 ꢁC; H NMR
d: 1.45 (3H, t, J¼7.0 Hz), 1.87 (3H, s), 4.20 (2H, br s),
4.36 (2H, q, J¼7.0 Hz), 7.58 (1H, br s) ppm; 13C NMR d:
10.3 (t, J¼2.4 Hz), 15.4, 48.4 (t, J¼2.9 Hz), 71.0 (t,
J¼3.6 Hz), 106.4 (t, 2J¼18.3 Hz), 136.5, 136.8 (t,
3J¼1.0 Hz), 137.9 (tt, 2J¼12.1 Hz, 3J¼3.6 Hz), 141.3 (dm,
1
1J¼248.2 Hz), 144.0 (dm, J¼248.2 Hz), 174.6 ppm; 19F
NMR d: ꢀ157.4 (m, 3J¼20.4 Hz), ꢀ141.6 (m, 3J¼
1
mp 205–206 ꢁC (dec); H NMR d: 1.77 (3H, q, J¼1.4 Hz),
20.4 Hz) ppm.
2.44 (3H, s), 5.72 (1H, br t, J¼1.5 Hz), 7.31 (2H, m,
3J¼8.4 Hz), 7.58 (2H, m, 3J¼8.4 Hz), 7.66 (1H, br s) ppm;
13C NMR d: 10.9 (t, J¼2.9 Hz), 21.7, 76.9 (t, J¼3.2 Hz),
106.7, 129.4, 129.9, 130.5, 130.9, 137.9 (dm,
1J¼251.4 Hz), 141.8, 142.1 (dm, 1J¼250.7 Hz), 144.0
Anal. Calcd for C13H11F4NO2 (289.2): C, 53.98; H, 3.83;
N, 4.84.
Found: C, 53.75; H, 4.07; N, 4.86.
1
(dm, J¼247.6 Hz), 146.5, 171.3; 19F NMR d: ꢀ160.9 (m,
3
3J¼20.5 Hz), ꢀ150.9 (m, J¼21.1 Hz), ꢀ138.5 (v br s),
4.2. General procedure for syntheses of dipyrrinones
ꢀ136.4 (v br s) ppm.
A mixture of 1 mmol of the corresponding pyrrolinone,
369 mg (3 mmol) of 3,5-dimethyl-2-formyl-1H-pyrrole,15
2.5 mL of anhyd CH3CN, and 1.1 mL (10 mmol) of N-methyl-
morpholine was heated under Ar at 90–95 ꢁC in a sealed
thick wall tube for 84 h (48 h for 9). After being cooled
and opened to atmospheric pressure, the mixture was chilled
at ꢀ20 ꢁC for 3 h, and the separated product was collected
by filtration. It was washed on the filter with CH3CN/H2O/
AcOH (5 mL/2 mL/2 mL), then dried under vacuum, and
purified in several portions by radial chromatography eluting
with gradient 1–3% CH3OH in CH2Cl2 (v/v). The fractions
containing pure pigment were combined, evaporated under
vacuum, and the residue was crystallized from CH3OH/
CH2Cl2 to give pure bright yellow dipyrrinone.
Anal. Calcd for C18H12F5NO3S (417.3): C, 51.80; H, 2.90;
N, 3.36.
Found: C, 52.25; H, 2.93, N, 3.23.
4.1.3. 3-Methyl-4-pentafluorophenyl-3-pyrrolin-2-one
(10). To a solution of 417 mg (1.0 mmol) of toluenesulfonyl-
pyrrolinone 13 in 60 mL of abs ethanol under N2 was added
sodium borohydride (114 mg, 3.0 mmol) during 10 min. The
mixture was stirred for 10 more minutes, the solvent was
evaporated under vacuum (<35 ꢁC), and the residue was
partitioned between 100 mL of 1% aq HCl and 100 mL of
CHCl3. The organic layer was washed with 3ꢂ50 mL of
H2O, dried over anhyd Na2SO4, filtered, and solvent was
evaporated under vacuum. The crude material was purified
by radial chromatography eluting with 1–2% CH3OH in
CH2Cl2 (v/v), and the polar band, after evaporation, was
crystallized from ethyl acetate/hexane to afford 249 mg
4.2.1. 3-Pentafluorophenyl-2,7,9-trimethyl-(10H)-dipyr-
rin-1-one (7). This compound was isolated in 84% yield.
1
It decomposed without melting at >321–324 ꢁC; H NMR
d: 1.94 (3H, s), 2.05 (3H, s), 2.46 (3H, s), 5.78 (1H, s),
1
4
(95%) of pyrrolinone 10. It had mp 133–134 ꢁC; H NMR
5.86 (1H, d, J¼1.8 Hz), 10.51 (1H, br s), 11.97 (1H, br s)
d: 1.88 (3H, m), 4.22 (2H, m), 7.78 (1H, br s) ppm;
ppm; 13C NMR d: 9.8 (br), 11.5, 13.6, 105.0, 107.3 (td,

S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
7053
2J¼19.2 Hz, 4J¼3.3 Hz), 111.1, 123.2, 124.9, 128.2, 129.4,
were evaporated under vacuum, and the residue was crystal-
lized from ethyl acetate/hexane or methanol/water to afford
pure bright yellow-orange tricyclic compounds.
1
130.8 (m), 136.4, 137.8 (dm, J¼252.5 Hz), 141.6 (dm,
1J¼250.1 Hz), 144.2 (dm, 1J¼248.5 Hz), 172.3 ppm; 1H
NMR ((CD3)2SO) d: 1.76 (3H, s), 1.92 (3H, s), 2.22 (3H,
4
4.3.1. 1-Pentafluorophenyl-2,7,9-trimethyl-3H,5H-dipyr-
rolo[1,2-c:20,10-f]pyrimidine-3,5-dione (4). This tricycle
was isolated in 67% yield. It had mp 255–256 ꢁC; 1H
NMR d: 1.98 (3H, s), 2.10 (3H, s), 2.70 (3H, s), 6.06 (1H,
br s), 6.16 (1H, s) ppm; 13C NMR d: 10.0 (br), 10.8, 15.7,
99.7, 105.3 (td, 2J¼18.7 Hz, 4J¼4.0 Hz), 117.9, 123.2,
126.9, 128.1, 129.0 (br), 132.5, 136.4, 138.1 (dm, 1J¼
250.3 Hz), 142.1 (dm, 1J¼252.8 Hz), 143.1, 144.1 (dm, 1J¼
250.5 Hz), 165.9 ppm; 19F NMR d: ꢀ160.4 (m), ꢀ151.1
(m, 3J¼21.0 Hz), ꢀ137.8 (m, 3J¼21.0 Hz) ppm.
s), 5.60 (1H, s), 5.74 (1H, d, J¼0.9 Hz), 10.48 (1H, s),
10.55 (1H, s) ppm; 13C NMR ((CD3)2SO) d: 9.2 (br), 11.1,
12.9, 101.0, 106.8 (td, 2J¼19.4 Hz, 4J¼3.3 Hz), 110.3,
122.4, 125.4, 125.7, 129.3, 129.9 (t, 3J¼1.9 Hz), 133.8,
1
137.5 (dm, 1J¼250.3 Hz), 140.9 (dm, J¼251.8 Hz), 143.6
1
(dm, J¼245.1 Hz), 170.1 ppm; 19F NMR d: ꢀ161.6 (m),
ꢀ153.0 (m, 3J¼21.0 Hz), ꢀ138.4 (m, 3J¼22.8 Hz) ppm.
Anal. Calcd for C18H13F5N2O (368.3): C, 58.70; H, 3.56;
N, 7.61.
Anal. Calcd for C19H11F5N2O2 (394.3): C, 57.87; H, 2.81;
N, 7.10.
Found: C, 58.46; H, 3.94; N, 7.54.
4.2.2. 3-(4-Ethoxytetrafluorophenyl)-2,7,9-trimethyl-
(10H)-dipyrrin-1-one (8). This pigment was obtained in
Found: C, 57.84; H, 3.19; N, 7.13.
1
74% yield. It had mp 287–289 ꢁC (dec); H NMR d: 1.49
4.3.2. 1-(4-Ethoxytetrafluorophenyl)-2,7,9-trimethyl-
3H,5H-dipyrrolo[1,2-c:20,10-f]pyrimidine-3,5-dione (5).
This compound was obtained in 74% yield. It had mp
192–193 ꢁC; 1H NMR d: 1.49 (3H, t, J¼7.1 Hz), 1.97 (3H,
s), 2.10 (3H, s), 2.69 (3H, s), 4.42 (2H, q, J¼7.1 Hz), 6.04
(1H, br s), 6.18 (1H, s) ppm; 13C NMR d: 10.0 (t,
J¼1.6 Hz), 10.8, 15.5, 15.6, 71.1 (t, J¼3.6 Hz), 99.7,
(3H, t, J¼7.1 Hz), 1.94 (3H, s), 2.05 (3H, s), 2.46 (3H, s),
4.42 (2H, q, J¼7.1 Hz), 5.82 (1H, s), 5.84 (1H, d,
4J¼2.3 Hz), 10.54 (1H, br s), 11.96 (1H, br, s) ppm; 13C
NMR d: 9.8, 11.4, 13.5, 15.5, 71.0, (t, J¼3.5 Hz), 104.9,
132.0 (t, 3J¼2.0 Hz), 136.1, 137.9 (tt, 2J¼12.1 Hz,
3J¼3.4 Hz), 141.3 (dm, 1J¼248.1 Hz), 144.3 (dm,
1J¼247.8 Hz), 172.6 ppm; 19F NMR d: ꢀ157.5 (m,
3J¼21.3 Hz), ꢀ140.5 (m, 3J¼21.3 Hz) ppm.
2
105.2 (t, J¼19.1 Hz), 110.9, 123.3, 125.3, 127.9, 128.9,
2
102.9 (t, J¼18.6 Hz), 117.7, 122.7, 126.9, 128.4, 130.2
(t, 3J¼2.2 Hz), 132.0, 136.1, 138.9 (tt, 2J¼11.9 Hz,
1
3J¼3.5 Hz), 141.4 (dm, J¼249.1 Hz), 143.2, 144.1 (dm,
1J¼249.1 Hz), 166.2 ppm; 19F NMR d: ꢀ156.5 (m,
Anal. Calcd for C20H18F4N2O2 (394.4): C, 60.91; H, 4.60;
N, 7.10.
3J¼20.1 Hz), ꢀ139.9 (m, 3J¼20.1 Hz) ppm.
Anal. Calcd for C21H16F4N2O3 (420.4): C, 60.00; H, 3.84;
N, 6.66.
Found: C, 60.99; H, 4.88; N, 7.11.
4.2.3. 3-Phenyl-2,7,9-trimethyl-(10H)-dipyrrin-1-one (9).
This compound was isolated in 64% yield. It had mp 293–
Found: C, 59.61; H, 4.16; N, 6.68.
1
295 ꢁC (dec); H NMR d: 2.01 (3H, s), 2.02 (3H, s), 2.47
4.3.3. 1-Phenyl-2,7,9-trimethyl-3H,5H-dipyrrolo[1,2-
c:20,10-f]pyrimidine-3,5-dione (6). This tricycle was iso-
lated in 93% yield. It had mp 196–197 ꢁC; H NMR d:
2.04 (3H, s), 2.08 (3H, s), 2.70 (3H, s), 6.03 (1H, br s),
6.34 (1H, s), 7.42 (2H, m), 7.53 (3H, m) ppm; 13C
NMR d: 9.4, 10.7, 15.6, 99.9, 117.4, 121.7, 126.9, 127.1,
128.8, 128.9, 129.4, 130.3, 130.5, 135.3, 143.5, 143.8,
167.3 ppm.
4
(3H, s), 5.82 (1H, d, J¼1.8 Hz), 6.06 (1H, s), 7.37 (2H,
1
m), 7.44 (1H, m), 7.49 (2H, m), 10.61 (1H, br s), 11.81
(1H, br s) ppm; 13C NMR d: 9.4, 11.4, 13.5, 105.5, 110.4,
123.4, 123.6, 127.3, 127.8, 128.2, 128.3, 129.8, 132.7,
135.1, 146.7, 173.3 ppm.
Anal. Calcd for C18H18N2O (278.3): C, 77.67; H, 6.52;
N, 10.06.
Anal. Calcd for C19H16N2O2 (304.3): C, 74.98; H, 5.30;
N, 9.21.
Found: C, 77.76; H, 6.28; N, 10.20.
4.3. General procedure for insertion of N,N0-carbonyl
bridge
Found: C, 75.00; H, 5.42; N, 9.29.
4.4. Sodium 2,7,9-trimethyl-1-pentafluorophenyl-
3H,5H-dipyrrolo[1,2-c:20,10-f]pyrimidine-3,5-dione-
8-sulfonate (1)
A mixture of 1 mmol of dipyrrinones 7–9, 1,10-carbonyldii-
midazole (0.81 g, 5 mmol), 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) (0.75 mL, 5 mmol), and 80 mL of anhydrous
CH2Cl2 was heated under N2 at reflux for 16 h. After cool-
ing, the mixture was washed with 100 mL of 1% aq HCl,
then with H2O (3ꢂ100 mL), and the solution was dried
over anhyd MgSO4. After filtration and evaporation of the
solvent under vacuum, the residue was purified by radial
chromatography on silica gel eluting with gradient 0.2–
0.8% CH3OH in CH2Cl2 (v/v). The fractions containing
non-polar fluorescent band were combined, the solvents
Finely powdered bridged dipyrrinone 4 (100 mg, 0.25 mmol)
was added to 6 mL of concd H2SO4, and the mixture was
stirred at 25 ꢁC for 4 h. Then the temperature was lowered
from ꢀ10 ꢁC to ꢀ15 ꢁC, and the solution was neutralized
to pH 7–8 with satd aq Na2CO3 while introducing a stream
of air in order to reduce foaming. When foaming became ex-
cessive, methanol was added in 1 mL portions on four occa-
sions. After dilution with H2O (50 mL), the mixture was

7054
S. E. Boiadjiev et al. / Tetrahedron 62 (2006) 7043–7055
extracted with CHCl3/CH2Cl2 1:1 (7ꢂ50 mL), adding 10 mL
portions of CH3OH after each extraction. The combined ex-
tracts were evaporated under vacuum, and the residue was
purified by radial chromatography by eluting with gradient
5–20% CH3OH/CH2Cl2 (v/v). The polar band was collected,
evaporated to dryness, and triturated with ethyl acetate. The
solid product was collected by filtration to afford 49 mg
previously described11,12 using absorbance at 434 nm for de-
tection. Studies were conducted in adult male homozygous
Gunn rats. Gunn rats were used to simplify the appearance
and integration of chromatograms since these rats lack
UGT1 isozymes and do not excrete bilirubin glucuronides
in bile.1,5 No metabolism of 1 or 2 by UGT1 isozymes would
be expected and control experiments indicated that hepatic
metabolism and excretion of 2 was qualitatively similar in
homozygous, heterozygous, and wild-type rats.
1
(39%) of sulfonate 1. It decomposed at >277–285 ꢁC; H
NMR ((CD3)2SO) d: 1.87 (3H, s), 2.19 (3H, s), 2.83 (3H,
s), 6.88 (1H, s) ppm; 13C NMR ((CD3)2SO) d: 9.5, 10.2,
2
13.5, 100.7, 104.9 (m, J¼18.7 Hz), 121.8, 125.9, 128.2,
1
128.8, 131.5, 132.7, 134.6 (br), 137.5 (dm, J¼251.4 Hz),
Acknowledgements
141.3 (dm, 1J¼250.5 Hz), 142.6, 143.8 (dm, 1J¼248.9 Hz),
165.3 ppm; 19F NMR ((CD3)2SO) d: ꢀ161.6 (m), ꢀ153.8
(m, 3J¼22.1 Hz), ꢀ138.2 (m, 3J¼22.1 Hz) ppm. FAB-
HRMS (3-NBA+Na) calcd for C19H10F5N2Na2O5S
(M+Na)+, m/z: 519.0026, found: 519.0021, D¼0.5 mDa,
error 1.0 ppm.
We thank the U.S. National Institutes of Health (HD-17779
and DK-26307) for generous support of this work, Dr. Justin
Brower for preliminary studies, and Wilma Norona for
excellent technical assistance with the metabolism studies.
Dr. S.E. Boiadjiev is on leave from the Institute of Organic
Chemistry, Bulgarian Academy of Sciences.
4.5. Sodium 1-(4-ethoxytetrafluorophenyl)-2,7,9-tri-
methyl-3H,5H-dipyrrolo[1,2-c:20,10-f]pyrimidine-3,5-
dione-8-sulfonate (2)
References and notes
Finely ground bridged dipyrrinone 5 (105 mg, 0.25 mmol)
was added to 5 mL of concd H2SO4 precooled at 0 ꢁC, and
the magenta colored mixture was stirred for 2 h. Then the
temperature was lowered from ꢀ10 ꢁC to ꢀ15 ꢁC, and the
solution was neutralized to pH 7–8 with concd aq Na2CO3
while introducing a stream of air in order to reduce foaming.
Methanol was added in 1 mL portions on four occasions to
wash the foam down. After dilution with H2O (50 mL), the
mixture was extracted with CHCl3/CH2Cl2 1:1 (7ꢂ50 mL)
adding 10 mL portions of CH3OH after each extraction.
The combined extracts were evaporated under vacuum and
the residue was purified by radial chromatography eluting
with gradient 5–20% CH3OH in CH2Cl2 (v/v). The polar
band was collected, evaporated to dryness, and triturated
with CH2Cl2/hexane. The solid product was collected by fil-
tration to give (after drying under vacuum at 80 ꢁC) 59 mg
(45%) of sulfonate 2. It had mp 258–261 ꢁC (dec); 1H
NMR ((CD3)2SO) d: 1.39 (3H, t, J¼7.0 Hz), 1.86 (3H, s),
2.20 (3H, s), 2.83 (3H, s), 4.40 (2H, q, J¼7.0 Hz), 6.87
(1H, s) ppm; 13C NMR ((CD3)2SO) d: 9.5, 10.2, 13.5,
15.3, 71.1, 100.7, 102.7 (t, 2J¼18.9 Hz), 121.7, 125.9,
128.4, 129.8 (br t), 131.0, 132.6, 134.7 (br), 137.9 (m),
141.1 (dm, 1J¼246.6 Hz), 142.7, 143.9 (dm,
1J¼243.3 Hz), 165.4 ppm; 19F NMR ((CD3)2SO) d:
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3
ꢀ156.7 (m, J¼21.9 Hz), ꢀ140.1 (m, J¼21.9 Hz) ppm.
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