Fluorophenyl bilirubins: Synthesis and stereochemistry

Article abstract of DOI:10.1007/s007060170010

Analogs of bilirubin with vinyl groups replaced by symmetrically-disposed o-fluorophenyls (1, bis-exo, and 2, bis-endo) were synthesized and characterized spectroscopically. Their ¹H NMR spectra and NOE data are consistent with an intramolecularly hydrogen-bonded ridge-tile conformation where each propionic acid group embraces an opposing dipyrrinone. Like bilirubin, 1 and 2 exhibit negative chirality induced circular dichroism (ICD) Cotton effects in chloroform containing quinine. Unlike bilirubin, however, in aqueous buffer containing human serum albumin, 2 exhibits a negative exciton chirality ICD, whereas that of 1 is positive.

Full text of DOI:10.1007/s007060170010

Monatshefte fuÈr Chemie 132, 1527±1546 (2001)
Fluorophenyl Bilirubins:
Synthesis and Stereochemistry
Ã
Justin O. Brower and David A. Lightner
Department of Chemistry, University of Nevada, Reno, NV 89557-0020, USA
Summary. Analogs of bilirubin with vinyl groups replaced by symmetrically-disposed o-¯uoro-
phenyls (1, bis-exo, and 2, bis-endo) were synthesized and characterized spectroscopically. Their
1H NMR spectra and NOE data are consistent with an intramolecularly hydrogen-bonded ridge-tile
conformation where each propionic acid group embraces an opposing dipyrrinone. Like bilirubin,
1 and 2 exhibit negative chirality induced circular dichroism (ICD) Cotton effects in chloroform
containing quinine. Unlike bilirubin, however, in aqueous buffer containing human serum albumin,
2 exhibits a negative exciton chirality ICD, whereas that of 1 is positive.
Keywords. Pyrroles; Conformation; Hydrogen-bonding; Circular dichroism.
Introduction
Bilirubin-IXꢀ (Fig. 1A), the yellow pigment of jaundice, is an important and
structurally intriguing mammalian natural product produced copiously in normal
human metabolism from hemoglobin and other heme proteins [1, 2]. Studies aiming
at understanding its properties and relating them to its metabolism have focussed
on the pigment's unique ability to fold into a conformation where its carboxylic
acids engage in hydrogen bonding to the opposing dipyrrinones (Fig. 1B) [3, 4].
Dipyrrinones are known to hydrogen bond very strongly to carboxylic acids [5±8],
and in bilirubin such hydrogen bonding profoundly decreases its polarity. This
makes bilirubin too lipophilic to be excreted in normal metabolism and thus
requires hepatic glucuronidation for elimination into bile [2, 9]. Analogs of
bilirubin with vinyl groups located symmetrically, such as bilirubin-IIIꢀ and XIIIꢀ
(3 and 4, Fig. 1C) exhibit solution and metabolism properties that are very nearly
the same as those of bilirubin. However, analogs with propionic acids displaced
from their natural locations at C(8) and C(12) cannot engage in conformation
stabilizing intramolecular hydrogen bonding, which makes them very polar and
thus differently behaved in solution and metabolism from bilirubin [10]. In order
to determine whether the properties and even the metabolism of bilirubin are
signi®cantly changed by replacing vinyl groups with aromatic rings (as when
vinyls are replaced with n-butyls [11]), we synthesized 1 and 2 (Fig. 1C), two
new symmetric bilirubins with o-¯uorophenyl groups replacing the vinyls of
Ã
Corresponding author. E-mail: lightner@unr.edu

1528
J. O. Brower and D. A. Lightner
Fig. 1. (A) Bilirubin-IXꢀ (bilirubin), shown with its two dipyrrinone chromophores arranged in a
linear shape, is nonsymmetric; (B) bilirubin in its energetically most stable, intramolecularly hydrogen-
bonded ridge-tile conformation; only one of two enantiomeric conformers is shown, and hydrogen
bonds are indicated by hatched lines; (C) linear representations for two new symmetric o-¯uoro-
phenyl rubins 1 and 2 and their symmetric bilirubin analogs (3 and 4)

Fluorophenyl Bilirubins
1529
mesobilirubin-IIIꢀ (3) and -XIIIꢀ (4). We chose the o-¯uorophenyl group because
of our interest in preparing bilirubin analogs possessing an MRI sensitive ¯uorine
label. In this paper, we describe the synthesis, stereochemistry, and solution pro-
perties of these novel bilirubins.
Results and Discussion
Synthesis aspects
Since o-¯uorophenyl rubins 1 and 2 share a common dipyrrylmethane central
core, the known dipyrrylmethane dialdehyde 16 [12] became the key synthetic
intermediate to be coupled with either pyrrolinone 7 or 8 (Scheme 1). Pyrrolinones 7
and 8 were prepared in a standard series of steps using Barton-Zard pyrrole-forming
reactions [11, 13]. Thus, reaction of 2-nitro-1-(2-¯uorophenyl)-ethyl acetate (15)
with p-toluenesulfonyl isocyanide (TosMIC) gave one (12) of the two required
monopyrrole precursors in 54% yield. Nitro acetate 15 was prepared by standard
acetylation methods from the corresponding nitro alcohol, which was prepared by a
Henry reaction between 1-nitroethane and 2-¯uorobenzaldehyde using KF as base.
A similar direct route to the monopyrrole (11) necessitated the synthesis of the
isomeric 1-nitro-2-(2-¯uorophenyl)-ethyl acetate, which in turn requires the less
available 2-¯uoro-ꢀ-nitrotoluene as starting material. Consequently, we decided to
convert 12 into 11 by means of an acid-catalyzed isomerization described earlier
for converting the phenyl analog of 12 to the phenyl analog of 11 [14]. We found
that 12 was smoothly isomerized to 11 in 95% isolated yield catalyzed by tri¯uoro-
acetic acid (TFA) in dichloromethane. Tosyl pyrroles 11 and 12 were converted
independently to pyrrolinones 9 and 10, respectively, in the conventional way [11].
Thus, bromination of 11 with phenyltrimethylammonium tribromide gave 13 in
97% yield, whereas the same reagents and conditions converted 12 to 14 in 87%
yield. Treatment of 13 with TFA and water gave tosylpyrrolinone 9 in 86% yield,
and similar treatment of 14 afforded 10 in 99% yield. The tosyl groups of 9 and 10
were removed by reduction with sodium borohydride in ethanol, affording 7 and 8,
respectively, in 98% and 90% isolated yields.
The ®nal, bilirubin-forming coupling steps involved the reaction of dipyrrole
dialdehyde 16 [12] with excess 7 in re¯uxing aqueous methanolic KOH and
afforded 1 as a bright yellow solid in 59% yield. The similar reaction of 16 with
8 proceeded less well and gave 2 in only 27% isolated yield. Presumably, the
location of the o-¯uorophenyl group on 8 sterically shields the adjacent reaction
center, thus slowing down the base-catalyzed condensation.
Solution properties
The bilirubin with o-¯uorophenyl groups replacing the exo vinyls of 3 was found
to give only slightly longer HPLC retention times using a reverse phase column
(1: 16.7 min, 3: 16.4 min). In contrast, substituting o-¯uorophenyl groups for endo
vinyls of 4 produced signi®cantly shorter retention times (2: 12.9 min, 4: 14.5 min).
These data suggest that whereas 1 and 3 might have similar polarities, 2 would
be noticeably more polar than 4 (and 1). Con®rmation comes from TLC on silica
gel using 1% CH3OH in CH2Cl2 as eluent: 1 (Rf ˆ 0.67) moved faster than 2

1530
J. O. Brower and D. A. Lightner
a
Scheme 1. Reagents and conditions: KOH/CH3OH-H2O/re¯., then HCl; bNaBH4/EtOH; cTFA-H2O
d
(5:1); PhNBMe3 Br3 /CH2Cl2; TFA/CH2Cl2; K2CO3/THF-iPrOH
e
g
‡
(Rf ˆ 0.55), 4 (Rf ˆ 0.53), and 3 (Rf ˆ 0.53), which had nearly identical Rf values
as 3 and 4. The data suggest that the presence of the large aromatic group at the exo
position of the lactam rings shields the polar lactam group from the silica surface
more effectively than an exo methyl group.
13C NMR spectroscopy and structure
The constitutional structures of bilirubins-IIIꢀ and -XIIIꢀ (3 and 4) are well-
established [1, 15], and the structures of 1 and 3 differ only in the exo substituents
(o-¯uorophenyl in 1, vinyl in 3), whereas the structures of 2 and 4 differ similarly

Fluorophenyl Bilirubins
1531
Table 1. Comparison of 13C NMR chemical shift assignments for o-¯uorophenyl mesobilirubin
analogs 1 (R1 ˆ 2-F-Ph, R2 ˆ Me) and 2 (R1 ˆ Me, R2 ˆ 2-F-Ph) with mesobilirubin-IIIꢀ (R1 ˆ Vn,
R2 ˆ Me; 3) and mesobilirubin-XIII ꢀ (R1 ˆ Me, R2 ˆ Vn; 4) in (CD3)2SOa
Position
1
2
3
4
1,19-CO
169.47
122.05
10.88
170.78
126.16
8.99
170.38
122.38
9.25
171.31
123.42
9.47
±
2,18
2,18-CH3 or 3,17-CH3
31,171 (or 21,181)
32,172 (or 22,182)
33,173 (or 23,183)
34,174 (or 24,184)
35,175 (or 25,185)
36,176 (or 26,186)
3,17
119.92
159.40
115.59
124.08
129.82
132.11
144.52
128.41
100.35
121.81
119.87
9.18
119.48
159.18
116.03
124.51
131.03
131.81
139.15
127.51
100.71
121.90
119.64
9.28
±
±
±
±
±
±
±
±
±
±
±
141.96
128.21
100.00
122.23
119.79
9.13
140.36
127.43
99.13
122.04
119.52
9.12
123.21
19.22
34.21
173.93
130.79
23.58
4,16
±
±
5,15-CH
6,14
7,13
7,13-CH3
8,12
81,121-CH2
82,122-CH2
83,123-COOH
9,11
124.36
19.26
123.46
19.16
124.11
19.21
34.17
173.91
131.34
23.67
34.19
34.18
173.99
131.45
23.78
173.91
131.03
23.61
10-CH2
a
Chemical shifts in ꢁ (ppm) down®eld from TMS
and only in the nature of the endo substituents. Consequently, it is not surprising
that the 13C NMR spectroscopic data (Table 1) for 1 and 2 are rather similar to
those of 3 and 4. Carbons bearing the altered substituents can be expected to
exhibit the largest chemical shift differences; however, ring carbons 2 and 18 of 1
and 3 exhibit only slight differences, whereas the methyl-bearing ring carbons 3
and 17 are by about 2.5 ppm more deshielded in 1 than in 3. Similarly, the methyl-
bearing ring carbons 2 and 18 of 2 are more deshielded than those of 4 by ca.
2.5 ppm, but ring carbons 3 and 17 are ꢀ1 ppm more shielded in 2 relative to 4.
Ring carbons 4/16, 6/4, 7/13, 8/12, and 9/11 of 1 and 3 and 2 and 4 do not show a
similar sensitivity; nor do bridging carbons 5/15 and 10. The chemical shifts of the
methyl groups at 2/18 and 3/17 are more shielded in 1 than in 3, but in 2 they are
more deshielded than in 4. The chemical shift differences observed between 1 and
2 are found in parallel to those of 3 and 4.

1532
J. O. Brower and D. A. Lightner
1
Analysis of hydrogen bonding and conformation by H NMR
Bilirubin and its symmetric analogs 3 and 4 are each composed of two dipyrrinones
(Fig. 1) which are known to participate in tight carboxylic acid to dipyrrinone
intramolecular hydrogen bonding [3, 4, 6, 16, 17]. Such hydrogen bonding strongly
deshields the dipyrrinone lactam and pyrrole NH (ꢀ7.5 and 8 ppm, respectively,
in the monomers [5]), causing large deshieldings in CDCl3. Similarly large
Table 2. 1H NMR chemical shift assignments for bilirubin analogs 1 (R1 ˆ 2-F-Ph, R2 ˆ Me) and 2
(R1 ˆ Me, R2 ˆ 2-F-Ph) with 3 (R1 ˆ Vn, R2 ˆ Me) and 4 (R1 ˆ Me, R2 ˆ Vn) in (CD3)2SOa and
a,b
CDCl3
Position
1
2
3
4
ꢀ,ꢀ0-COOH
11.93
13.53
10.13
10.88
10.55
9.36
11.87
13.60
10.32
10.98
10.49
9.25
11.89
13.71
9.90
10.70
10.49
9.30
2.16
2.18
±
11.89
13.67
10.03
10.79
10.43
9.26
2.00
2.16
±
21,24-NHCO
22,23-NH
2,18 (or 3,17)-CH3
33,173 (or 23,183)-ArH
34,174 (or 24,184)-ArH
35,175 (or 25,185)-ArH
36,176 (or 26,186)-ArH
2.11
1.74
2.14
1.86
7.40
7.39
7.31
7.42
±
±
7.29
7.38
±
±
7.37
7.43
±
±
7.43
7.55
±
±
7.19
7.23
±
±
7.27
7.37
±
±
7.12
7.21
±
±
±
5,15-CH
6.19
5.57
6.09
6.21
2.13
2.15
2.42 e
3.01 d
2.58 d
1.93 e
2.90 d
2.81 d
4.00
4.08
6.09
6.14
1.92
1.99
2.42 f
3.02 d
2.58 d
1.94 f
2.90 d
2.82 d
3.98
4.08
±
6.26
5.84
7,13-CH3
2.06
1.77
2.20
2.02
81,121-CH2
2.45 c
3.04 d
2.60 d
1.96 c
2.92 d
2.82 d
4.03
2.39 c
3.01 d
2.56 d
1.93 c
2.89 d
2.80 d
3.98
82,122-CH2
10-CH2
4.11
4.08
a
Chemical shifts in ꢁ (ppm) down®eld from TMS; b values in italics are from CDCl3.; c t, J ˆ 7.8 Hz;
d
e
f
multiplets, see Table 3 for coupling constants; t, J ˆ 7.5 Hz; t, J ˆ 8.5 Hz

Fluorophenyl Bilirubins
1533
deshieldings are found in dipyrrinone±dipyrrinone intermolecularly hydrogen-
bonded dimers in CDCl3 [5, 6]. In bilirubins, the dipyrrinone NH chemical shifts
have been used to detect and distinguish dipyrrinone to dipyrrinone intermolecular
hydrogen bonding [5, 6, 8, 16] from dipyrrinone to carboxylic acid intramolecular
1
hydrogen bonding [4, 6±8, 16±18]. Such H NMR studies have shown that the
pyrrole NH resonance of bilirubin in CDCl3 is found at ca. 9.2 ppm when the
dipyrrinone and carboxylic acid groups are linked by intramolecular hydrogen
bonding, which is ꢀ1 ppm up®eld relative to the pyrrole NH chemical shift
(ꢀ10.2 ppm) found for planar dipyrrinone to dipyrrinone dimers [5, 6, 8].
The data of Table 2 show chemical shift of ca. 9.3 ppm for the pyrrole NH in
1±4 in CDCl3, which we believe to be consistent with dipyrrinone to carboxylic
acid intramolecular hydrogen bonding of the type shown in Fig. 1B. In contrast, in
(CD3)2SO, a solvent thought to insert into the bilirubin hydrogen bonding matrix
[16±21], the CO2H and lactam NH signals become more shielded than in CDCl3,
whereas the pyrrole NH typically becomes more deshielded.
1
The stereochemical conclusions reached above were con®rmed by H{1H}
homonuclear Overhauser effect (NOE) measurements in CDCl3 (Fig. 2).
Signi®cantly, and consistent with a ridge-tile conformation (Fig. 1B), NOEs are
observed between the carboxylic acid hydrogen and the lactam NH of 1±4. The
NOE measurements also con®rm syn-Z-dipyrrinone conformation: moderately
strong NOEs found between (i) the pyrrole and lactam NHs within individual
dipyrrinones and (ii) the vinylic hydrogens at C(5) and C(15) and the pyrrole
methyls at C(7) and C(13) and the lactam methyls at C(3) and C(17).
Fig. 2. 1H{1H} Homonuclear Overhauser effects (NOEs) found in 1 and 2 in CDCl3 are shown by
solid, double-headed curved arrows; signi®cant, albeit weak, NOEs are found between the CO2H
protons and the lactam NH protons (indicated by dashed arrows)

1534
J. O. Brower and D. A. Lightner
Table 3. 1H NMR chemical shiftsa and coupling constantsb for the propionic acid ±CꢂHAHX±
CꢀHBHC±CO2H segments of 1±4 in CDCl3 at 22ꢂC
ꢂ-CH2
ꢀ-CH2
MBR
HX
HA
HB
HC
1
ꢁ:
ꢁ:
ꢁ:
ꢁ:
2.60
3.04
2.92
2.82
JBX
JCX
JAX
2.7
3.2
JAB
JAC
JAX
13.8
2.1
JAB
JBX
JBC
13.8
2.7
JAC
JCX
JBC
2.1
3.2
13.2
13.2
18.5
18.5
2
3
2.56
3.01
2.89
2.80
JBX
JCX
JAX
2.5
3.1
JAB
JAC
JAX
13.7
2.7
JAB
JBX
JBC
13.7
2.5
JAC
JCX
JBC
2.7
3.1
14.0
14.0
16.0
16.0
2.58
3.01
2.90
2.81
JBX
JCX
JAX
2.0
3.5
JAB
JAC
JAX
14.5
2.5
JAB
JBX
JBC
14.5
2.0
JAC
JCX
JBC
2.5
3.5
13.5
13.5
17.0
17.0
4
2.58
3.02
2.90
2.82
JBX
JCX
JAX
2.0
4.0
JAB
JAC
JAX
14.0
2.5
JAB
JBX
JBC
14.0
2.0
JAC
JCX
JBC
2.5
4.0
14.0
14.0
18.5
18.5
a
b
ꢁ (ppm) down®eld from TMS; J in Hz from 500 MHz spectra
Further evidence for intramolecular hydrogen bonding between propionic acid
and dipyrrinone groups in the bilirubins can be elicited from an examination
of vicinal coupling constants (Table 3). In CDCl3, the well-de®ned ABCX
(ddd) coupling pattern is characteristic of restricted mobility [16, 17, 22, 23] in the
±CHAHXCHBHC±COOH segment which is constrained to adopt a ®xed staggered
3
geometry due to strong intramolecular hydrogen bonding. Analysis of JHH in the
propionic acid chains clearly shows the ABCX pattern of the ®xed staggered
propionic acid geometry in 1±4, thus providing strong supporting experimental
evidence for folded ridge-tile structures. On the other hand, the less complicated
A2B2 pattern found in (CD3)2SO (Table 2) indicates more motional freedom in
the propionic acid segment, whose CO2H groups are thought to be linked to the
dipyrrinones via bound solvent molecules [17, 19, 21, 22]. Taken collectively, the
NMR data support intramolecularly hydrogen-bonded ridge-tiles for 1±4 (Figure 3)
and a ®xed staggered conformation of the propionic acid segments.
UV/Vis spectroscopic analysis
The UV/Vis spectra of 1±4 in a wide range of solvents of different polarity and
hydrogen bonding ability are rather similar (Table 4). In polar solvents, 1±4
invariably show lmax ꢁ 440±450 nm and a shoulder near 430 nm. In nonpolar
solvents, lmax lies at about 450±460 nm, with a shoulder near 430±440 nm. The
band shape is due to two exciton components originating in an electric transition

Fluorophenyl Bilirubins
1535
Fig. 3. Ball and Stick [20] representations of the Sybyl molecular dynamics energy-minimized
conformations of o-¯uorophenyl rubins 1 (left) and 2 (right) illustrating the greater proximity of the
exo o-¯uorophenyl group of 1 to its lactam carbonyl hydrogen bonding region; hatched lines represent
hydrogen bonds
dipole±dipole interaction between the two proximal dipyrrinone chromophores
oriented at an angle of 90ꢂ (as in Fig. 4) [4]. The data are consistent with the ridge-
tile conformation for 1±4 derived from NMR studies and support the confor-
mational analysis by circular dichroism spectroscopy given below.
Induced circular dichroism and conformation
Stereochemical investigations of bilirubin and its analogs have repeatedly indicated
that (i) the most stable conformation is one where the two dipyrrinones are rotated
into a ridge-tile shape, (ii) considerable stabilization of the ridge-tile is achieved
by intramolecular hydrogen bonding, and (iii) the pigment adopts either of
two interconverting enantiomeric ridge-tiles as depicted in Fig. 3 [4, 17, 23, 24].
The component dipyrrinone chromophores of 1±4 have strongly-allowed long-
wavelength electronic transitions (Table 4), making them excellent candidates for
transition dipole±dipole interaction (exciton coupling) [24, 25] (Fig. 4). Such
intramolecular exciton interaction or resonance splitting produces two long wave-
length transitions in the UV/Vis spectrum and two corresponding but oppositely-
signed bands in the CD spectrum. According to exciton chirality theory [26], a
long-wavelength negative and short-wavelength positive CD couplet exhibits

1536
J. O. Brower and D. A. Lightner
Table 4. Solvent dependence of UV/Vis spectroscopic data of rubins 1±4a
max
"
Rubin
(ꢃmax/nm)
Benzene
CHCl3
(CH3)2CO
CH3OH
CH3CN
(CH3)2SO
62000 (458)
57000 (444)sh
58000 (451)
53000 (435)sh
61690 (460)
48070 (426)sh
61000 (456)
58300 (444)sh
57400 (449)
55000 (437)sh
64610 (456)
49560 (424)sh
60400 (452)
56000 (436)sh
56600 (445)
52400 (429)sh
62360 (451)
50890 (424)sh
46580 (447)
34610 (415)sh
59800 (449)
52300 (428)sh
55700 (442)
48700 (421)sh
60700 (451)
49120 (417)sh
45310 (446)
37540 (416)sh
61400 (449)
57000 (434)sh
56500 (442)
52700 (426)sh
62360 (451)
51220 (423)sh
46980 (445)
39430 (417)sh
61600 (452)
50800 (421)sh
60100 (444)
47800 (413)sh
67760 (459)
48810 (416)sh
47830 (446)
37670 (415)sh
1
2
3
4
45580 (447)
46920 (450)
a
22ꢂC, ꢀ1.4 Â 10 5 M; shoulders or in¯ections were determined by ®rst and second derivative spectra
sh
Fig. 4. Interconverting intramolecularly hydrogen-bonded enantiomeric conformers of bilirubins
1±4 (see Fig. 1 for R1, R2); the double headed arrows represent the dipyrrinone long-wavelength
electric transition moments (dipoles); the relative helicities (M, minus or P, plus) of the dipole vectors
are shown (inset) for each enantiomer; hydrogen bonds are indicated by hatched lines
negative exciton chirality and corresponds to a negative helical disposition of the
relevant dipyrrinone long-wavelength transition dipoles. Usually, this corresponds to
the M-helical enantiomeric type of Fig. 4 [4, 24].
In a nonpolar, aprotic solvent such as chloroform, the conformational
equilibrium between bilirubin enantiomers can be displaced from 1:1 by adding
a chiral recognition agent [11, 24]. This leads typically to an intense bisignate
induced circular dichroism (ICD) Cotton effect, as has been noted previously
for bilirubin in aqueous buffers with added albumin [11, 27] and in chloroform

Fluorophenyl Bilirubins
1537
Fig. 5. Circular dichroism spectra of 1.4 Â 10 5 M 1±4 in CHCl3 in the presence of ca. 4.2 Â 10 3 M
quinine at 22ꢂC; the compound's number is indicated on each CD curve
with added quinine [11, 24] or other optically active amines [28]. Similarly, for
the bilirubins discussed here, in CHCl3 strong negative exciton chirality ICDs
are observed in the presence of quinine (Fig. 5), as is characteristic of an exciton
system in which the two dipyrrinone chromophores of the bichromophoric rubin
Ã
molecule interact by coupling locally excited ꢄ ! ꢄ transitions (electric dipole
transition moment coupling). At a molar ratio of quinine:pigment ꢁ 300 : 1, the
Cotton effect intensities reach a maximum for 3, giving an intense bisignate
max
456
max
408
CD ꢃÁ"
ˆ
89; Á"
ˆ ‡58† of exactly the same signed order but larger
magnitudes than those seen for bilirubin or 4 [24]. The Cotton effect intensities
of 1 are nearly identical to those of its counterpart 3, whereas those of 2 are very
similar; only 4 exhibits smaller magnitudes. Interestingly, the presence of the larger
unsaturated group (o-¯uorophenyl or vinyl) in the exo positions leads to more
intense Cotton effects (1 and 3) than when they are located at the endo sites (2 and
4; Table 5). Whether this re¯ects ineffective complexation/recognition or an altered
pigment conformation is not immediately clear, but the presence of the unsaturated
Table 5. Comparison of circular dichroism and UV/Vis spectroscopic data for 1±4 in CHCl3 solu-
tions containing quininea
Pigment
CD
UV/Vis
max
"
Á"max (ꢃ)1
ꢃ2 at Á" ˆ 0
Á"max (ꢃ)3
ꢃ(nm)
1
2
3
4
‡ 52 (403)
‡ 46 (403)
‡ 58 (408)
‡ 39 (405)
422
419
427
425
82 (452)
80 (446)
89 (456)
64 (453)
54910
57140
63330
56790
456
448
458
455
a
Pigment conc.: ꢀ 1.4  10 5 M; quinine conc.: ꢀ 4.2  10 3 M; pigment:quinine molar ratio ˆ
ꢀ1:300

1538
J. O. Brower and D. A. Lightner
Fig. 6. Circular dichroism spectra of 1.41 Â 10 5 M 1±4 in the presence of ca. 2.8 Â 10 5 M HSA
in pH 7.4 Tris buffer at 22ꢂC; the compound's number is indicated on each CD curve
substituent nearer the lactam carbonyl leads to larger ICDs and perhaps promotes
stronger complexation with quinine.
When human serum albumin (HSA) is used as the chiral complexation agent,
aqueous buffered (pH ˆ 7.4, Tris) solutions of 1, 3, and 4 exhibit strong positive
chirality bisignate ICD Cotton effects as observed for bilirubin [27]. In contrast, 2
exhibits a strong negative bisignate ICD Cotton effect (Fig. 6). The CD intensities
of 1 are similar to those of 3, and both are more than twice as large as those of
4. The data suggest a greater enantioselective chiral complexation by HSA for 1 and
3 than 4 and implicate the larger exo groups to produce a favorable binding
interaction with the chiral complexation agent. The data are qualitatively similar to
those seen with the corresponding mesobilirubins-IIIꢀ and -XIIIꢀ (vinyls reduced
to ethyls) and their n-methyl analogs [11]. The ICD data for 1, 3, and 4 suggest
rather similar conformations on HSA, whereas the contrasting ICD data for 2
suggest a mirror image or different conformation.
Adding 5 mm3 of CHCl3 to 5 cm3 of a pH 7.4 buffered aqueous HSA solution
of 2 leads to a very striking intensi®cation of its Cotton effect (Fig. 7). Addition of
a second 5 mm3 aliquot effects essentially no change. In contrast, and as noted
previously for bilirubin [29], addition of 5 mm3 of CHCl3 to pH 7.4 aqueous
buffered HSA solutions of 1 and 4 leads to Cotton effect sign inversions (Figs. 8
and 9). The Cotton effect magnitudes increase in intensity upon addition of a
second 5 mm3 aliquot of CHCl3, but not strongly. Although the ®rst 5 mm3 of
CHCl3 do not invert the CD of 3 (Fig. 10), the second do. At a total of 10 mm3/
5 cm3, the solutions containing 1 and 2 are saturated, but solutions of 3 and 4
are able to dissolve more CHCl3. Addition of 15 mm3 of CHCl3 again increases
the magnitude of the Cotton effects of 3 and 4, and at this point the solutions become
saturated and the Cotton effects reach their maximum values (Table 6). The
sluggishness of 3 to invert, coupled with the less intense Cotton effect of 1 when
compared to 2 at CHCl3 saturation, suggests that 1 and 3 are initially more tightly
bound to HSA in a favorable binding site than their counterparts. Conversely, rubin

Fluorophenyl Bilirubins
1539
Fig. 7. Circular dichroism spectra of 1.40 Â 10 5 M 2 in the presence of 2.80 Â 10 5 M HSA in pH
7.4 Tris buffer at 22ꢂC upon addition of CHCl3; the amounts of CHCl3 added (mm3/5 cm3 buffered
pigment-albumin solutions) are indicated on each CD curve (mm3)
Fig. 8. Circular dichroism spectra of 1.43 Â 10 5 M 1 in the presence of 2.90 Â 10 5 M HSA in
pH 7.4 Tris buffer at 22ꢂC upon addition of CHCl3; the amounts of CHCl3 added (mm3/5 cm3
buffered pigment-albumin solutions) are indicated on each CD curve (mm3)
2 initially displays a negative chirality Cotton effect and greatly increases in
magnitude with increasing amounts of CHCl3. The Cotton effects for rubin 4 in-
vert immediately with only a trace of CHCl3, and at the saturation limit the Cotton
effect is greater than for its counterpart 3. This suggests that endo aromatic and
vinyl rubins 2 and 4 are more tightly bound to HSA than their exo counterparts
after the addition of CHCl3. Apparently, it is the exo location of the unsaturated
groups rather than their presence that is responsible for the initial favorable binding
to HSA, and the endo location of these groups that is responsible for favored
binding after treatment with CHCl3.

1540
J. O. Brower and D. A. Lightner
Fig. 9. Circular dichroism spectra of 1.47 Â 10 5 M 4 in the presence of 2.86 Â 10 5 M HSA in
pH 7.4 Tris buffer at 22ꢂC upon addition of CHCl3; the amounts of CHCl3 added (mm3/5 cm3
buffered pigment-albumin solutions) are indicated on each CD curve (mm3)
Fig. 10. Circular dichroism spectra of 1.42 Â 10 5 M 3 in the presence of 2.84 Â 10 5 M HSA in pH
7.4 Tris buffer at 22ꢂC upon addition of CHCl3; the amounts of CHCl3 added (mm3/5 cm3 buffered
pigment-albumin solutions) are indicated on each CD curve (mm3)
Concluding comments
In this paper we describe the synthesis and characterization of two new aromatic
bilirubin analogs, 1 and 2. The exo vs. endo location of their 2 o-¯uorophenyl
groups might not be expected to play a signi®cant role in the linear (Fig. 1) or
porphyrin-like conformations, but in the intramolecularly hydrogen-bonded ridge-
tile conformation, the exo o-¯uorophenyl acts to shield the lactam hydrogen
bonding region (Fig. 3). The endo o-¯uorophenyl is too far away to exert a similar
effect. The studies show that introduction of the aromatic groups can have a
marked effect on the polarity of the pigments, making 1 and 2 expectedly less polar

Fluorophenyl Bilirubins
1541
Table 6. In¯uence of CHCl3 on the circular dichroism spectra of 1±4a in pH 7.4 Tris buffer contain-
ing HSA
Pigment
CHCl3
(mm3)
CD
UV/Vis
ꢃ(nm)
Á"max (ꢃ)1
ꢃ2 at Á" ˆ 0 Á"max (ꢃ)3
"
max
1
2
3
4
41 (400)
‡ 60 (406)
63 (408)
8 (393)
417
434
354
411
‡ 63 (447)
43200
52200
54470
37670
452
454
462
455
0 mm3
5 mm3
10 mm3
15 mm3
29 (459)
‡ 67 (449)
‡ 27 (447)
14 (454)
233 (448)
‡ 23 (443)
15 (460)
1
2
3
4
‡ 9 (404)
‡ 149 (400)
17 (397)
424
419
418
431
41600
62400
55570
39570
449
452
461
451
‡ 12 (404)
1
2
3
4
‡ 29 (406)
‡ 154 (400)
‡ 21 (406)
‡ 39 (399)
b
426
419
425
422
b
40 (456)
238 (448)
42 (455)
69 (452)
44680
62250
56520
40930
b
452
452
460
449
b
b
1
2
3
4
b
b
b
b
b
‡ 40 (406)
‡ 51 (400)
425
421
72 (454)
94 (452)
58130
43830
460
449
a
CHCl3 was added to 5 cm3 solutions that were ꢀ1.4 Â 10 5 M in pigment and had a 1:2 molar ratio
b
of pigment to HSA (human serum albumin) at 22ꢂC; solubility limit of CHCl3 exceeded
(based on HPLC and TLC) than the parent bilirubins (3 and 4). The increased
lipophilicity has some effect on the CD spectroscopic properties of 1 and 2, from
which one can discern that the presence of an exo o-¯uorophenyl appears to interfere
with complexation to human serum albumin. The energetically most stable
conformation of 1 and 2 is still the folded ridge-tile structure preferred by bilirubin
and it symmetric analogs, bilirubins 3 and 4. In preliminary metabolic experiments
in rats (carried out by Dr. A. F. McDonagh at the University of California, San
Francisco), the presence of the o-¯uorophenyl groups does not appear to prevent
rapid uptake of the compound from blood into the liver, but the exo/endo location
of the aromatic ring seems to affect glucuronidation by bilirubin glucuronosyl
transferase. Thus, 2 (with endo o-¯uorophenyl groups) exhibits less ef®cient
glucuronide formation and excretion than 1.
Experimental
General
All UV/Vis spectra were recorded on a Perkin-Elmer ꢃ-12 spectrophotometer, and all circular
dichroism (CD) spectra on a Jasco J-600 instrument. Nuclear magnetic resonance (NMR) spectra
were obtained on a GE QE-300 spectrometer operating at 300 MHz, or on a Varian Unity Plus
500 MHz spectrometer in CDCl3 unless otherwise speci®ed. Chemical shifts are reported in ꢁ/ppm
referenced to the residual CHCl3 1H signal at 7.26 ppm and the CHCl3 13C signal at 77.230 ppm. J-
modulated spin-echo (APT) and HMQC experiments were used to assign 13C NMR spectra. Melting

1542
J. O. Brower and D. A. Lightner
points were taken on a MelTemp capillary apparatus and are uncorrected. Combustion analyses of the
new compounds were carried out by Desert Analytics, Tucson, AZ; the results were within 0.40%
of the calculated values. Analytical thin layer chromatography was carried out on J. T. Baker silica
gel IB-F plates (125 ꢅ layers). Flash column chromatography was carried out using Woelm silica gel
F, thin layer chromatography grade. HPLC analyses were carried out on a Perkin-Elmer Series 4 high
performance liquid chromatograph with an LC-95 UV/Vis spectrophotometric detector (410 nm).
The column was a Beckman-Altex ultrasphere-IP 5 ꢅm C-18 ODS column (25 Â 0.46 cm) ®tted with
a similarly-packed precolumn (4.5 Â 0.46 cm). The ¯ow rate was 0.75±1.0 cm3/minute; the elution
solvent was 0.1 M di-n-octylamine acetate in 5% aqueous MeOH, the column temperature was
ꢀ 34ꢂC.
Commercial reagents were used as received from Aldrich or Acros, HPLC grade MeOH was
from Fisher, human serum albumin (defatted) was obtained from Sigma. Spectroscopic data were
obtained in spectral grade solvents (Aldrich or Fisher).
CD and UV measurements
Stock solutions of 1±4 (ꢀ 7.0 Â 10 4 M) were prepared by dissolving an appropriate amount of the
desired compound in 2 cm3 of DMSO. Next, 100 mm3 of the stock solution were diluted to 5 cm3
(volumetric ¯ask) with an HSA solution (ꢀ 2.8 Â 10 5 M in pH 7.4 Tris buffer). The ®nal concen-
tration of the solution was ꢀ 1.4 Â 10 5 M in pigment. Up to four 5 cm3 solutions of each pigment
were prepared, as needed, in 5 cm3 volumetric ¯asks. To each ¯ask, the indicated volume (mm3) of
CHCl3 was added. Gentle shaking was occasionally required to dissolve all of the CHCl3 in the
aqueous solution, at which point CD and UV/Vis measurements were recorded.
5,50-Diformyl-3,30-bis-(2-methoxycarbonylethyl)-4,40-dimethyl-2,20-dipyrrylmethane (16)
16 was prepared according to a literature procedure [12].
1-(o-Fluorophenyl)-2-nitro-1-propanol (C9H8N11O4F2)
o-Fluorobenzaldehyde (116.00 g, 0.93 mol) and KF (2.7 g, 0.05 mol eq.) was stirred mechanically
with 400 cm3 of 2-propanol while a solution of nitroethane (71.00 g, 0.93 mol) in 100 cm3 of 2-
propanol was added dropwise with stirring over several hours. Stirring was continued for 2 days at
room temperature. The mixture was then diluted with CH2Cl2 and washed with H2O (3 Â 300 cm3)
and brine (300 cm3). The organic extracts were dried over anhydrous. MgSO4, ®ltered, and the
solvent was removed. The resulting yellow oil was suf®ciently pure for the next step.
Yield: 186.1 g (quantitative); IR (neat): ꢆ ˆ 3511, 2993, 1617, 1555, 1490, 1458, 1226,
1
1053 cm 1; H NMR: ꢁ ˆ 7.58±7.03 (4H, m), 5.72±5.39 (2  d, 1H), 4.89±4.76 (2  dq, 1H), 2.93
(d, J ˆ 3.0 Hz, 1H), 2.80 (d, J ˆ 3.0 Hz, 1H), 1.47 (d, J ˆ 6.0 Hz, 3H), 1.41 (d, J ˆ 6.0 Hz, 3H) ppm;
13C NMR: ꢁ ˆ 161.44, 160.78, 158.18, 157.55, 130.36, 130.26, 129.91, 129.79, 128.22, 128.18,
127.73, 125.92, 125.75, 125.64, 125.47, 124.64, 124.60, 124.32, 124.29, 115.48, 115.19, 114.92,
87.75, 85.44, 69.88, 68.64, 15.48, 11.83 ppm; MS: m/z (%) ˆ 152 (31), 125 (100), 109 (19), 97 (56),
77 (39), 51 (27) amu.
1-(o-Fluorophenyl)-2-nitro-propyl acetate (15; C11H12NO4F)
1-(o-Fluorophenyl)-2-nitro-1-propanol (50.00 g, 0.25 mol) and DMAP (200 mg) were dissolved in
150 cm3 of acetic anhydride and stirred at room temperature overnight. The solution was then chilled
in an ice bath, and H2O (150 cm3) was added. Stirring was continued for several hours, after which the
mixture was diluted with CH2Cl2 (200 cm3) and washed successively with H2O (2 Â 500 cm3), aqueous

Fluorophenyl Bilirubins
1543
NaHCO3 solution (2 Â 500 cm3), and brine (500 cm3). The organic layer was dried over anhydrous
MgSO4, ®ltered, and the solvent was removed to yield a light green oil.
Yield: 56.83 g (94%); b.p.: ꢀ128ꢂC (0.45 mm Hg); IR (neat): ꢆ ˆ 2996, 1752, 1559, 1457,
1
1391, 1221 cm 1; H NMR: ꢁ ˆ 7.40±7.06 (m, 4H), 6.62 (d, J ˆ 5.0 Hz, 1H), 6.35 (d, J ˆ 9.0 Hz,
1H), 5.09±4.90 (2  dq, 1H), 2.22 (s, 3H), 2.03 (s, 3H), 1.54 (d, J ˆ 7.0 Hz, 3H), 1.41 (d,
J ˆ 7.0 Hz, 3H) ppm; 13C NMR: ꢁ ˆ 168.50, 168.41, 166.29, 166.14, 161.76, 161.06, 158.42,
157.74, 131.00, 130.90, 130.58, 128.67, 127.75, 124.67, 124.42, 122.79, 122.67, 122.63, 122.60,
122.35, 122.28, 122.25, 122.19, 115.78, 115.60, 115.48, 115.32, 84.80, 83.42, 70.77, 69.43,
21.48, 19.97, 15.48, 12.80 ppm; MS: m/z (%) ˆ 241 (6), 194 (20), 152 (100), 135 (60), 109 (25),
57 (9) amu.
3-(o-Fluorophenyl)-4-methyl-2-p-toluenesulfonyl-1H-pyrrole (12; C18H16NO2F)
p-Toluenesulfonyl methyl isocyanide (TosMIC) (20.00 g, 0.10 mol) and K2CO3 (60 g) were partially
dissolved in 100 cm3 of a mixture of THF:2-propanol ˆ 1:1 and stirred magnetically at room tem-
perature. A solution of 1-(o-¯uorophenyl)-2-nitro-propyl acetate (15; 20.00 g, 0.08 mol) was added
dropwise, and the mixture was stirred at room temperature for 5 days. It was then diluted with
200 cm3 of CH2Cl2, washed with H2O (2 Â 200 cm3) and brine (200 cm3), and dried over MgSO4.
The solvent was removed on a rotary evaporator, and the residue was crystallized from CH2Cl2/
hexane to yield a tan solid. An analytical sample was prepared by recrystallization from CH2Cl2/
hexane. The colorless crystals were collected and dried overnight in a drying pistol over P2O5.
Yield: 14.8 g (54%); m.p.: 168±169ꢂC; IR (KBr): ꢆ ˆ 3301, 1596, 1471, 1213, 1140, 1082,
1
753 cm 1; H NMR: ꢁ ˆ 9.27 (s, 1H), 7.35±6.98 (m, 8H), 6.81 (d, J ˆ 2.5, 1H), 2.34 (s, 3H), 1.87
(s, 3H) ppm; 13C NMR: ꢁ ˆ 160.19 (JCF ˆ 246 Hz), 143.65, 139.27, 133.60 (JCF ˆ 3.3 Hz), 129.78
(JCF ˆ 8.8 Hz), 129.43, 126.91, 125.13, 123.56 (JCF ˆ 3.3 Hz), 123.27, 122.19, 120.61, 120.41
(JCF ˆ 6.8 Hz), 115.16 (JCF ˆ 22 Hz), 21.61, 1033 (JCF ˆ 2.2 Hz) ppm.
5-Bromo-2-p-toluenesulfonyl-3-(o-¯uorophenyl)-4-methyl-5-bromo-1H-pyrrole
(14; C18H15NO2SBrF)
3-(o-Fluorophenyl)-4-methyl-2-p-toluenesulfonyl-1H-pyrrole (12; 0.50 g, 1.51 mmol) was dissolved
in 10 cm3 of CH2Cl2 and stirred magnetically in an ice bath. A solution of phenyl trimethyl
ammonium tribromide (PTT) (1.20 g, 2 eq.) in 20 cm3 of CH2Cl2 was added dropwise, and the result-
ing yellow-orange solution was stirred at 0ꢂC for 30 min. Subsequently, it was washed with aqueous
NaHSO3 solution (2 Â 30 cm3) and brine (30 cm3) and dried over MgSO4. The solvent was removed
using a rotary evaporator to yield a colorless solid (0.54 g, 87%). An analytical sample was prepared
by the addition of hexane to a solution of the pyrrole in CH2Cl2. Colorless crystals were collected
and dried over P2O5 in a drying pistol overnight.
Yield: 0.54 g (87%); m.p.: 157±158ꢂC; IR (KBr): ꢆ ˆ 3285, 1596, 1465, 1317, 1240, 1186,
762 cm 1; 1H NMR: ꢁ ˆ 9.23 (s, 1H), 7.41±6.99 (m, 8H), 2.35 (s, 3H), 1.80 (s, 3H) ppm; 13C NMR:
ꢁ ˆ 160.15 (JCF ˆ 247.2 Hz), 144.10, 138.76, 133.60 (JCF ˆ 3.3 Hz), 130.35 (JCF ˆ 7.7 Hz), 129.59,
127.16, 126.34, 123.97, 123.79 (JCF ˆ 3.3 Hz), 122.30, 119.76 (JCF ˆ 16.5 Hz), 104.61, 21.76,
10.46 ppm; MS: m/z (%) ˆ 409 (100), 343 (77), 269 (15), 160 (39), 91 (25) amu.
4-(o-Fluorophenyl)-3-methyl-5-p-toluenesulfonylpyrrolin-2-one (10; C18H16NO3SF)
5-Bromo-2-p-toluenesulfonyl-3-(o-¯uorophenyl)-4-methyl-5-bromo-1H-pyrrole (14; 5.00 g, 12.25
mmol) was dissolved in 45 cm3 of TFA and 8 cm3 of H2O and then stirred at room temperature for 2
days. The resulting dark solution was diluted with 50 cm3 of CH2Cl2 and washed sequentially with
H2O (2 Â 50 cm3), aqueous NaHCO3 solution (2 Â 50 cm3), and brine (50 cm3), then dried over

1544
J. O. Brower and D. A. Lightner
anhydrous MgSO4. The solvent was removed on a rotary evaporator to yield a light grey powder.
Colorless crystals were obtained by the addition of hexane to a solution of the pyrrolinone in CH2Cl2.
They were collected and dried overnight in a drying pistol over P2O5.
Yield: 4.18 g (99%); m.p.: 148±150ꢂC; IR (KBr): ꢆ ˆ 3419, 1706, 1490, 1321, 1137, 811,
761 cm 1; 1H NMR: ꢁ ˆ 7.65 (d, J ˆ 8 Hz, 2H), 7.24 (d, J ˆ 8 Hz, 2H), 7.43 (br t, 1H), 7.17 (dt, J ˆ 3,
3H), 6.82 (br s, 1H), 5.82 (br s, 1H), 2.42 (s, 3H), 1.78 (s, 3H) ppm; 13C NMR: ꢁ ˆ 172.81, 159.92
(JCF ˆ 250 Hz), 145.85, 139.78, 136.82, 131.68 (JCF ˆ 8 Hz), 131.00, 130.86 (JCF ˆ 3 Hz), 129.73,
129.62, 124.30 (JCF ˆ 3 Hz), 119.39 (JCF ˆ 14 Hz), 116.37 (JCF ˆ 22 Hz), 77.75, 21.90, 10.34 ppm.
4-(o-Fluorophenyl)-3-methylpyrrolin-2-one (8; C11H10NOF)
4-(o-Fluorophenyl)-3-methyl-5-p-toluenesulfonyl-pyrrolin-2-one (10; 0.10 g, 0.29 mmol) and NaBH4
(23.0 mg, 0.60 mmol) were stirred in 10 cm3 of absolute EtOH at room temperature for 15 min.
The solution was then diluted with 15 cm3 of CH2Cl2, washed with H2O (2 Â 15 cm3) and brine
(15 cm3), and dried over MgSO4. The solvent was evaporated on a rotary evaporator, and the residue
was crystallized from CH2Cl2/hexane to afford a light tan powder (55.0 mg, 90%). An analytical
sample was prepared by precipitation from a solution in CH2Cl2 with hexane. The colorless crystals
were collected and dried over P2O5 in a drying pistol overnight.
1
M.p.: 142±143ꢂC; IR (KBr): ꢆ ˆ 3210, 1687, 1490, 1360, 1267, 1236, 124 cm 1; H NMR:
ꢁ ˆ 8.07 (br s, 1H), 7.33 (dt, J ˆ 2, 7 Hz, 2H), 7.20 (t, J ˆ 7 Hz, 1H), 7.14 (t, J ˆ 7 Hz, 1H), 4.26
(s, 3H), 1.95 (d, JHF ˆ 1 Hz, 3H) ppm; 13C NMR: ꢁ ˆ 175.78, 159.63 (JCF ˆ 250 Hz), 144.89, 132.22,
130.55 (JCF ˆ 22 Hz), 130.00 (JCF ˆ 3 Hz), 124.33 (JCF ˆ 3 Hz), 121.68 (JCF ˆ 14 Hz), 116.30
(JCF ˆ 22 Hz), 49.13 (JCF ˆ 5 Hz), 10.16 ppm.
3,17-Desvinyl-3,17-bis-(o-¯uorophenyl)bilirubin-XIIIꢀ (2; C41H38N4O6F2)
In a 50 cm3 round bottom ¯ask, 4-(o-¯uorophenyl)-3-methyl-pyrrolin-2-one (8; 0.10 g, 0.52 mmol)
and diformyl-dipyrrylmethane [12, 30] (42.0 mg, 0.10 mmol) was dissolved in 20 cm3 of MeOH and
13 cm3 of 6 M aqueous KOH. The solution was heated at re¯ux for 48 h. The resulting reddish solu-
tion was then chilled in an ice bath and poured into 50 cm3 of ice water. Acidi®cation by slow addition
of concentrated HCl yielded a brown precipitate that was collected by centrifugation and ®ltration.
It was dissolved in CH2Cl2 and triturated with MeOH to afford a bright yellow precipitate that was
collected by ®ltration.
Yield: 20.3 mg (27%); m.p.: 254±258ꢂC (dec); IR (KBr): ꢆ ˆ 3412, 1685, 1491, 1400, 1251, 1105,
754 cm 1; 1H NMR: ꢁ ˆ 1.86 (s, 6H), 2.02 (s, 6H), 2.56 (ddd, J ˆ 2.48, 3.16, 14.01 Hz, 2H), 2.80
(ddd, J ˆ 2.74, 3.16, 16.00 Hz, 2H), 2.89 (ddd, J ˆ 13.73, 2.48, 16.00 Hz, 2H), 3.01 (ddd, J ˆ
13.73,2.74, 14.01 Hz, 2H), 4.08(s, 2H), 5.84(s, 2H), 7.451 7.192(m, 8H), 9.26(s, 2H), 10.98(s, 2H),
13.59 (s, 2H) ppm; 13C NMR: ꢁ ˆ 9.37, 10.30, 18.77, 22.50, 32.75, 104.13, 116.40, 119.87, 119.88,
124.24, 124.71, 125.04, 126.97, 128.38, 130.83, 131.84, 134.04, 140.21, 160.11, 173.99, 179.85 ppm.
4-(o-Fluorophenyl)-3-methyl-2-p-toluenesulfonyl-1H-pyrrole (11; C18H16NO2SF)
3-(o-Fluorophenyl)-4-methyl-2-p-toluenesulfonyl-1H-pyrrole (12; 6.0 g, 18.2 mmol) was dissolved
in 18 cm3 of TFA and 164 cm3 of CH2Cl2 to make a 0.1 M solution. The solution was stirred at room
temperature for 4 days, during which time it darkened. It was washed with H2O (2 Â 200 cm3),
aqueous NaHCO3 solution (2 Â 200 cm3), and brine (200 cm3); then it was dried over MgSO4. The
solvent was removed on a rotary evaporator and the residue crystallized from CH2Cl2/hexane to
afford a tan solid. Colorless crystals were obtained by precipitation from CH2Cl2/hexane. They were
collected and dried overnight in a drying pistol over P2O5.
Yield: 5.54 g (92%); m.p.: 158±159ꢂC; IR (KBr): ꢆ ˆ 3366, 1315, 1229, 1188, 1086, 814 cm
1
;
1H NMR: ꢁ ˆ 9.25 (br s, 1H), 7.82 (d, J ˆ 8.4 Hz, 2H), 7.31 (d, J ˆ 8.4 Hz, 2H), 7.27 7.02 (m, 4H),

Fluorophenyl Bilirubins
1545
7.04 (d, J ˆ 2 Hz, 1H), 2.42 (s, 3H), 2.22 (d, JHF ˆ 1 Hz, 3H) ppm; 13C NMR: ꢁ ˆ 159.91 (JCF
ˆ
246 Hz), 143.93, 139.66, 131.46 (JCF ˆ 3.3 Hz), 129.97, 129.66 (JCF ˆ 8.8 Hz), 126.81, 124.61,
124.51, 124.05 (JCF ˆ 3.3 Hz), 122.54, 121.98 (JCF ˆ 15.4 Hz), 120.97, 115.83 (JCF ˆ 23 Hz),
21.68, 10.41 (JCF ˆ 3.3 Hz) ppm.
5-Bromo-4-(o-¯uorophenyl)-3-methyl-2-p-toluenesulfonyl-1H-pyrrole (13; C18H18NO2SBrF)
Compound 13 was prepared as described for 5-bromo-2-p-toluenesulfonyl-3-(o-¯uorophenyl)-4-
methyl-5-bromo-1H-pyrrole (14) using 5.69 g (17.3 mmol) of 2-(p-tosyl)-3-methyl-4-(o-¯uorophe-
nyl)-1H-pyrrole and 13.0 g (2.0 eq.) of PTT.
1
Yield: 6.85 g (97%); m.p.: 140±141ꢂC; IR (KBr): ꢆ ˆ 3287, 1464, 1232, 1183, 1067, 817 cm
;
1H NMR: ꢁ ˆ 9.64 (br s, 1H), 7.85 (d, J ˆ 8 Hz, 2H), 7.33 (d, J ˆ 8 Hz, 2H), 7.20 7.09 (m, 4H), 2.43
(s, 3H), 2.14 (s, 3H) ppm; 13C NMR: ꢁ ˆ 160.26 (JCF ˆ 249 Hz), 144.22, 139.472, 132.66 (JCF
ˆ
2.2 Hz), 130.07, 129.92 (JCF ˆ 8.8 Hz), 127.01, 126.61, 125.76, 124.07 (JCF ˆ 3.3 Hz), 121.70,
120.35 (JCF ˆ 15.4 Hz), 115.92 (JCF ˆ 22 Hz), 105.89, 21.71, 10.56 (JCF ˆ 2.2 Hz) ppm; MS: m/z
(%) ˆ 409 (100), 344 (15), 188 (45), 172 (96), 133 (81), 91 (24) amu.
3-(o-Fluorophenyl)-4-methyl-5-p-toluenesulfonyl-pyrrolin-2-one (9; C18H16NO3SF)
Compound 9 was prepared as described for 4-(o-¯uorophenyl)-3-methyl-5-p-toluenesulfonyl-pyrro-
lin-2-one (10) using 2.00 g (4.90 mmol) of 5-bromo-4-(o-¯uorophenyl)-3-methyl-2-p-toluene-
sulfonyl-1H-pyrrole (13).
Yield: 1.45 g (86%); m.p.: 188±189ꢂC; IR (KBr): ꢆ ˆ 3426, 1700, 1496, 1387, 1318, 1207, 1137,
1
1080, 819, 760, 630, 588 cm
;
1H NMR: ꢁ ˆ 7.72 (d, J ˆ 8 Hz, 2H), 7.31 (d, J ˆ 8 Hz, 2H),
7.14 6.96 (m, 4H), 6.86 (s, 1H), 5.21 (br s, 1H), 2.40 (s, 3H), 2.24 (d, JHF ˆ 2.2 Hz, 3H) ppm; 13C
NMR: ꢁ ˆ 171.1, 159.81 (JCF ˆ 250 Hz), 147.80, 146.30, 132.01, 131.15 (JCF ˆ 3.3 Hz), 130.87
(JCF ˆ 8.8 Hz), 130.05, 129.88, 129.79, 124.15 (JCF ˆ 3.3 Hz), 117.54 (JCF ˆ 15.4 Hz), 115.95
(JCF ˆ 22 Hz), 79.54, 21.92, 14.99 (JCF ˆ 4.4 Hz) ppm.
3-(o-Fluorophenyl)-4-methyl-pyrrolin-2-one (7; C11H10NOF)
Compound 7 was prepared as described for 4-(o-¯uorophenyl)-3-methyl-pyrrolin-2-one (8) using
1.00 g (2.90 mmol) of 3-(o-¯uorophenyl)-4-methyl-5-p-toluenesulfonyl-pyrrolin-2-one (9) and 0.23 g
(2 eq.) of NaBH4.
Yield: 0.54 g (98%); m.p.: 158±159ꢂC; IR (KBr): ꢆ ˆ 3198, 1678, 1660, 1490, 1456, 1211, 755,
688 cm 1; 1H NMR: ꢁ ˆ 7.74 (br s, 1H), 7.41 7.09 (m, 4H), 4.01 (br s, 2H), 2.04 (s, 3H) ppm; 13
C
NMR: ꢁ ˆ 174.54, 160.08 (JCF ˆ 250 Hz), 154.54, 131.65 (JCF ˆ 3 Hz), 129.95 (JCF ˆ 9 Hz), 127.65,
124.17 (JCF ˆ 3 Hz), 119.27 (JCF ˆ 15 Hz), 115.88 (JCF ˆ 22 Hz), 50.93, 14.82 (JCF ˆ 3 Hz) ppm.
2,18-Desvinyl-2,18-bis-(o-¯uorophenyl)bilirubin-IIIꢀ (1; C41H38N4O6F2)
Compound 1 was prepared exactly as described for 3,17-desvinyl-3,17-bis-(o-¯uorophenyl)-
bilirubin-XIIIꢀ (2).
Yield: 44.3 mg (59%); m.p.: 275±280ꢂC (dec); IR (KBr): ꢆ ˆ 3417, 2924, 1685, 1617, 1498,
1399, 1248, 778 cm 1; 1H NMR: ꢁ ˆ 2.14 (d, JHF ˆ 2.10 Hz, 6H), 2.20 (s, 6H), 2.60 (ddd, J ˆ 2.74,
3.20, 13.23 Hz, 2H), 2.82 (ddd, J ˆ 2.74, 3.20, 18.48 Hz, 2H), 2.92 (ddd, J ˆ 13.81, 2.74,
18.48 Hz, 2H), 3.04 (ddd, J ˆ 13.81, 3.20, 13.23 Hz, 2H), 4.11 (s, 2H), 6.26 (s, 2H), 7.12 (m, 2H),
7.19 (m, 2H), 7.31 (m, 2H), 7.37 (m, 2H), 9.36 (s, 2H), 10.88 (s, 2H), 13.53 (s, 2H) ppm; 13C NMR:
ꢁ ˆ 10.43, 11.27, 18.80, 22.55, 32.78, 102.86, 116.10, 119.39, 120.09, 122.32, 124.26, 124.60,
125.38, 129.26, 129.97, 131.92, 134.32, 145.61, 160.15, 172.99, 179.87 ppm.

1546
J. O. Brower and D. A. Lightner: Fluorophenyl Bilirubins
Acknowledgments
We thank the National Institutes of Health (HD-17779) for support of this research.
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Received May 22, 2001. Accepted May 29, 2001