Novel benzoic acid congeners of bilirubin

Article abstract of DOI:10.1021/jo030091t

Three regioisomeric bilirubins and biliverdins with propionic acids replaced by benzoic acids were synthesized from the corresponding xanthobilirubic acids by oxidative coupling. The rubins were found to exhibit widely varying polarity, spectroscopic properties, and stereochemistry. The isomer with ortho benzoic acids (1o) was much less polar than either the meta (1m) or para (1p) because 1o (but not 1m and 1p) can adopt a folded conformation with both carboxylic acids intramolecularly hydrogen bonded to the opposing dipyrrinones. The consequences of such conformational differentiation are found in the varying ¹H NMR, UV-vis, and circular dichroism spectral properties.

Full text of DOI:10.1021/jo030091t

Novel Ben zoic Acid Con gen er s of Bilir u bin
Stefan E. Boiadjiev and David A. Lightner*
Chemistry Department, University of Nevada, Reno, Nevada 89557
lightner@scs.unr.edu
Received March 13, 2003
Three regioisomeric bilirubins and biliverdins with propionic acids replaced by benzoic acids were
synthesized from the corresponding xanthobilirubic acids by oxidative coupling. The rubins were
found to exhibit widely varying polarity, spectroscopic properties, and stereochemistry. The isomer
with ortho benzoic acids (1o) was much less polar than either the meta (1m ) or para (1p) because
1o (but not 1m and 1p) can adopt a folded conformation with both carboxylic acids intramolecularly
hydrogen bonded to the opposing dipyrrinones. The consequences of such conformational dif-
ferentiation are found in the varying 1H NMR, UV-vis, and circular dichroism spectral properties.
In tr od u ction
both of bilirubin’s propionic acids to glucuronide esters
in the livers of both humans and rats.1,12-14
J aundice appears in humans who cannot efficiently
eliminate bilirubin (Figure 1), which is a yellow tetra-
pyrrole dicarboxylic acid formed in normal adult metabo-
lism, principally from the heme of red cells, at the rate
of 250-300 mg per day.1,2 Perhaps the most important
aspect of the bilirubin molecular structure, concluded
from numerous investigations, is its strong propensity
to adopt a ridge-tile shape3 with both carboxylic acid
groups firmly engaged in intramolecular hydrogen bond-
ing, each to a dipyrrinone.3-8 Such a structure (Figure
1) explains many of the chemical properties of the
pigment, especially its lipophilicity, e.g., soluble in CHCl3
but insoluble in CH3OH.9 It also helps to understand why
bilirubin is not excreted intact by the liver (in contrast
to biliverdin or more polar bilirubin isomers where the
propionic acids are displaced from C(8) and C(12), e.g.,
to C(7) and C(13)).10,11 For excretion, a specific glucu-
ronosyl transferase enzyme (UGT1A1) converts one or
Effective intramolecular hydrogen bonding between the
bilirubin dipyrrinones and its carboxylic acid groups
requires that the propionic acids (i) stem from C(8) and
C(12) and (ii) be capable of orienting their CO2H termini
toward the dipyrrinones.3,4 Mesobilirubin-XIIIR (Figure
2) has these essential components and orientation, and
its most stable conformation is the intramolecularly
hydrogen-bonded ridge-tile (as in Figure 1).4 Molecular
modeling studies indicate that a bilirubin with propionic
acids replaced by acrylic acids with cis (but not trans)
carbon-carbon double bonds should adopt an intramo-
lecularly hydrogen-bonded ridge tile conformation. Bi-
lirubins with ortho benzoic acid groups replacing propi-
onic acids have their CO2H groups locked more firmly
into the stereochemistry required for intramolecular
hydrogen bonding15 and are predicted to behave similarly.
In contrast, the meta (1m ) and para (1p) regioisomers
have longer carbon chain paths connecting C(8)/C(12) to
the CO2H groups, longer carbon chain paths that have
their counterparts in (i) the lipophilic rubin with butyric
acids replacing propionic, and (ii) in the more polar
pentanoic acid rubin.16 Unlike butyric acid and pentanoic
acid rubins, the carbon chain paths of 1m and 1p are
very inflexible and no more flexible than that of 1o. In
the following we describe the syntheses of 1o, 1m , and
1p and compare their polarity and spectroscopic proper-
ties and stereochemistry.
(1) Chowdhury, J . R.; Wolkoff, A. W.; Chowdhury, N. R.; Arias, I.
M. Hereditary J aundice and Disorders of Bilirubin Metabolism. In The
Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R.,
Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York,
2001; Vol. 2, pp 3063-3101.
(2) McDonagh, A. F. Bile Pigments: Bilatrienes and 5, 15-Bila-
dienes. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New
York, 1979; Vol. 6, pp 293-491.
(3) Bonnett, R.; Davies, J . E.; Hursthouse, M. B.; Sheldrick, G. M.
Proc. R. Soc. London, Ser. B 1978, 202, 249-268.
(4) Person, R. V.; Peterson, B. R.; Lightner, D. A. J . Am. Chem. Soc.
1994, 116, 42-59.
(5) For leading references, see Sheldrick, W. S. Isr. J . Chem. 1983,
23, 155-166.
(11) Ramonas, L. M.; McDonagh, A. F.; Palma, L. A. J . Pharmacol.
Methods 1981, 5, 149-164.
(6) Do¨rner, T.; Knipp, B.; Lightner, D. A. Tetrahedron 1997, 53,
2697-2716.
(12) Bosma, P. J .; Seppen, J .; Goldhoorn, B.; Bakker, C.; Oude
Elferink, R. P.; Chowdhury, J . R.; Chowdhury, N. R.; J ansen, P. L. J .
Biol. Chem. 1994, 269, 17960-17964.
(7) For leading references, see: Kaplan, D.; Navon, G. Isr. J . Chem.
1983, 23, 177-186.
(8) Navon, G.; Frank, S.; Kaplan, D. J . Chem. Soc., Perkin Trans. 2
1984, 1145-1149.
(13) Coffman, B. L.; Green, M. D.; King, C. D.; Tephly, T. R. Mol.
Pharmacol. 1995, 47, 1101-1105.
(9) Lightner, D. A.; McDonagh, A. F. Acc. Chem. Res. 1984, 17, 417-
(14) Owens, I. S.; Ritter, J . K.; Yeatman, M. T.; Chen, F. J .
Pharmacokinet. Biopharm. 1996, 24, 491-508.
(15) Boiadjiev, S. E.; Lightner, D. A. Tetrahedron 2002, 58, 7411-
7421.
424.
(10) McDonagh, A. F.; Lightner, D. A. The Importance of Molecular
Structure in Bilirubin Metabolism and Excretion. In Hepatic Metabo-
lism and Disposition of Endo and Xenobiotics; Bock, K. W., Gerok, W.,
Matern, S., Eds.; Falk Symposium No. 57; Kluwer: Dordrecht, The
Netherlands, 1991, Chapter 5, 47-59.
(16) (a) Lightner, D. A.; McDonagh, A. F. J . Perinatol. 2001, 21,
S13-S16. (b) Shrout, D. P.; Puzicha, G.; Lightner, D. A. Synthesis 1992,
328-332.
10.1021/jo030091t CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/09/2003
J . Org. Chem. 2003, 68, 7591-7604
7591

Boiadjiev and Lightner
F IGURE 1. Enzymic conversion of heme to biliverdin and
biliverdin to bilirubin. The most stable (ridge-tile) conforma-
tion of bilirubin (lower left) is one of many possible conforma-
tions that arise by rotation of the dipyrrinones about C(10)
and the only conformation where nonbonded intramolecular
steric interactions are minimized and where both carboxylic
acid groups engage in intramolecular hydrogen bonding to the
opposing dipyrrinones. The ridge-tile conformation is defined
by torsion angles about both C(10) C-C single bonds [φ1
)
N(22)-C(9)-C(10)-C(11) and φ2 ) C(9)-C(10)-C(11)-N(23)],
the C(5)-C(6) single bond [θ1 ) C(4)-C(5)-C(6)-N(22)] and
the C(14)-C(15) single bond [θ2 ) N(23)-C(14)-C(15)-C(16)],
where φ1 ) φ2 ) -60° and θ1 ) θ2 ) 0°. (In the porphyrin-like
shape of bilirubin (right), φ1 ) φ2 ) 0°.)
F IGURE 2. Mesobilirubin-XIIIR and its analogue with an
alkanoic acid chain-stiffening double bond, and the regioiso-
meric benzoic acid rubins with carboxyphenyl groups replacing
propionic acids.
2,4-dione with sodium o-bromobenzoate has been re-
ported,18 and the product successfully used in pyrrole
synthesis,15 the m- and p-bromobenzoates do not react
in the same manner.18a An alternative approach to
pyrrole ring formation involved base-catalyzed Barton-
Zard reaction of R-isocyano esters with nitroalkenes (or
â-acetoxy nitroalkanes as convenient surrogates).19 The
initial stages of such an approach, condensing methyl m-
or p-formylbenzoates and nitroethane, followed by con-
densation with benzyl isocyanoacetate were explored.
This route leads to 9-benzyloxycarbonyl dipyrrinones20
and thus necessitates further elaboration of the resulting
9-ester group in order to convert the dipyrrinone to a
linear tetrapyrrole. In light of this perceived encum-
brance, we were more inclined toward a Suzuki coupling
reaction21 on a preformed pyrrole nucleus.
Resu lts a n d Discu ssion
Syn th eses. As in other reported syntheses of rubins
and verdins, the tetrapyrroles of this study were obtained
following oxidative coupling of dipyrrinones using chlo-
ranil in dichloromethane and formic acid at 40 °C
(Scheme 1). Thus, 5o gave verdin ester 3o, 5m gave 3m ,
and 5p gave 3p, all in good yield. The isobutyl ester was
chosen for the para series to overcome the extreme
insolubility of the dipyrrinone methyl ester and to
facilitate handling of the rubin ester (2p). Reduction of
the verdin esters using sodium borohydride converted the
deep blue verdin color to the bright yellow rubin color,
and the surprisingly stable rubin ester benzoic acids were
isolated in 91-93% yield following radial chromatogra-
phy and crystallization. Saponification of rubin esters (2)
afforded the benzoic acid rubins (1), of which 1m and 1p
were decidedly more polar than 1o.
(17) Fischer, H.; Orth, H. Die Chemie des Pyrroles; Akademische
Verlag GmbH: Leipzig, 1934; Vol. I.
(18) (a) Bacon, R. G. R.; Murray, J . C. F. J . Chem Soc., Perkin Trans.
1 1975, 1267-1272. (b)Aalten, H. L.; van Koten, G.; Vrieze, K.; van
der Kerk-van Hoof, A. Recl. Trav. Chim. Pays-Bas 1990, 109, 46-54.
(19) Barton, D. H. R.; Kervagoret, J .; Zard, S. Z. Tetrahedron 1990,
46, 7587-7598.
(20) (a) Kinoshita, H.; Ngwe, H.; Kohori, K.; Inomata, K. Chem. Lett.
1993, 1441-1442. (b) Kohori, K.; Hashimoto, M.; Kinoshita, H.;
Inomata, K. Bull. Chem. Soc. J pn. 1994, 67, 3088-3093.
Biaryls containing conjoined sterically hindered 3-pyr-
rolyl and meta or para carboxy (or carboalkoxy) phenyl
moieties are not known. In principle their syntheses
might be achieved by a classical Fischer-Knorr pyrrole
ring closure17 using the appropriate 3-arylpentane-2,4-
dione. Although arylation of the sodium salt of pentane-
7592 J . Org. Chem., Vol. 68, No. 20, 2003

Benzoic Acid Congeners of Bilirubin
SCHEME 1a
SCHEME 2a
a
Reagents and conditions: (a) NaOH/EtOH-H2O, ∆, then HCl;
(b) CH2N2; (c) (CH3)2CHCH2I/Cs2CO3/DMF; (d) CH3OH, ∆; (e)
NaOH/EtOH-H2O, ∆, then HNO3/aq. NaNO3; (f) HONdC(CO2Et)2/
Zn/HOAc/NaOAc, ∆; (g) Pd(PPh3)4, DMF, aq. Na2CO3, ∆; (h) KI,
H2O2, EtOH-H2O, ∆.
a
Reagents and conditions: (a) NaOH/EtOH-H2O, then HCl;
(b) NaBH4/CH3OH-CHCl3; (c) chloranil/HCO2H, ∆.
Suzuki coupling of a â-bromopyrrole with phenylbo-
ronic acid has been reported,22 but carbo(alko)xy-substi-
tuted boronic acids were not known to be participants in
the Suzuki coupling reaction with pyrroles. The precur-
sors, m-(methoxycarbonyl)phenyl boronic acid (12m )23
and its p-isomer (12p),24 were prepared inexpensively by
a combination of literature methods. Commercially avail-
able m- or p-bromotoluene easily formed Grignard re-
agents, which were reacted (inverse addition at -78 °C)
with trimethyl borate. During the acidic workup, the
borate ester hydrolyzed to furnish the corresponding
tolylboronic acids. Permanganate oxidation of the methyl
group to a carboxylic acid was followed by acid-catalyzed
esterification to afford 12m and 12p (Scheme 2). Methyl
3,5-dimethyl-1H-pyrrole-2-carboxylate (10)25 was iodi-
boronic acid 12 for 16-18 h at elevated temperature,
following the conditions suggested for phenylboronic
acid,22 yielded only 15-20% of the desired biaryl 8 after
tedious chromatographic separation. Numerous optimi-
zation efforts, including changing the solvent and base,
and using conditions for hindered substrates21b (38%
yield) led to little or no improvement. Examining the
composition of the reaction mixture with time revealed
that the coupling is essentially complete within a very
short time, 15-25 min at 100-105 °C when 4 mol % of
27
Pd(PPh3)4 catalyst and aqueous Na2CO3 were used in
oxygen-free dimethylformamide solvent. Under these
conditions both 12m and 12p reacted similarly with
iodopyrrole 11 to afford consistent 75-78% yields of
arylpyrroles 8m and 8p, respectively. The â-bromopyrrole
analogue of 11 showed an identical reactivity in a Suzuki
reaction under the same conditions.
26
nated at the â-position using KI/H2O2 to give almost
quantitatively the iodopyrrole 11. Reaction of 11 with
(21) (a) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457-2483.
(b) Watanabe, T.; Miyaura, N.; Suzuki, A. Synlett 1992, 207-210 (for
hindered substrates). (c) Suzuki, A. J . Organomet. Chem. 1999, 576,
147-168.
(22) Chang, C. K.; Bag, N. J . Org. Chem. 1995, 60, 7030-7032.
(23) (a) Suenaga, H.; Nakashima, K.; Mikami, M.; Yamamoto, H.;
J ames, T. D.; Samankumara Sandanayake, K. R. A.; Shinkai, S. Recl.
Trav. Chim. Pays-Bas 1996, 115, 44-48. (b) Haino, T.; Matsumura,
K.; Harano, T.; Yamada, K.; Saijyo, Y.; Fukazawa, Y. Tetrahedron
1998, 54, 12185-12196.
(24) (a) Matsubara, H.; Seto, K.; Tahara, T.; Takahashi, S. Bull.
Chem. Soc. J pn. 1989, 62, 3896-3901. (b) Hylarides, M. D.; Wilbur,
D. S.; Hadley, S. W.; Fritzberg, A. R. J . Organomet. Chem. 1989, 367,
259-265.
Standard saponification of the dimethyl esters 8m or
8p to their diacids (7m and 7p, respectively), followed
by condensation with 5-bromomethylene-4-ethyl-3-meth-
yl-2-oxo-1H-pyrrole (6),28 afforded crude yellow dipyr-
rinone acids 4m and 4p, respectively (Scheme 2). Curi-
ously, when this type of condensation is performed using
pyrrole diacids with an alkanoic side chain,28,29 the
dipyrrinones formed are esterified in the alkanoic acid
side chain from reaction with methanol solvent under
(25) Boiadjiev, S. E.; Lightner, D. A. Tetrahedron: Asymmetry 2002,
13, 1721-1732.
(26) Treibs, A.; Kolm, H. G. Liebigs Ann. Chem. 1958, 614, 176-
198.
(27) Coulson, D. R. Inorg. Synth. 1990, 28, 107-109.
(28) Shrout, D. P.; Lightner, D. A. Synthesis 1990, 1062-1065.
(29) Puzicha, G.; Shrout, D. P.; Lightner, D. A. J . Heterocycl. Chem.
1990, 27, 2117-2123.
J . Org. Chem, Vol. 68, No. 20, 2003 7593

Boiadjiev and Lightner
TABLE 1. Com p a r ison of 13C NMR Sp ectr a l
Assign m en ts of Ca r boxyp h en yl-xa n th obilir u bic Acid s 4
a n d Th eir Ester s 5a
TABLE 2. Com p a r ison of 1H NMR Sp ectr a l Assign m en ts
of Ca r boxyp h en yl-xa n th obilir u bic Acid s 4 a n d Th eir
Ester s 5a
acids
esters
acids (δ)
esters (δ)
ortho meta
para ortho meta
para
ortho meta
para
ortho meta
para
position
1-CO
(4o)
(4m )
(4p)
(5o)
(5m )
(5p)
position
(4o)
(4m )
(4p)
(5o)
(5m )
(5p)
172.0 172.1
122.4 122.6
8.1
147.2 147.3
17.2
14.9
127.7 128.57 127.7 127.9 128.6
97.6 97.3 97.2 97.5 97.2
122.2 121.6
122.8 123.4
10.0
121.9 120.7
134.9 135.8
172.0 172.0 172.0
122.7 122.0 122.6
8.1
147.3 147.2 147.3
17.2
14.8
172.0
122.8
8.1
147.3
17.1
14.8
126.7
97.1
121.4
123.5
10.2
120.7
140.8
129.1
pyrrole NH
lactam NH
2-CH3
10.53 10.66 10.68 10.54 10.66 10.69
2
9.84
1.79
9.84
1.79
9.84
1.79
9.83
1.79
9.84
1.79
9.84
1.79
2-CH3
3
3-CH2CH3
3-CH2CH3
4
5
6
7
7-CH3
8
i-
o-
o′-
m-
m′-
p-
8.1
8.1
8.1
3-CH2CH3
3-CH2CH3
5-CH)
2.51b 2.52b 2.52b 2.51b
2.52b 2.52p
17.2
14.8
17.1
14.8
17.2
14.8
1.09c 1.10c 1.10c 1.09c
1.10c 1.10q
5.96
1.90
6.00
2.07
7.79f
6.00
2.09
7.36i
5.97
1.87
6.00
2.06
6.00
2.09
7-CH3
o-H
o′-H
m-H
7.79n 7.40r
7.52o 7.40r
7.98r
121.5 121.8 121.4
123.5 123.0 123.4
10.2
120.7 121.8 120.7
140.3 135.3 136.0
7.18d 7.49g 7.36i
7.22j
7.75k
7.56l
7.73d
7.95i
10.1
9.8
10.1
m′-H
7.50e 7.52e 7.95i
7.36e 7.82h
7.55e 7.98r
p-H
7.40m 7.83o
-OCH3
3.64
3.85
133.9 128.56 129.2 131.9 128.8
-OCH2CH(CH3)2
-OCH2CH(CH3)2
-OCH2CH(CH3)2
-CO2H
4.07s
2.02n
0.98t
129.0 129.9
130.4 129.6
132.1 126.5
126.4 133.7
169.5 167.4
129.2 129.2 129.71 129.1
129.3 131.2 129.6
129.3 132.3 126.3
128.7 126.5 134.1
167.3 168.0 166.3
129.3
129.3
128.8
165.7
12.57 12.94 12.79
2.06
9-CH3
2.25
2.27
2.04
2.24
2.27
CO2R
-OCH3
-OCH2CH(CH3)2
-OCH2CH(CH3)2
-OCH2CH(CH3)2
9
Acquired in 2 × 10-2 M solutions in (CD3)2SO solvent at
a
51.9
52.2
b
d
25 °C. q, J ) 7.6 Hz. c t, J ) 7.6 Hz. d, 3J ) 7.6 Hz. e dd, 3J )
70.2
27.4
18.9
7.6, 7.6 Hz. f br. dd, J ) 1.5, 1.6 Hz. ddd, J ) 7.6, J ) 1.5, 1.6
4
g
3
4
h
i
j
Hz. ddd, 3J ) 7.6, 4J ) 1.6, 1.6 Hz. br. d, 3J ) 8.2 Hz. dd,
3J ) 7.7, 4J ) 0.9 Hz. dd, 3J ) 7.7, 4J ) 1.1 Hz. ddd, 3J ) 7.7,
k
l
129.5 130.8
11.6
130.0 129.4 129.67 130.1
12.0 11.5 11.8 11.9
7.6, 4J ) 1.1 Hz. ddd, 3J ) 7.7, 7.6; 4J ) 0.9 Hz. m. o br. d,
m
n
9-CH3
11.8
3J ) 7.6 Hz. q, J ) 7.7 Hz. t, J ) 7.7 Hz. br. d, J ) 8.3 Hz.s d,
p
q
r
a
Acquired in 2 × 10-2 M solutions in (CD3)2SO solvent at
t
J ) 6.4 Hz. d, J ) 6.7 Hz.
25 °C.
soluble in DMSO. The corresponding sodium or am-
monium salts are rather soluble in both methanol and
water.
acidic reaction conditions. In contrast, only a partial
esterification (<10%) occurred in the syntheses of 4m and
4p, consistent with what was detected earlier in the
preparation of 4o.15 Possibly the dipyrrinones 4 are much
less soluble in methanol than their counterparts with
alkanoic acid chains and precipitate soon after their
formation, thereby slowing any subsequent esterification.
The m-methyl ester 5m was prepared in 75% yield from
crude acid 4m by esterification with an excess of diazo-
methane in ether followed by purification using chroma-
tography. Similar treatment of the crude p-acid 4p gave
a surprisingly insoluble methyl ester that was not
suitable for purification by chromatography. Therefore,
an SN2 esterification process from reaction of isobutyl
iodide with the cesium carboxylate salt30 of 4p was
followed. This reaction afforded a 58% yield of isobutyl
ester 5p (based on the amount of starting 8p), which
showed favorable solubility and was amenable to chro-
matographic purification.
The synthesis of the ortho compounds has been re-
ported previously15 and for purposes of comparison is
outlined briefly here and in Scheme 2. At the time the
work was initiated, pyrroles substituted with â o-car-
boethoxyphenyl, o-carbomethoxyphenyl, or o-carboxy-
phenyl groups were unknown. For purposes of Suzuki
coupling to make the diester of 7, although m- and
p-carboxy (or carboalkoxy) phenylboronic acids had been
described in the literature, the o-isomer had not. We
therefore decided to pursue a more classical Fischer-
Knorr synthesis from 3-(o-carboxyphenyl)-pentane-2,4-
dione. 3-(o-Carboxyphenyl)-pentane-2,4-dione (9) was
prepared in high yield by reaction of sodium o-bromoben-
zoate with the sodium salt of pentane-2,4-dione.18 The
resulting acid (9) under the same Fischer-Knorr pyrrole-
forming condensation conditions afforded a 65% isolated
yield of pyrrole R-ester of acid 7o, which was saponified
to 7o and condensed smoothly with 6 to afford 4o.
Attempted oxidative coupling of dipyrrinone acid 4o gave
no verdin, apparently because of interference (by proton
transfer) from the free CO2H group; however, oxidative
coupling of dipyrrinone ester 5o gave a mixture of verdins
2o in 91% yield.
The purified bright yellow esters (5) were saponified
quantitatively with aqueous sodium hydroxide in a
homogeneous mixture with ethanol. The precipitated
acids (4) are nearly insoluble in nonpolar solvents and
only partially soluble in methanol. They are, however,
(30) (a) Dijkstra, G.; Kruizinga, W. H.; Kellogg, R. M. J . Org. Chem.
1987, 52, 4230-4234. (b) Kunz, H.; Lerchen, H.-G. Tetrahedron Lett.
1987, 28, 1873-1876.
Molecu la r Str u ctu r e. The constitutional structures
of 4 and 5 follow from the methods of synthesis and from
7594 J . Org. Chem., Vol. 68, No. 20, 2003

Benzoic Acid Congeners of Bilirubin
TABLE 3. Com p a r ison of 13C NMR Sp ectr a l
TABLE 4. Com p a r ison of 1H NMR Sp ectr a l Assign m en ts
of Ca r boxyp h en yl-m esobilir u bin s 1 a n d 2a
Assign m en ts of Ca r boxyp h en yl-m esobilir u bin s 1 a n d 2a
R ) H (δ)
R ) alkyl (δ)
R ) H
ortho meta para ortho meta para
(1o)b (1m ) (1p) (2o)c (2m ) (2p)
168.9 167.3 167.2 167.4 166.1 165.6
169.1 167.5
172.0 172.1 172.1 172.0 172.0 172.1
172.0 172.0
123.1 123.6 123.8 123.1 123.5 123.8
R ) alkyl
ortho
meta
(1m )
para
(1p)
ortho
(2o)c
meta
(2m )
para
position
(1o)b
(2p)
position
COOR
2,18-CH3
1.78
1.79
1.79
1.79 1.78
1.79
1.79
1.79
3,17-CH2CH3 2.50d
2.51d
2.50d 2.50k 2.51d
2.52d
2.50d 2.50d
1.10e 1.09e
1,19-CONH
2,18
3,17-CH2CH3 1.09e
1.10e 1.09l 1.09e
1.10e
1.10e
123.1
8.1
(br.)
123.2
8.07
8.08
5,15-CH)
5.91
5.93
1.73
1.77
5.91
1.89
5.93 5.91
5.92
1.93 1.71
1.72
5.87
1.84
5.92
1.92
2,18-CH3
3,17
8.1
8.1
8.1
8.1
7,13-CH3
147.1 147.2 147.2 147.1 147.2 147.1
147.2 147.2
17.17 17.2 17.2 17.15 17.2 17.2
o,o
7.54h 7.06m
7.16h 7.06m 6.72n
6.78o
7.46s 7.08w
3,17-CH2CH3
o′,o′
6.63f
6.82f
7.66g
7.70g
7.23-
7.15t
7.08w
17.18
14.9
17.16
14.8 14.8 14.9
3,17-CH2CH3
4,16
14.8 14.7
m,m
7.75m 7.67p
7.69q
7.75w
128.2 129.2 129.6 128.3 129.0 129.6
128.4
97.8
97.9
128.5
97.5 97.3 97.7
(br.)
m′,m′
7.27h 7.75m 7.26-
7.29u 7.75w
7.66v
5,15
97.2 97.2
7.31h
7.33h
7.37r
6,14
122.0 120.8 120.6 121.8 120.9 120.6
122.1 121.9
122.1 122.4 122.4 122.0 122.2 122.2
122.2 122.1
9.86 9.68
9.89 9.70
122.6 122.9 123.2 122.2 122.8 123.1
122.7 122.3
134.8 135.2 139.7 135.2 135.4 140.1
134.9 135.3
132.9 130.1 128.8 131.2 129.9 128.5
133.0 131.4
128.8 133.6 128.8 129.0 133.9 128.5
129.0 129.1
130.2 130.3 129.2 130.7 129.6 129.3
130.4 131.0
132.0 127.8 129.2 132.2 127.9 129.3
132.1 (br.)
126.18 126.4 127.7 126.2 126.2 126.8
p,p
7.23-
7.31h
7.68h
7.26-
7.33h
7,13
COOH
CO2CH3
12.32
12.73 12.66
3.51
3.54
7,13-CH3
8, 12
i, i
9.9 10.1
9.8 10.0
3.75
CO2CH2-
CH(CH3)2
CO2CH2-
CH(CH3)2
CO2CH2-
CH(CH3)2
10-CH2
4.01x
1.99h
0.96y
o, o
3.57;
4.03
9.72
4.06 3.56,
4.04
9.68
4.08
9.72
o′,o′
3.78i
3.75i
3.74j
9.71
3.67j
9.76 9.72
9.79
m,m
21,24-
lactam NH 9.77
10.08
pyrrole NH 10.24
m′,m′
p,p
22,23-
10.39 10.46 10.13
10.20
10.40 10.47
126.24
126.4
51.68 51.9
51.72
a
Acquired in 2 × 10-2 M solutions in (CD3)2SO solvent at
R ) CH3
25 °C. Italicized signals dominate 65:35. c Ratio ∼1:1. q, J )
b
d
7.6 Hz. e t, J ) 7.6 Hz. f dd, 3J ) 7.0, 4J ) 1.0 Hz. dd, 3J ) 7.5,
g
R ) CH2CH(CH3)2
R ) CH2CH(CH3)2
R ) CH2CH(CH3)2
9,11
70.1
27.4
18.9
4J ) 1.4 Hz. m. AB, 2J ) 16.8 Hz. s. q, J ) 7.5 Hz. t, J )
h
i
j
k
l
7.5 Hz. d, J ) 8.0 Hz. dd, J ) 7.5, J ) 1.1 Hz. o dd, J ) 7.6,
m
n
3
4
3
4J ) 1.1 Hz. dd, 3J ) 7.5, 4J ) 1.5 Hz. dd, 3J ) 7.7, 4J ) 1.5
p
q
130.1 129.6 129.8 129.9 129.1 130.0
Hz. ddd, 3J ) 7.5; 7.5, 4J ) 1.5 Hz. dd, 4J ) 1.7; 1.6 Hz. ddd,
r
s
t
130.5
23.9
24.2
130.3
23.9 24.1 23.8
24.0
3J ) 7.8, 4J ) 1.7; 1.6 Hz. dd, 3J ) 7.8; 7.7 Hz. v ddd, 3J ) 7.7;
u
10
24.1 24.1
4J ) 1.7; 1.6 Hz. d, J ) 8.1 Hz. x d, J ) 6.5 Hz. y d, J ) 6.7 Hz.
w
a
Acquired in 2 × 10-2 M solutions in (CD3)2SO solvent at
4m , 4p, 5m , and 5p) may be attributed to the magnetic
anisotropy of the benzene ring, which is more constrained
to be perpendicular to the attached pyrrole ring in order
to minimize nonbonded steric interaction between sets
of three “ortho” groups (2 × CH3 + 1 × CO2H in 4o and
5o).
In complete correspondence, the constitutional struc-
tures of 1 and 2 follow from their syntheses (from 4 and
5) and from their 13C NMR spectra (Table 3), which show
the expected characteristic carbon resonances for the
pyrrolinone and pyrrole units, and their â-substituents.
Again the distinctive ortho, meta, and para substitution
patterns from the benzoic acid resonances find good
correlation with the indicated structures.
25 °C. Italicized signals dominate 65:35. c Ratio ∼1:1.
b
their 13C NMR spectra. Thus, the 13C resonances assign-
able to the dipyrrinone skeleton of the ortho, meta, and
para isomers of dipyrrinones 4 and 5 (Table 1) all
correlate nicely, with only slight differences in chemical
shifts. The main differences, as expected, lie with reso-
nances coming from the benzoic acid moiety, due to the
differing substitution patterns of the aromatic ring. A
similarly good correlation is found in their 1H NMR
spectra (Table 2), with the main distinguishing features
coming from the benzoic acid rings. The more shielded
C(7) and C(9) methyls of 4o or 5o (relative to those in
J . Org. Chem, Vol. 68, No. 20, 2003 7595

Boiadjiev and Lightner
F IGURE 3. Ball and Stick (ref 33) conformational representations of the most stable P -helical conformers of 1 with two
dipyrrinones shaped like a ridge-tile. In 1o (upper left) considerable conformational stabilization is afforded by intramolecular
hydrogen bonding between the dipyrrinones and the opposing o-carboxyphenyl groups. Such hydrogen bonding (and hence
conformational stabilization) is spatially impossible in 1m (upper right) and 1p (lower). The shape of each isomer of 1 may be
defined by the torsion angles φ1, φ2, θ1, and θ2 (see Figure 1), where in 1o, φ1 ≈ φ2 ≈ 60° and θ1 ≈ θ2 ≈ -14°; in 1m , φ1 ≈ φ2 ≈ 70°
and θ1 ≈ θ2 ≈ 45°; and in 1p, φ1 ≈ φ2 ≈ 80° and θ1 ≈ θ2 ≈ -41°.
Small but perhaps characteristic differences may be
found in the chemical shifts of (i) the C(4/16) resonances,
which are more shielded by ∼1 ppm in 1o and 2o than
in 1m , 1p, 2m , or 2p and (ii) the C(6/14) resonances,
which are more deshielded by ∼1 ppm in 1o and 2o than
in 1m , 2m , 1p, or 2p. As detected previously,15 the
doubled carbon signals in 1o and 2o give clear evidence
of diastereotopicity (due to restricted rotation about the
pyrrole to benzoic acid bond). Similar evidence for atrop-
isomerism is not found in 1m or 2m . Consistent with the
properties than those of the meta (1m ) and para (1p). It
is soluble in CHCl3; the latter two are insoluble. It is
insoluble in CH3OH; the latter two are slightly soluble.
Its HPLC retention time (26.8 min) using a reverse phase
column31 is much longer than that of 1m (8.0 min) or 1p
(7.1 min). In TLC on silica gel using 2% (by vol) CH3OH
in CH2Cl2 as eluent: 1o (Rf ) 0.93) moved faster than
1m (Rf ) 0.01) and 1p (Rf ) 0.02). In 8% CH3OH-
CH2Cl2: 1o (Rf ) 1.00), 1m (Rf ) 0.08), and 1p (Rf )
0.09). The data indicate that 1m and 1p exhibit very
similar polarity and solubility properties, that these
isomers are much more polar than 1o. Among bilirubins,
such large differences are in accord with a structural
model in which the polar carboxylic acid groups of 1o are
hydrogen-bonded intramolecularly to the dipyrrinones,
whereas in 1m and 1p, such intramolecular hydrogen
bonding is absent. No such large differences in solubility
1
conclusions from 13C NMR, the H NMR spectra (Table
4) of 1 and 2 show few differences, except for those
attending the differing substitution pattern of the benzoic
acid rings and the evidence for atropisomerism found in
1o and 2o. Again, as in dipyrrinones 4 and 5, the C(7/
13)-CH3 and C(10)-CH2 resonances of 1o and 2o show
a greater shielding than in 1m , 1p, 2m , and 2p because
the aromatic rings are more constrained to remain
orthogonal to the pyrrole rings in 1o and 2o.
Solu tion P r op er ties. The bilirubin with o-benzoic
acid groups (1o) exhibits considerably different solubility
(31) McDonagh, A. F.; Palma, L. A.; Lightner, D. A. J . Am. Chem.
Soc. 1982, 104, 6867-6869.
7596 J . Org. Chem., Vol. 68, No. 20, 2003

Benzoic Acid Congeners of Bilirubin
F IGURE 4. Long-wavelength UV-vis absorption curves of
the isomeric benzoic-xanthobilirubic acids 4o (s), 4m (- - -),
and 4p (‚‚‚‚) in CHCl3, CH3OH, and (CH3)2SO solvent run at
3.5-4.0 × 10-5 M at 23 °C. The CHCl3 and CH3OH solutions
contained 2% (by vol) (CH3)2SO.
F IGURE 5. Long-wavelength UV-vis absorption curves of
benzoic acid rubins 1o (s), 1m (- - -), and 1p (‚‚‚‚) in CHCl3,
CH3OH, and (CH3)2SO solvent run at 1.6-2.2 × 10-5 M at
23 °C. The CHCl3 and CH3OH solutions contained 2% (by vol)
(CH3)2SO.
the expected folded, ridge-tile structures. They differ only
slightly in overall shape, as determined by torsion angles
about C(10), C(5)/C(15) (see Figure 1). In 1o, φ1 ≈ φ2 ≈
60° and θ1 ≈ θ2 ≈ -14°; in 1m , φ1 ≈ φ2 ≈ 70° and θ1 ≈
θ2 ≈ 45°; in 1p, φ1 ≈ φ2 ≈ 80° and θ1 ≈ θ2 ≈ -41°. The
conformation of 1o is greatly stabilized by intramolecular
hydrogen bonding; those of 1m and 1p arise by minimi-
zation of nonbonding steric repulsions. In 1, the benzoic
acid ring is not coplanar with the conjoined pyrrole. As
a result of intramolecular hydrogen bonding in 1o, the
twist angle of the phenyl ring is ∼75°, but in 1m and 1p
it is 142-148°. Some weak residual hydrogen bonding
between the CO2H and lactam carbonyl appears in 1m
(∼2.2 Å H‚‚‚‚O) but the CO2H carbonyl to NH hydrogen
bonds are absent. While it may be noted that the
representations of 1o, 1m , and 1p all belong to the same
molecular chirality, the helicity represented is opposite
to that shown for bilirubin in Figure 1. In fact the
bilirubin and benzoic acid rubins 1 shown all have
isoenergetic nonsuperimposable mirror images.
properties and polarity are evident for the isomeric rubin
esters (2) or the isomeric verdins (3).
Molecu la r Con for m a tion fr om Molecu la r Dyn a m -
ics Ca lcu la tion s. Substantial evidence from investiga-
tions of bilirubin stereochemistry has been marshaled to
show that (i) the most stable conformation is shaped like
a ridge-tile, with the planes of the two dipyrrinones
intersecting at an angle of ∼100° and (ii) considerable
conformational stabilization is achieved by intramolecu-
lar hydrogen bonding.4 The computed (Sybyl)32,33 global
energy minimum conformations of 1 (Figure 3) all show
(32) Boiadjiev, S. E.; Person, R. V.; Puzicha, G.; Knobler, C.;
Maverick, E.; Trueblood, K. N.; Lightner, D. A. J . Am. Chem. Soc. 1992,
114, 10123-10133.
(33) The molecular dynamics calculations used to find the global
energy minimum conformations of 1 were run on an SGI Octane
workstation using vers. 6.4 of the Sybyl forcefield as described in ref
4. The Ball and Stick drawings were created from the atomic
coordinates using Mu¨ller and Falk’s “Ball and Stock” program for the
Macintosh (http://www.orc.uni-Linz.ac.at/mueller/ball_stick.html).
J . Org. Chem, Vol. 68, No. 20, 2003 7597

Boiadjiev and Lightner
clear evidence that the long-wavelength absorption con-
sists of two overlapping components. Substantial differ-
ences attend the change of solvents from nonpolar CHCl3
to the more polar solvents. As seen in the UV-vis spectra
of other rubins, the band shape consisting of overlapping
curves has its origin in exciton coupling4,34b,36 and is due
to two exciton components originating in an interaction
between the electric dipole transition moments in the two
proximal dipyrrinone chromophores. The exact shape of
the composite curve and the transcendency of one com-
ponent over the other is determined by the relative
orientation of the two dipyrrinones and can thus provide
an insight into conformation of the rubin.4 The data are
consistent with a conformation where the dipyrrinones
are oriented at ∼100° angle (as in Figures 1 and 3), for
the ridge-tile conformation for 1o and 2o, even for 1m ,
2m , 1p, and 2p in polar solvents such as CH3OH and
(CH3)2SO. In CHCl3, which promotes hydrogen bonding,
the shape of the UV-vis curves of 1o and 2o differ
considerably from the others, which show an exaggera-
tion of the shorter wavelength exciton component. Such
an exaggeration has been seen previously in the UV-
vis spectra of bilirubin dimethyl ester and other rubins
incapable of intramolecular hydrogen bonding. It has
been attributed to a change in conformation, from ridge-
tile to helical, to accommodate effective intermolecular
hydrogen bonding in a dimer.35 Such rubins are known
to be dimeric in CHCl3; so, it is not surprising that 2m
and 2p exhibit the UV-vis curves shown in CHCl3. It is
surprising that 2o does not, and the data suggest that
this ester continues to adopt a ridge-tile shape and would
be monomeric in CHCl3. The UV-vis data in CHCl3 for
the benzoic acid rubins are consistent with a ridge-tile
conformation (Figure 3) in 1o, and a tendency toward the
ridge-tile in 1m , but very likely an intermolecularly
hydrogen-bonded dimer for 1p.
In d u ced Cir cu la r Dich r oism a n d Con for m a tion .
As observed previously with bilirubin and its analogues,
circular dichroism (CD) is induced in the presence of
chiral solvating agents.37,38 The origin of the CD can be
deduced from two observations: (i) bilirubin behaves like
a molecular exciton consisting of two dipyrrinones inter-
acting by excited-state dipole-dipole coupling, and (ii)
the pigment adopts either of two interconverting enan-
tiomeric ridge-tiles, as depicted in Figure 7.4 The com-
ponent dipyrrinone chromophores of 1 have strongly
allowed long-wavelength electric dipole transition mo-
ments (Table 5), making them excellent candidates for
induced electric dipole-electric dipole (Figure 7) interac-
F IGURE 6. Long-wavelength UV-vis absorption curves of
benzoic ester rubins 2o (s), 2m (- - -), and 2p (‚‚‚‚) in CHCl3,
CH3OH, and (CH3)2SO solvent run at 1.6-2.2 × 10-5 M at
23 °C. The CH3OH and (CH3)2SO solutions contained 2%
(by vol) CHCl3.
UV-Visible Sp ectr a l An a lysis. The UV-vis spectral
data of 1-5 in a wide range of solvents of different
polarity and hydrogen bonding ability are reported in the
Experimental Section and illustrated in Figures 4 (for
4), 5 (for 1), and 6 (for 2). The dipyrrinones (4) show
characteristic absorption near 400-410 nm, with ꢀ values
35,000-40,000, as is usually seen in other dipyrrinone
constituents of bilirubin.34 The ꢀ values of 1o are gener-
ally lower than those of 1m and 1p, due possibly to steric
interference of conjugation between the dipyrrinone and
benzene chromophores. In CHCl3, the curves are slightly
broader than in CH3OH or (CH3)2SO, presumably be-
cause intermolecularly hydrogen-bonded dimerization is
much favored in CHCl3.35 The UV-vis spectra of 1 and
2 clearly differ from those of 4: the curves of Figures 5
and 6 are generally broader than those of 4, and there is
(35) (a) Huggins, M. T.; Lightner, D. A. Monatsh. Chem. 2001, 132,
203-221. (b) Boiadjiev, S. E.; Lightner, D. A. J . Heterocycl. Chem. 2000,
37, 863-870.
(36) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy-
Exciton Coupling in Organic Stereochemistry; University Science
Books: Mill Valley, CA, 1983.
(37) Boiadjiev, S. E.; Lightner, D. A. Tetrahedron: Asymmetry 1999,
10, 607-655.
(38) (a) Reisinger, M.; Lightner, D. A. J . Inclusion Phenom. 1985,
3, 479-486. (b) Lightner, D. A.; An, J . Y.; Pu, Y.-M. Tetrahedron 1987,
43, 4287-4296. (c) Lightner, D. A.; An, J . Y.; Pu, Y. M. Arch. Biochem.
Biophys. 1988, 262, 543-559. (d) Pu, Y. M.; Lightner, D. A. Croat.
Chem. Acta 1989, 62, 301-324. (e) Gawron´ski, J . K.; Polonski, T.;
Lightner, D. A. Tetrahedron 1990, 46, 8053-8066. (f) Trull, F. R.;
Shrout, D. P.; Lightner, D. A. Tetrahedron 1992, 48, 8189-8198. (g)
Kar, A.; Lightner, D. A. Tetrahedron 1998, 54, 12671-12690. (h)
Brower, J . O.; Lightner, D. A.; McDonagh, A. F. Tetrahedron 2000,
56, 7869-7883. (i) Brower, J . O.; Lightner, D. A.; McDonagh, A. F.
Tetrahedron 2001, 57, 7813-7827.
(34) (a) Byun, Y. S.; Lightner, D. A. J . Org. Chem. 1991, 56, 6027-
6033. (b)Lightner, D. A.; Gawron´ski, J . K.; Wijekoon, W. M. D. J . Am.
Chem. Soc. 1987, 109, 6354-6362.
7598 J . Org. Chem., Vol. 68, No. 20, 2003

Benzoic Acid Congeners of Bilirubin
F IGURE 7. Bilirubin 3-D conformational structures shaped like ridge-tiles of left (M)- and right (P )-handed chirality are
isoenergetic, nonsuperimposable mirror images (enantiomers). Dashed lines are hydrogen bonds, and double-headed arrows
represent the approximate locations of the dipyrrinone long-wavelength electric dipole transition moments. Inset boxes show the
relative orientation of the transition dipoles, (+) for P helicity and (-) for M.
TABLE 5. UV-vis Sp ectr a l Da ta of Ca r boxyp h en yl-m esobilir u bin s 1 a n d Th eir Ester s 2a
ꢀmax (λmax, nm)
pigment
C6H6
CHCl3
CH3OH
CH3CN
(CH3)2SO
ortho (1o)b
58,500 (434)
55,800 (419)sh
54,400 (420)
64,900 (393)
56,600 (418)sh
72,300 (393)
53,900 (412)sh
69,200 (390)
21,200 (429)sh
85,600 (383)
26,600 (428)sh
93,300 (389)
59,300 (431)
57,400 (419)sh
47,300 (431)sh
58,000 (409)
36,700 (420)sh
79,400 (387)
62,300 (415)
61,800 (399)
20,700 (428)sh
85,900 (381)
25,500 (428)sh
90,100 (383)
62,300 (421)
57,800 (401)sh
62,200 (420)
58,200 (397)sh
64,400 (413)
64,600 (400)
61,900 (411)
61,400 (399)sh
67,400 (421)
61,100 (398)sh
66,300 (419)
65,200 (401)sh
58,900 (422)
59,700 (416)
60,600 (399)
57,600 (415)
59,400 (396)
61,800 (415)sh
66,200 (396)
59,900 (418)sh
62,500 (397)
62,100 (419)
61,300 (399)
65,200 (414)sh
67,600 (397)
meta (1m )c
para (1p)c
ortho (2o)b
meta (2m )b
para (2p)b
33,900 (417)sh
69,100 (380)
31,900 (418)sh
83,600 (380)
39,600 (409)sh
76,400 (379)
21,200 (421)sh
88,600 (376)
26,200 (419)sh
94,800 (380)
a
b
Concentration 1.6-2.2 × 10-5 M. All solutions contained 2% v/v of CHCl3. c All solutions contained 2% v/v of (CH3)2SO.
tion (exciton coupling). Although one would expect only
a weak, monosignate CD associated with a π f π*
excitation from a dipyrrinone chromophore perturbed by
dissymmetric vicinal action, when two dipyrrinone chro-
mophores interact by coupling locally excited π f π*
transitions (electric dipole transition moment cou-
pling),4,32,34b,39 one would expect to observe bisignate
spectra characteristic of an exciton system.36 The com-
ponent dipyrrinone chromophores of the bichromophoric
dipyrrinone electric dipole transition moments (Figure
7). Observed by UV-vis spectroscopy, the two electronic
transitions overlap to give the typically broadened and
sometimes split long-wavelength absorption band (Fig-
ures 4-6), but in the CD spectra the two exciton
transitions are oppositely signed and thus bisignate
spectra are typically seen, as predicted by theory.4,34b,36
According to exciton chirality theory,36 the signed order
of the bisignate CD Cotton effects may be used to predict
the relative orientation of the two electric dipole transi-
tion moments, one from each dipyrrinone of the rubin.
Thus, a negative exciton chirality (long-wavelength nega-
tive Cotton effect followed by a positive short wavelength
Cotton effect) corresponds to a negative torsion angle
between the transition dipoles. On the other hand, a
positive exciton chirality (long-wavelength positive Cot-
ton effect followed by a negative short-wavelength Cotton
effect) corresponds to a positive torsion angle. Accord-
ingly, the P -helical conformer of Figure 3 is predicted to
have a positive exciton chirality, and the M-helical is
predicted to have a negative exciton chirality. In a
nonpolar, aprotic solvent (such as chloroform) that pro-
motes hydrogen bonding and association with chiral
complexation agents such as quinine, the conformational
equilibrium between bilirubin enantiomers can be dis-
placed from 1:1 by adding a chiral recognition agent. This
rubins have strongly allowed long-wavelength electronic
max
410
transitions (ꢀ
∼40,000) but only a small interchro-
mophoric orbital overlap in the ridge-tile conformation
(Figure 1). They are nicely oriented to interact by
electrostatic interaction of the local transition moment
dipoles, which are polarized along the long axis of each
dipyrrinone, i.e., resonance splitting. Such intramolecular
exciton interaction produces two long-wavelength transi-
tions in the UV-vis spectrum and two corresponding
bands in the CD spectrum. One band is higher in energy,
and one is lower in energy, with the splitting being
dependent on the strength and relative orientation of the
(39) (a) McDonagh, A. F.; Lightner, D. A.; Reisinger, M.; Palma, L.
A. J . Chem. Soc., Chem. Commun. 1986, 249-250. (b) Lightner, D.
A.; Wijekoon, W. M. D.; Zhang, M. H. J . Biol. Chem. 1988, 263, 16669-
16676. (c) Pu, Y.-M.; McDonagh, A. F.; Lightner, D. A. J . Am. Chem.
Soc. 1993, 115, 377-380.
J . Org. Chem, Vol. 68, No. 20, 2003 7599

Boiadjiev and Lightner
F IGURE 8. CD (upper) and UV-vis (lower) spectra of benzoic
acid rubins 1o (s), 1m (- - -), and 1p (‚‚‚‚) in CHCl3 with added
quinine [A] and added cinchonidine [B]. The pigment/alkaloid
F IGURE 9. CD (upper) and UV-vis (lower) spectra of benzoic
acid rubins 1o (s), 1m (- - -), and 1p (‚‚‚‚) in CHCl3 with added
quinidine [A] and added cinchonine [B]. The pigment/alkaloid
ratio is 1:300 for pigment concentrations of 1.6-1.9 × 10-5
M
ratio is 1:300 for pigment concentrations of 1.6-1.9 × 10-5
M
at 23 °C.
at 23 °C.
leads typically to an intense bisignate induced circular
dichroism (ICD) Cotton effect, as has been noted previ-
ously for bilirubin in chloroform with added quinine34b
or other optically active amines38a-d,g-i and in aqueous
buffers with added serum albumin.39 Similarly, for the
benzoic acid rubins of 1o and 1m of this work, in CHCl3
comparably intense negative exciton chirality ICDs are
observed in the presence of quinine (Figure 8A), as is
characteristic of an exciton system in which the two
dipyrrinone chromophores of the bichromophoric rubin
molecule interact by coupling locally excited π f π*
transitions (electric dipole transition moment coupling).40
In contrast, only a weaker, broader positive exciton
7600 J . Org. Chem., Vol. 68, No. 20, 2003

Benzoic Acid Congeners of Bilirubin
TABLE 6. Com p a r ison of Cir cu la r Dich r oism a n d
UV-vis Sp ectr a l Da ta of Ca r boxyp h en yl-m esobilir u bin s
1 in th e P r esen ce of Cin ch on a Alk a loid sa
CD
UV
ꢀmax
+40.0 (394) 64,500
pigment +
alkaloid
λ2 at
∆ꢀ ) 0
∆ꢀmax (λ1)
∆ꢀmax (λ3)
λmax
1o +
-94.5 (437) 408
-101.2 (437) 411
+8.6 (439) 419
430
quinine
1m +
+56.5 (395) 42,400sh 427
55,600 383
quinine
1p +
-28.1 (376) 26,800sh 430
quinine
1o +
92,200
383
431
+142.9 (436) 410
-40.2 (401) 386
-42.6 (432) 400
-81.5 (441) 415
+30.6 (448) 403
+132.1 (430) 399
+103.7 (441) 414
+37.4 (425) 389
-105.4 (435) 400
-82.1 (393) 65,100
quinidine
1m +
quinidine
1p +
+56.9 (370) 38,500sh 426
70,500 381
+70.1 (375) 26,600sh 430
quinidine
1o + cin-
chonidine
1m + cin-
chonidine
1p + cin-
chonidine
1o + cin-
chonine
1m + cin-
chonine
1p + cin-
chonine
91,800
383
431
+45.4 (400) 63,200
-40.4 (381) 34,100sh 429
79,500 388
-189.1 (377) 25,200sh 433
93,900
383
433
-60.6 (396) 63,600
-24.1 (367) 34,900sh 427
77,800 386
+153.0 (378) 29,600sh 431
92,300 386
F IGURE 10. CD (upper) and UV-vis (lower) spectra of 1.6-
1.9 × 10-5 M benzoic acid rubins 1o (s), 1m (- - -), and 1p
(‚‚‚‚) in aqueous buffered (pH 7.4, Tris) human serum albumin
(HSA) at 23 °C. The pigment/HSA ratio is 1:2.
Concentration 1.6-1.9 × 10-5 M of a chloroform solution
a
containing 4.8-5.7 × 10-3 M concentration of each alkaloid.
chirality CD is observed for 1p. The UV-vis curves also
show evidence of exciton coupling, but the CD curves
indicate chiral stereochemistry: a preponderance of the
M helical rubin enantiomers 1o and 1m and a mild
preference for P in 1p due to enantioselection by quinine.
Interestingly, in the presence of a related alkaloid
(cinchonidine) with the same absolute configuration, only
the CD and UV spectra (Figure 8B) of 1o and the UV of
1p remain relatively unchanged. The CD couplet of 1m
weakens and inverts signs and the UV spectrum shifts,
whereas the CD of 1p intensifies greatly to give a well-
defined positive exciton chirality couplet. The exciton CD
couplet signs of 1o invert in the presence of quinidine
and cinchonine (Figure 9) with UV-vis spectra remain-
ing similar to those of Figure 8. With sign inversions,
the CD spectra of 1p remain similar to those in Figure
8A, and those in Figure 9B remain similar to those in
Figure 8B. Oddly, the CD spectra (Figure 9) of 1m vary
considerably: those of Figure 8B and 9B are similar (as
are the UVs), and those of Figure 8A and 9A have the
same signed order but rather different magnitudes. Yet
the UV-vis profiles for 1m in Figures 8A and 9A remain
similar. In some cases (quinidine, cinchonine) the mag-
nitudes are larger; in others (cinchonidine) they are
smaller, apparently all dictated by the stability of the
complex formed. It is tempting to link the “well-behaved”
exciton curves seen for 1o to their ability to fold into
ridge-tile conformations and maintain them by intramo-
lecular hydrogen bonding. The CD curves of 1m show
nice behavior only with quinine; those of 1p show intense
bisignate Cotton effects only with cinchonidine and
cinchonine. At present the reasons for the varying
TABLE 7. Com p a r ison of Cir cu la r Dich r oism a n d
UV-vis Sp ectr a l Da ta of Ca r boxyp h en yl-m esobilir u bin s
1 in th e P r esen ce of Hu m a n Ser u m Albu m in a
CD
UV
ꢀmax
pigment
(buffer)
λ2 at
∆ꢀ ) 0
∆ꢀmax (λ1)
∆ꢀmax (λ3)
λmax
1o (A)
1o (B)
1o (C)
1m (A) +50.2 (448)
1m (B) +53.5 (448)
1m (C) +56.1 (445)
+32.1 (425)
+36.2 (429)
+71.3 (425)
413
413
409
425
-80.9 (389) 64,700
-83.1 (389) 65,800
-92.0 (386) 65,000
-116.2 (400) 65,800
437
437
433
451
41,900sh 413
425
424
393
386
392
-124.7 (401) 65,200
451
42,700sh 415
-129.0 (401) 67,100
451
44,300sh 414
1p (A)
1p (B)
1p (C)
-10.4 (417)
-9.5 (398)
-7.8 (418)
+11.2 (377) 34,500sh 435
60,300
389
+6.4 (369) 34,100sh 435
60,500 390
+12.0 (371) 36,500sh 434
57,300 392
a
Concentration of pigment 1.6-1.9 × 10-5 M; concentration of
HSA 3.2-3.8 × 10-5 M. Buffers: A, 0.1 M phosphate buffer,
pH 7.42; B, 0.1 M phosphate buffer, pH 8.02; C, 0.1 M Tris buffer,
pH 7.40.
behaviors are not clear. The UV-vis curves of 1o vary
little from one alkaloid solution to another and differ from
those of 1m and 1p. In Figures 8 and 9, the UV-vis
curves of 1m are very much like those of 1p, showing a
strong tilt toward the higher energy exciton component
near 380 nm and are thus consistent with a conformation
dominated by a more helix-like structure involved in
intermolecular hydrogen bonding. The data are sum-
marized in Table 6.
When human serum albumin (HSA) is used as the
chiral complexation agent, aqueous buffered (pH 7.4 Tris
(40) Bauman, D.; Killet, C.; Boiadjiev, S. E.; Lightner, D. A.;
Scho¨nhofer, A.; Kuball, H.-G. J . Phys. Chem. 1996, 100, 11546-11558.
J . Org. Chem, Vol. 68, No. 20, 2003 7601

Boiadjiev and Lightner
s) ppm; 13C NMR (CDCl3) δ 14.3, 14.6, 51.3, 72.1, 118.1, 130.9,
134.6, 161.6 ppm; MS m/z (relative abund.) 279 (M+•, 100%),
247 (85%), 220 (9%), 120 (8%). Anal. Calcd for C8H10INO2
(279.1): C, 34.43; H, 3.61; N, 5.02. Found: C, 34.38; H, 3.69;
N, 4.94.
or phosphate) solutions of 1o and 1m exhibit strong
positive exciton chirality bisignate ICD Cotton effects,
effectively the same as observed for bilirubin. In contrast,
1p exhibits a much weaker negative chirality Cotton
effect that is broadly bisignate (Figure 10). The CD
intensities of 1 are comparable in magnitude to those of
bilirubin, apparently due to a similar enantioselective
chiral complexation by HSA. Interestingly, for reasons
yet unclear, a change in pH, from 7.4 to 8.0 leads to more
intense Cotton effects for both 1o and 1m but not for 1p.
The ICD data for 1o and 1m suggest rather similar
conformations on HSA, whereas the contrasting ICD data
for 1p suggest a different conformation or a mixture of
conformations.
Gen er a l P r oced u r e for Syn th eses of Ar ylp yr r oles 8m
a n d 8p. To an argon-protected solution of 2.79 g (10 mmol) of
iodopyrrole 11 and 2.70 g (15 mmol) of boronic acid 12m 23 or
12p24 in 70 mL of purified and deoxygenated dimethylforma-
mide was added 462 mg (0.4 mmol) of Pd(PPh3)427 followed by
a hot solution of 2.12 g (20 mmol) of anh. Na2CO3 in 13 mL of
deoxygenated water. The mixture was placed in a preheated
oil bath at 100-105 °C and stirred under Ar for 20-25 min
(until sudden color change from yellow to dark brown-black).
After cooling with an ice bath, the mixture was diluted with
350 mL of diethyl ether and washed with 10% aqueous NH4OH
(3 × 50 mL) and then with water (4 × 50 mL). The organic
layer was dried (MgSO4) and filtered, and the solvent was
evaporated under vacuum. The residue was purified by radial
chromatography (eluent hexane-ethyl acetate ) 93:7 to 85:
15) collecting the bright fluorescent band of arylpyrroles 8m
or 8p. After solvent evaporation, the material was recrystal-
lized from hexanes-ethyl acetate.
Meth yl 3,5-Dim eth yl-4-(m -m eth oxyca r bon ylp h en yl)-
1H-p yr r ole-2-ca r boxyla te (8m ). The meta-isomer was ob-
tained in 77% yield: mp 140-141 °C; 1H NMR (CDCl3) δ 2.25
(3H, s), 2.28 (3H, s), 3.87 (3H, s), 3.93 (3H, s), 7.43 (1H, m),
7.47 (1H, m), 7.93 (1H, m), 7.97 (1H, m), 8.78 (1H, br. s) ppm;
13C NMR (CDCl3) δ 11.3, 12.1, 51.1, 52.1, 117.4, 123.7, 126.6,
127.4, 128.3, 130.16, 130.21, 131.1, 134.5, 135.4, 162.2, 167.2
ppm; MS m/z (relative abund.) 287 (M+•, 81%), 256 (44%), 255
(100%), 224 (9%), 195 (12%), 168 (31%). Anal. Calcd for
Con clu sion s
Novel analogues (1) of bilirubin with propionic acids
replaced by benzoic acids offer three different isomers and
three different fixed orientations of the carboxylic acid
groups relative to the two dipyrrinone components of the
pigment. In only one isomer, the ortho (1o), can intramo-
lecular hydrogen bonding (Figure 3) effectively stabilize
the ridge-tile conformation. Consequently, 1o is less polar
than either the meta (1m ) or para (1p) isomers and
exhibits uniquely different chromatographic and spec-
troscopic properties.
Exp er im en ta l Section
C
16H17NO4 (287.3): C, 66.88; H, 5.96; N, 4.88. Found: C, 66.78;
H, 5.74; N, 4.96.
Gen er a l P r oced u r es. Nuclear magnetic resonance spectra
were acquired at 11.75 T magnetic field strength instrument
operating at 1H frequency of 500 MHz and 13C frequency of
Met h yl 3,5-Dim et h yl-4-(p -m et h oxyca r b on ylp h en yl)-
1H-p yr r ole-2-ca r boxyla te (8p). Following the procedure
above, the para-isomer was synthesized in 78% yield: mp
155-156 °C; 1H NMR (CDCl3) δ 2.27 (3H, s), 2.30 (3H, s), 3.87
1
125 MHz in solutions of CDCl3 (referenced at 7.26 ppm for H
and 77.00 ppm for 13C) or (CD3)2SO (referenced at 2.49 ppm
for 1H and 39.50 ppm for 13C). J -modulated spin-echo (At-
tached Proton Test) and gHMBC experiments were used to
assign the 13C NMR spectra. Gas chromatography-mass
spectrometry analyses were carried out on a gas chromato-
graph (30 m DB-1 column) equipped with a mass selective
detector. Radial chromatography was carried out on silica gel
PF254 with CaSO4 binder preparative layer grade, using 1-, 2-,
or 4-mm thick rotors, and analytical thin-layer chromatogra-
phy was carried out on silica gel IB-F plates (125-µm layer).
Melting points are uncorrected. The combustion analyses were
carried out by Desert Analytics, Tucson, AZ. High-resolution
FAB mass spectra were obtained at the Nebraska Center for
Mass Spectrometry, University of Nebraska, Lincoln, for
samples which were >98% pure by NMR.
3
(3H, s), 3.94 (3H, s), 7.31 (2H, m, J ) 8.5 Hz), 8.07 (2H, m,
3J ) 8.5 Hz), 8.83 (1H, br. s) ppm; 13C NMR (CDCl3) δ 11.3,
12.2, 51.1, 52.0, 117.7, 123.7, 126.5, 127.8, 129.5, 129.8, 130.3,
140.1, 162.1, 167.1 ppm; MS m/z (relative abund.) 287 (M+•
,
94%), 255 (100%), 224 (25%), 196 (11%), 168 (21%). Anal. Calcd
for C16H17NO4 (287.3): C, 66.88; H, 5.96; N, 4.88. Found: C,
67.20; H, 6.02; N, 4.92.
3-Eth yl-8-(o-m eth oxyca r bon ylp h en yl)-2,7,9-tr im eth yl-
1,10-d ih yd r o-11H-d ip yr r in -1-on e (5o) was prepared as
reported previously15 from reaction of 6 and 7o: mp 289-
sh
435
max
410
291°C (lit.15 mp 289-291°C); UV-vis ꢀ 27,700, ꢀ
45,900
max
407
(benzene), ꢀ
40,500 (CHCl3), ꢀmax 42,000 (CH3OH), ꢀmax
413
400
38,100 (CH3CN), ꢀmax 39,900, ꢀmax 38,100 (DMSO); 13C NMR
412
399
and 1H NMR in Tables 1 and 2.
The spectral data were obtained in spectral grade solvents,
used as provided, and dimethylformamide was purified by a
standard procedure.41
3-Eth yl-8-(m -m eth oxyca r bon ylp h en yl)-2,7,9-tr im eth yl-
1,10-d ih yd r o-11H-d ip yr r in -1-on e (5m ). A mixture of 1.44
g (5 mmol) of monopyrrole 8m , 2.00 g (50 mmol) of sodium
hydroxide, 30 mL of ethanol, and 9 mL of water was heated
at reflux for 4 h. After cooling, the ethanol solvent was
evaporated under vacuum, and the residue was diluted with
10 mL of 50% aqueous NaNO3. The solution was cooled at
-20 °C and acidified by slow addition of a solution of
concentrated HNO3 in 50% aqueous NaNO3 (1:5 v/v). The
precipitated product was collected by filtration, washed with
cold water and dried overnight under vacuum. This crude
diacid (7m ) was obtained in nearly quantitative yield and was
used immediately in the following step without further char-
acterization.
A mixture of diacid from above, 1.17 g (5.4 mmol) of
5-bromomethylene-4-ethyl-3-methyl-2-oxo-1H-pyrrole (6),28 35
mL of anhydrous methanol, and 1 drop of 48% aqueous HBr
was heated at reflux for 9 h. Then the mixture was chilled
overnight at -20 °C. The precipitated crude dipyrrinone acid
4m was collected by filtration, suspended in 50 mL of CH3OH
Meth yl 3,5-Dim eth yl-4-iod o-1H-p yr r ole-2-ca r boxyla te
(11). According to Treibs and Kolm26 for iodination of the ethyl
ester of 10, to a solution of 15.32 g (100 mmol) of methyl 3,5-
dimethyl-1H-pyrrole-2-carboxylate (10)25 in 150 mL of ethanol,
6 mL (105 mmol) of acetic acid, and 22 mL (200 mmol) of 30%
aqueous hydrogen peroxide was added a solution of 18.26 g
(110 mmol) of potassium iodide in 25 mL of water during 10
min at 75-80 °C. The mixture was stirred at 75 °C for 10 min.
Then it was cooled slowly while adding 60 mL of water and
chilled at -15 °C for 2 h. The product was collected by
filtration, washed with cold aqueous ethanol, and dried under
vacuum. Recrystallization from ethyl acetate-hexane afforded
26.12 g (94%) of iodopyrrole 11: mp 188-189 °C; 1H NMR
(CDCl3) δ 2.28 (3H, s), 2.29 (3H, s), 3.85 (3H, s), 9.14 (1H, br.
(41) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pergamon Press: England, 1988.
7602 J . Org. Chem., Vol. 68, No. 20, 2003

Benzoic Acid Congeners of Bilirubin
max
415
and 30 mL of CHCl3, and treated for 10 min with ethereal
diazomethane (generated from 30 mmol of N-nitroso-N-meth-
ylurea). Excess CH2N2 was destroyed with acetic acid, and the
solvents were evaporated under vacuum. The residue was
purified by radial chromatography (2-4% CH3OH in CHCl3-
CH2Cl2 ) 1:1 v/v) and after recrystallization from CH2Cl2-
CH3OH, 1.36 g (75%) of bright yellow dipyrrinone 5m was
posed without melting at 309-331 °C; UV-vis ꢀ
38,000,
max
401
max
408
ꢀ
37,400 (benzene), ꢀ
38,300 (CHCl3), ꢀmax 39,100 (CH3-
411
OH), ꢀ4m0a0x 37,400 (CH3CN), ꢀmax 37,000 (DMSO); NMR data in
Tables 1 and 2; HRMS (FAB, 3-NBA) calcd for C21H22N2O3
350.1630; found 350.1628, ∆ ) 0.2 mDa, error 0.6 ppm.
8-(p-Ca r boxyp h en yl)-3-eth yl-2,7,9-tr im eth yl-1,10-d ih y-
d r o-11H-d ip yr r in -1-on e (4p). This dipyrrinone acid was
prepared in 95% yield. It decomposed without melting at 322-
409
max
430
max
408
obtained: mp 272-273 °C; UV-vis ꢀ
30,300, ꢀ
45,900
max
(benzene), ꢀ
41,300 (CHCl3), ꢀmax 42,500 (CH3OH), ꢀmax
max
416
max
401
max
408
405
409
397
343 °C; UV-vis ꢀ
39,600, ꢀ
39,100 (benzene), ꢀ
36,900 (CH3CN), ꢀmax 40,000 (DMSO); 1H NMR (CDCl3) δ
1.21 (3H, t, J ) 7.6 4H08z), 1.97 (3H, s), 2.18 (3H, s), 2.48 (3H, s),
2.58 (2H, q, J ) 7.6 Hz), 3.94 (3H, s), 6.22 (1H, s), 7.48 (1H,
m), 7.49 (1H, m), 7.96 (1H, m), 7.98 (1H, m), 10.63 (1H, br. s),
11.39 (1H, br. s) ppm; 13C NMR (CDCl3) δ 8.6, 10.3, 12.3, 15.0,
18.0, 52.1, 101.0, 122.7, 122.9, 123.0, 123.8, 127.0, 127.9, 128.2,
130.2, 130.9, 131.9, 134.3, 136.2, 148.5, 167.3, 174.3 ppm. Anal.
Calcd for C22H24N2O3 (364.4): C, 72.50; H, 6.64; N, 7.69.
Found: C, 72.24; H, 6.48; N, 7.44.
39,800 (CHCl3), ꢀmax 41,000 (CH3OH), ꢀmax 39,900 (CH3CN),
411
398
ꢀm41a1x 38,100, ꢀsh 37,500 (DMSO); NMR data in Tables 1 and 2;
403
HRMS (FAB, 3-NBA) calcd for C21H22N2O3 350.1630; found
350.1629, ∆ ) 0.1 mDa, error 0.3 ppm.
Gen er a l P r oced u r e for Syn th eses of Mesobiliver d in
Ester s An a logu es 3. To a solution of 2.00 mmol of the
corresponding dipyrrinone ester (5o, 5m or 5p) in 440 mL of
CH2Cl2 was added p-chloranil (1.23 g, 5.00 mmol) followed by
22 mL of 97% formic acid, and the mixture was heated at
reflux for 24 h. The reaction volume was reduced by distillation
to one-half, and reflux was continued for 6 h. Then the mixture
was chilled overnight at -20 °C. The separated yellow solid
was removed by filtration and discarded. The blue filtrate was
carefully neutralized with 5% aqueous NaHCO3 and then
washed with 4% aqueous NaOH (2 × 100 mL) and H2O (4 ×
100 mL). After drying (anhydrous Na2SO4), filtration, and
evaporation of the solvent under vacuum, the residue was
purified by radial chromatography (gradient CH2Cl2-CH3-
CO2H-CH3OH ) 100:3.2 to 100:3:7 v/v/v). The combined pure
fractions were washed with 1% aqueous NaHCO3 and H2O and
then dried (anhydrous Na2SO4). After filtration, the solvent
was evaporated under vacuum and the residue was recrystal-
lized from CHCl3-hexane to afford pure mesobiliverdin esters
3.
3-Eth yl-8-(p-isobu toxycar bon ylph en yl)-2,7,9-tr im eth yl-
1,10-d ih yd r o-11H-d ip yr r in -1-on e (5p). Following the pro-
cedure from above 1.44 g (5 mmol) of monopyrrole 8p was
converted to 1.42 g (81%) of crude dipyrrinone acid 4p
(containing ∼10% of methyl ester). This amount was treated
for 2 h at reflux with a mixture of 25 mL of 10% aqueous
NaOH and 50 mL of ethanol. After cooling, the ethanol solvent
was evaporated under vacuum. The residue was diluted with
cold water (25 mL) and acidified at 0 °C with 10% aqueous
HCl. The reprecipitated 4p was collected by filtration, washed
with water, and dried for 48 h under vacuum (P2O5).
A mixture of 1.30 g (3.7 mmol) of this purified acid, 1.79 g
(5.5 mmol) of cesium carbonate, 30 mL of anhydrous dimeth-
ylformamide and 1.3 mL (11.1 mmol) of isobutyl iodide was
heated for 18 h at 80 °C. After cooling the mixture was
partitioned between 300 mL of CHCl3 and 300 mL of H2O. The
organic layer was washed with water (3 × 100 mL) and dried
(Na2SO4), and after filtration, the solvent was evaporated
under vacuum. The residue was purified by radial chroma-
tography (eluent 2-3% CH3OH in CH2Cl2) and the pure
material was recrystallized from CH3OH-CH2Cl2 to afford
1.18 g (58% based on 8p) of bright yellow isobutyl ester 5p:
3,17-Dieth yl-8,12-bis(o-m eth oxyca r bon ylp h en yl)-2,7,-
13,18-tetr a m eth yl-(21H,24H)-bilin -1,19-d ion e (3o). This
mesobiliverdin ester was synthesized in 84% yield as previ-
ously described from dipyrrinone 5o:15 mp 284-288 °C (lit.15
mp 284-8°C); UV-vis ꢀm65a0x 18,300, ꢀ3m7a5x 65,100 (benzene), ꢀmax
646
max
17,200; ꢀ
OH), ꢀ
66,400 (CHCl3), ꢀmax 17,100, ꢀmax 66,300 (CH3-
375
max
648
370
max
367
sh
431
max
410
17,400, ꢀ
67,700 (CH3CN), ꢀmax 19,500, ꢀmax
648 375
mp 263-264 °C; UV-vis ꢀ
33,900, ꢀ
49,000 (benzene),
647
max
43,900 (CHCl3), ꢀmax 46,100 (CH3OH), ꢀmax 40,400 (CH3-
66,400 (DMSO).
ꢀ
406
411
397
CN), ꢀ4m1a2x 43,200, ꢀsh 42,300 (DMSO); 1H NMR (CDCl3) δ 1.05
3,17-Dieth yl-8,12-bis(m -m eth oxyca r bon ylp h en yl)-2,7,-
13,18-tetr a m eth yl-(21H,24H)-bilin -1,19-d ion e (3m ). This
mesobiliverdin ester was synthesized in 85% yield: mp 269-
404
(6H, d, J ) 6.7 Hz), 1.20 (3H, t, J ) 7.7 Hz), 1.97 (3H, s), 2.11
(1H, m), 2.20 (3H, s), 2.50 (3H, s), 2.58 (2H, q, J ) 7.7 Hz),
4.13 (2H, d, J ) 6.5 Hz), 6.21 (1H, s), 7.37 (2H, d, J ) 8.3 Hz),
8.09 (2H, d, J ) 8.3 Hz), 10.65 (1H, br. s), 11.38 (1H, br. s)
ppm; 13C NMR (CDCl3) δ 8.6, 10.4, 12.5, 15.0, 18.0, 19.2, 28.0,
70.9, 100.9, 122.8, 123.0, 123.2, 123.7, 127.8, 128.0, 129.5,
129.6, 132.1, 140.8, 148.6, 166.8, 174.3 ppm. Anal. Calcd for
max
647
max
375
max
639
271 °C; UV-vis ꢀ
19,900, ꢀ
67,500 (benzene), ꢀ
max
18,100, ꢀ
70,100 (CHCl3), ꢀmax 18,500, ꢀmax 71,000 (CH3-
375
max
632
638
370
max
366
OH), ꢀ
18,200, ꢀ
70,500 (CH3CN), ꢀmax 22,500, ꢀmax
630 375
1
68,100 (DMSO); H NMR (CDCl3) δ 1.24 (6H, t, J ) 7.6 Hz),
1.84 (6H, s), 2.14 (6H, s), 2.54 (4H, q, J ) 7.6 Hz), 3.93 (6H,
s), 6.00 (2H, s), 6.47 (1H, s), 7.45 (2H, m), 7.48 (2H, m), 7.96
(2H, m), 7.97 (2H, m), 8.47 (2H, br. s) ppm; 13C NMR (CDCl3)
δ 8.4, 10.2, 14.4, 17.9, 52.2, 96.1, 118.5, 127.9, 128.4, 128.5,
128.8, 130.4, 130.8, 134.0, 134.2, 139.3, 140.6 141.5, 146.8,
150.0, 166.8, 172.6 ppm. Anal. Calcd for C43H42N4O6 (710.8):
C, 72.66; H, 5.96; N, 7.88. Found: C, 72.49; H, 5.78; N, 7.88.
C
25H30N2O3 (406.5): C, 73.86; H, 7.44; N, 6.89. Found: C,
74.22; H, 7.64; N, 6.90.
Gen er a l P r oced u r e for Syn th eses of Dip yr r in on e
Acid s 4. A mixture of 1 mmol of the corresponding ester 5o,15
5m , or 5p, 10 mL of 10% aqueous NaOH, and 25 mL of ethanol
was heated at reflux for 3 h. After cooling, the ethanol solvent
was removed under vacuum. The residue was diluted with 10
mL of water and acidified at 0 °C with 10% aqueous HCl. The
precipitated product was collected by filtration, washed with
water (3 × 10 mL), and dried under vacuum for 24 h (P2O5).
3,17-Dieth yl-8,12-bis(p-isobu toxyca r bon ylp h en yl)-2,7,-
13,18-tetr a m eth yl-(21H,24H)-bilin -1,19-d ion e (3p). This
compound was obtained in 80% yield: mp 265-266 °C; UV-
vis ꢀm65a4x 19,700, ꢀ3m7a8x 70,300 (benzene), ꢀm65a1x 18,200, ꢀ3m7a5x 74,300
(CHCl3), ꢀmax 18,500, ꢀmax 77,300 (CH3OH), ꢀmax 18,400, ꢀmax
8-(o-Ca r boxyp h en yl)-3-eth yl-2,7,9-tr im eth yl-1,10-d ih y-
d r o-11H-d ip yr r in -1-on e (4o). The acid 4o was isolated in
94% yield. It decomposed without melting at 287-314 °C; UV-
644
371
639
371
76,200 (CH3CN), ꢀmax 21,700, ꢀmax 72,100 (DMSO); 1H NMR
(CDCl3) δ 1.03 (12H, d, J ) 6.73H78z), 1.24 (6H, t, J ) 7.6 Hz),
1.83 (6H, s), 2.10 (2H, m), 2.14 (6H, s), 2.54 (4H, q, J ) 7.6
Hz), 4.12 (4H, d, J ) 6.6 Hz), 6.02 (2H, s), 6.53 (1H, s), 7.34
(4H, d, J ) 8.2 Hz), 8.08 (4H, d, J ) 8.2 Hz), 8.40 (2H, br. s)
ppm; 13C NMR (CDCl3) δ 8.3, 10.3, 14.4, 17.9, 19.2, 27.9, 71.1,
95.9, 118.4, 128.3, 128.8, 129.4, 129.66, 129.70, 138.4, 139.4,
140.6, 141.5, 146.8, 150.0, 166.3, 172.5 ppm. Anal. Calcd for
637
max
405
max
405
vis ꢀ
35,900 (benzene), ꢀ
35,000 (CHCl3), ꢀmax 36,000
411
(CH3OH), ꢀmax 35,400 (CH3CN), ꢀmax 35,100 (DMSO); NMR
data in Ta3b9l8es 1 and 2; HRMS40(5FAB, 3-NBA) calcd for
C
21H22N2O3 350.1630; found 350.1635, ∆ ) -0.5 mDa, error
-1.3 ppm.
8-(m -Ca r b oxyp h en yl)-3-et h yl-2,7,9-t r im et h yl-1,10-d i-
h yd r o-11H-d ip yr r in -1-on e (4m ). Following the general pro-
cedure above, acid 4m was obtained in 98% yield. It decom-
C
49H54N4O6 (795.0): C, 74.03; H, 6.85; N, 7.05. Found: C,
74.00; H, 6.78; N, 7.00.
J . Org. Chem, Vol. 68, No. 20, 2003 7603

Boiadjiev and Lightner
Gen er a l P r oced u r e for Syn th eses of Mesobilir u bin
Ester An a logu es (2). To an oxygen-free solution of 0.5 mmol
of the corresponding mesobiliverdin ester (3o, 3m , or 3p) in 8
mL of CHCl3 and 80 mL of anhydrous CH3OH kept at ∼10 °C
was slowly added sodium borohydride (1.13 g, 30.0 mmol)
during 15 min while purging the mixture with N2. After the
mixture had turned from deep blue to bright yellow, ice-water
(250 mL) was added, and the reaction was quenched with 8
mL of acetic acid followed by enough 10% aqueous HCl to bring
the pH to ∼3. The product was extracted with CHCl3 (4 × 100
mL), and the combined extracts were washed with water until
neutral. After drying (anhydrous Na2SO4) and evaporation of
the solvent under vacuum, the residue was purified by radial
chromatography (gradient 0.5-3.0% of CH3OH in CH2Cl2 v/v)
and recrystallization from CH2Cl2-CH3OH to afford pure
bright yellow mesobilirubin esters 2.
1-2% of CH3OH in CH2Cl2 v/v) and recrystallized from
CH3OH-CH2Cl2 to afford bright yellow 1o. In the cases of
much less soluble meta (1m ) and para (1p) isomers, the
ethanol solvent was partially (15 mL) removed by distillation
while simultaneously adding 10 mL of water. After cooling,
the mixture was further diluted with 20 mL of H2O and slowly
acidified at 0 °C with 10% aqueous HCl until pH ∼3. The
precipitated product was separated by filtration (or centrifuga-
tion), washed with H2O (5 × 5 mL), and dried under vacuum
to afford an almost quantitative yield of dark green 1m or 1p.
Recrystallization from warm (CH3)2SO-CHCl3 afforded pure
yellow rubins 1m and 1p.
8,12-Bis(o-ca r b oxyp h en yl)-3,17-d iet h yl-2,7,13,18-t et -
r a m eth yl-(10H,21H,23H,24H)-bilin -1,19-d ion e (1o). This
mesobilirubin was synthesized in 91% yield as previously
described.15
3,17-Dieth yl-8,12-bis(o-m eth oxyca r bon ylp h en yl)-2,7,-
13,18-t et r a m et h yl-(10H ,21H ,23H , 24H )-b ilin -1,19-d ion e
(2o). This mesobilirubin ester was synthesized in 92% yield
as previously described.15
8,12-Bis(m -ca r b oxyp h en yl)-3,17-d iet h yl-2,7,13,18-t et -
r a m eth yl-(10H,21H,23H,24H)-bilin -1,19-d ion e (1m ). This
mesobilirubin was obtained in 55% yield. It decomposed at
1
>318 °C; H NMR ((CD3)2SO) δ 1.10 (6H, t, J ) 7.6 Hz), 1.79
3,17-Dieth yl-8,12-bis(m -m eth oxyca r bon ylp h en yl)-2,7,-
13,18-t et r a m et h yl-(10H ,21H ,23H , 24H )-b ilin -1,19-d ion e
(2m ). This mesobilirubin ester was synthesized in 93% yield:
mp 249-251 °C (decomp); 1H NMR (CDCl3) δ 1.06 (6H, t, J )
7.6 Hz), 1.60 (6H, s), 2.06 (6H, s), 2.39 (4H, q, J ) 7.6 Hz),
3.93 (6H, s), 4.22 (2H, s), 6.02 (2H, s), 7.13 (4H, m), 7.80 (4H,
m), 10.68 (2H, br. s), 10.87 (2H, br. s) ppm; 13C NMR (CDCl3)
δ 8.0, 10.3, 14.8, 17.9, 23.3, 52.0, 100.4, 122.6, 123.0, 123.9,
124.3, 127.0, 128.1, 129.5, 129.8, 130.9, 131.0, 134.7, 135.8,
147.3, 167.2, 174.6 ppm. Anal. Calcd for C43H44N4O6 (712.8):
C, 72.45; H, 6.22; N, 7.86. Found: C, 72.16; H, 6.07; N, 7.71.
3,17-Dieth yl-8,12-bis(p-isobu toxyca r bon ylp h en yl)-2,7,-
13,18-tetr am eth yl-(10H,21H,23H,24H)-bilin -1,19-dion e (2p).
This compound was obtained in 91% yield: mp 278-282 °C
(decomp); 1H NMR (CDCl3) δ 1.05 (6H, t, J ) 7.7 Hz), 1.06
(12H, d, J ) 6.7 Hz), 1.54 (6H, s), 2.11 (6H, s), 2.12 (2H, m),
2.39 (4H, q, J ) 7.7 Hz), 4.12 (4H, d, J ) 6.6 Hz), 4.25 (2H, s),
6.03 (2H, s), 7.18 (4H, br. d, J ) 8.0 Hz), 7.86 (4H, br. d, J )
8.0 Hz), 10.72 (2H, s), 10.90 (2H, s) ppm; 13C NMR (CDCl3) δ
7.8, 10.4, 14.8, 17.8, 19.3, 23.8, 27.9, 70.8, 100.3, 122.4, 123.3,
124.0, 124.6, 127.9, 129.3, 129.7, 129.8, 130.9, 140.3, 147.4,
166.4, 174.5 ppm. Anal. Calcd for C49H56N4O6 (797.0): C, 73.84;
H, 7.08; N, 7.03. Found: C, 74.17; H, 7.00; N, 7.06.
Gen er a l P r oced u r e for Syn th eses of Mesobilir u bin
An a logu es (1). A mixture of 0.2 mmol of mesobilirubin diester
2o, 2m , or 2p and 20 mL of ethanol was saturated for 30 min
with argon. Then 3.0 mL of 1 M aqueous NaOH (3 mmol) was
added and the mixture was heated at reflux for 1 h. In the
case of the ortho-isomer (1o), after cooling the mixture was
diluted with 100 mL of H2O and 100 mL of CHCl3 and then
poured into 100 mL of 1% aqueous HCl, and the product was
extracted with CHCl3 (4 × 25 mL). The combined extracts were
washed with H2O (4 × 50 mL) and dried (anhydrous Na2SO4).
After filtration and evaporation of the solvent under vacuum,
the residue was purified by radial chromatography (gradient
(6H, s), 1.89 (6H, s), 2.50 (4H, q, J ) 7.6 Hz), 4.03 (2H, s),
5.91 (2H, s), 7.16 (2H, m), 7.27 (2H, m), 7.54 (2H, m), 7.68
(2H, m), 9.72 (2H, s), 10.39 (2H, s), 12.73 (2H, br. s) ppm; 13
C
NMR ((CD3)2SO) δ 8.1, 9.9, 14.8, 17.2, 23.9, 97.5, 120.8, 122.4,
122.9, 123.6, 126.4, 127.8, 129.2, 129.6, 130.1, 130.3, 133.6,
135.2, 147.2, 167.3, 172.1 ppm. Anal. Calcd for C41H40N4O6
(684.8): C, 71.91; H, 5.89; N, 8.18. Found: C, 71.64; H, 6.01;
N, 8.13.
8,12-Bis(p -ca r b oxyp h en yl)-3,17-d iet h yl-2,7,13,18-t et -
r a m eth yl-(10H,21H,23H,24H)-bilin -1,19-d ion e (1p). This
compound was prepared in 62% yield. It decomposed at
1
>328 °C; H NMR ((CD3)2SO) δ 1.09 (6H, t, J ) 7.5 Hz), 1.79
(6H, s), 1.93 (6H, s), 2.50 (4H, q, J ) 7.5 Hz), 4.06 (2H, s),
5.93 (2H, s), 7.06 (4H, d, J ) 8.0 Hz), 7.75 (4H, d, J ) 8.0 Hz),
9.76 (2H, s), 10.46 (2H, s), 12.66 (2H, br. s) ppm; 13C NMR
((CD3)2SO) δ 8.1, 10.1, 14.8, 17.2, 24.1, 97.3, 120.6, 122.4, 123.2,
123.8, 127.7, 128.8, 129.2, 129.6, 129.8, 139.7, 147.2, 167.2,
172.1 ppm. Anal. Calcd for C41H40N4O6 (684.8): C, 71.91; H,
5.89; N, 8.18. Found: C, 72.12; H, 5.95; N, 8.14.
Ack n ow led gm en t. We thank the U.S. National
Institutes of Health (HD-17779) for support of this work.
S.E.B. is on leave from the Institute of Organic Chem-
istry, Bulgarian Academy of Sciences, Sofia.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra, 500 and 125 MHz, respectively, of compounds 1o, 1m ,
and 1p in (CD3)2SO and 1o in CDCl3; compounds 2o, 2m , 2p,
3o, 3m , 3p, 5o, 5m , 5p, 8m , and 8p in CDCl3; compounds 4o,
4m , and 4p in (CD3)2SO; and compound 7o in (CD3)2SO. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O030091T
7604 J . Org. Chem., Vol. 68, No. 20, 2003