Synthesis and properties of mesobilirubins XIIγ and XIIIγ and their mesobiliverdins

Article abstract of DOI:10.1007/s00706-014-1160-6

The title pigments, with propionic acid groups displaced to the lactam end rings, were synthesized for the first time by the "1 + 2 + 1" approach, coupling two equivalents of a monopyrrole to a dipyrrylmethane (to give XIIγ), or the "2 + 2" approach, self-coupling two equivalents of a dipyrrinone (to give XIIIγ). Using the "1 + 2 + 1" approach, mesobilirubin IIIα was also prepared. Mesobilirubins XIIγ and XIIIγ are more polar than mesobilirubin IIIα and unlike IIIα cannot effectively engage the propionic acid groups in intramolecular hydrogen bonding to the dipyrrinone components. The new mesobilirubins give exciton coupled circular dichroism spectra in the presence of human serum albumin or quinine, with the XIIγ isomer exhibiting Cotton effect intensities nearly as strong as those from the IIIα isomer; whereas, the XIIIγ isomer exhibits far weaker intensities. Mesobilirubin IIIα requires glucuronidation for hepatobiliary elimination; whereas, XIIγ and XIIIγ do not, and they are excreted intact across the liver into bile. The corresponding biliverdins XIIγ and XIIIγ are reduced only slowly by biliverdin IXα reductase, in contrast to the fast reduction of the natural IXα isomer. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-014-1160-6

Monatsh Chem (2014) 145:775–789
DOI 10.1007/s00706-014-1160-6
ORIGINAL PAPER
Synthesis and properties of mesobilirubins XIIc and XIIIc
and their mesobiliverdins
Portia Mahal G. Sabido David A. Lightner
•
Received: 31 October 2013 / Accepted: 16 January 2014 / Published online: 13 March 2014
Ó Springer-Verlag Wien 2014
Abstract The title pigments, with propionic acid groups
displaced to the lactam end rings, were synthesized for the
first time by the ‘‘1 ? 2 ? 1’’ approach, coupling two
equivalents of a monopyrrole to a dipyrrylmethane (to give
XIIc), or the ‘‘2 ? 2’’ approach, self-coupling two equiv-
alents of a dipyrrinone (to give XIIIc). Using the
Introduction
In his successful quest to determine the chemical structure
of heme, for which he was awarded the Nobel Prize in
Chemistry in 1930, Hans Fischer recognized 15 possible
position isomers for two vinyl (V), four methyl (M), and
two propionic acid (P) groups distributed around the por-
phyrin macrocyclic periphery and located at the pyrrole b-
carbons. In 1927, he labeled these isomers I–XV in a long
paper published with Stangler [1] and showed 11 non-
symmetric and the four symmetric structures (in Fig. 1) of
mesoporphyrins, wherein the vinyl (V) groups are reduced
to ethyl (E). Two years later together with Zeile [2] he
proved by synthesis that the heme of nature possesses the
nonsymmetric structure called protoporphyrin IX. In the
current work, we are interested in bilirubin analogues with
a symmetric ordering of the b-substituents as found in three
of the four symmetric mesoporphyrins: III, IV, XII, and
XIII (Fig. 1).
‘‘1 ? 2 ? 1’’ approach, mesobilirubin IIIa was also pre-
pared. Mesobilirubins XIIc and XIIIc are more polar than
mesobilirubin IIIa and unlike IIIa cannot effectively
engage the propionic acid groups in intramolecular
hydrogen bonding to the dipyrrinone components. The new
mesobilirubins give exciton coupled circular dichroism
spectra in the presence of human serum albumin or qui-
nine, with the XIIc isomer exhibiting Cotton effect
intensities nearly as strong as those from the IIIa isomer;
whereas, the XIIIc isomer exhibits far weaker intensities.
Mesobilirubin IIIa requires glucuronidation for hepatobil-
iary elimination; whereas, XIIc and XIIIc do not, and they
are excreted intact across the liver into bile. The corre-
sponding biliverdins XIIc and XIIIc are reduced only
slowly by biliverdin IXa reductase, in contrast to the fast
reduction of the natural IXa isomer.
By 1929, it was becoming recognized that the yellow
pigment of jaundice arose in nature from heme catabolism
(Fig. 2). Yet its structure as bilirubin IXa was not estab-
lished until 13 years later by Fischer and Plieninger [3],
again by total synthesis. Along the twisting road to its
structure determination, Fischer and co-workers [4] pre-
pared two different symmetric derivatives of bilirubin:
mesobilirubin IIIa (1) and XIIIa (Fig. 1). The latter was
synthesized again 54 years later [5, 6]. Although neither
pigment was prepared from macrocyclic ring opening of
two of the four symmetric mesoporphyrins, III and XIII,
they might be viewed as having been derived from them by
cleaving the porphyrin macrocycle at the a-carbon on the
symmetry plane orthogonal to the plane of the macrocycle
Keywords Dipyrrole ꢀ Tetrapyrrole ꢀ Hydrogen bonds ꢀ
Circular dichroism
P. M. G. Sabido
Institute of Chemistry, University of the Philippines-Diliman,
Quezon City, Philippines
D. A. Lightner (&)
Department of Chemistry, University of Nevada, Reno,
NV 89557-0216, USA
e-mail: lightner@unr.edu
(Fig. 1), with loss of the a-carbon. These two mesobiliru-
bins exhibit many of the solubility and solution properties
123

7
76
P. M. G. Sabido, D. A. Lightner
P
P
γ
E
M
3
M
7
P
8
P
12
M
13 15 17
M
E
18
19
O
P
M
M
E
E
M
M
P
18
M
M
2
5
2
1
0
N
HN
1
CH2 11
CH2
O
9
β
δ
O
O
6
14
16
N
H
4
N
H
N
H
N
H
N
H
N
H
N
H
N
H
H
N
N
M
M
1
: Mesobilirubin III
α
Mesobilirubin IIIγ
α
E
E
III
M
M
γ
M
E
P
7
M
M
P
13
E
M
M
P
3
E
M
M
E
P
17
M
P
P
N
N
H
CH2
β
δ
CH2
O
O
O
O
N
H
N
H
N
H
N
H
H
N
H
N
H
N
H
N
H
N
N
E
E
Mesobilirubin IV
α
Mesobilirubin IVγ
α
M
M
IV
M
P
M
E
E
M
P
M
2
3
5
15 17
18
M
M
γ
O
CH2
O
N
H
N
H
N
N
H
E
M
P
7
M
M
P
13
M
E
P
P
H
N
N
H
CH2
2: Mesobilirubin XII
γ
O
O
β
δ
N
H
N
H
N
H
N
H
H
N
N
M
2
P
3
M
E
E
M
P
15 17
M
18
5
M
M
Mesobilirubin XII
α
α
O
O
E
E
N
H
N
H
N
N
H
4: Mesobiliverdin XIIγ
XII
P
M
E
M
M
E
M
P
2
3
5
15 17
18
P
P
γ
CH2
O
O
M
E
M
P
8
P
12
M
E
M
N
H
N
H
N
H
N
H
M
M
N
N
H
CH2
3: Mesobilirubin XIIIγ
O
O
β
δ
N
H
N
H
N
H
N
H
H
N
N
P
M
E
M
M
E
M
15 17
P
18
Mesobilirubin XIII
α
E
E
2
3
5
α
M
M
O
O
N
H
N
H
N
N
H
XIII
5: Mesobiliverdin XIIIγ
Fig. 1 Center the four symmetric mesoporphyrins (III, IV, XII, and
XIII) and the corresponding (left) mesobilirubins a and mesobiliru-
bins c due to opening of the macrocyclic ring at and with loss of the
a- or c-carbon. A dashed line through the a and c carbons lies at the
intersection of the plane of the macrocycle and an orthogonal plane of
symmetry represented by the dashed vertical line. E ethyl, M methyl,
P propionic acid
of bilirubin IXa and have been used as bilirubin IXa
analogues in studies of metabolism, photochemistry,
C(12), at C(7) and C(13). In fact, although mesobilirubin
XIIa (Fig. 1) is not known, the properties of synthetic
mesobilirubin IVa [5] (Fig. 1) support the contention that
the location of the acid groups at C(8) and C(12) is
essential for intramolecular hydrogen bonding. It was
found to have very different solubility properties and
metabolism from bilirubin IXa [7].
Similarly, one can imagine macrocyclic ring opening of
symmetric mesoporphyrins III, IV, XII, and XIII at the
c-carbon lying on the out-of-plane symmetry plane to
produce the corresponding mesobilirubins IIIc, IVc, XIIc,
spectroscopy, and pK . The key structural element relating
a
these three pigments is the location of their propionic acid
groups at carbons 8 and 12, which permits tight
intramolecular hydrogen bonding (Fig. 2b). Although eli-
sion of the a-carbon of mesoporphyrins III and XIII leads
to mesobilirubins fully capable of intramolecular hydrogen
bonding, elision of the a-carbon of mesoporphyrins IV and
XII does not, because the derived mesobilirubins (IVa and
XIIa) have propionic acids located away from C(8) and
1
23

Synthesis and properties of mesobilirubins XIIc and XIIIc
777
M
V
A
M
V
M
V
O
H
O
H
O
H
O
H
H
α
Fe
γ
V
M
V
M
V
M
N
N
N
N
N
N
Heme
Biliverdin
β
δ
5
15
Oxygenase
Reductase
H
H
N
N
N
N
N
N
M
M
M
M
M
M
8
12
P
P
P
P
P
P
HEME
BILIVERDIN IXα
BILIRUBIN IXα
B
C
V
O
H
M
M
7
β
O H
O
N
N
8
O
H
5
O
15
O
V
V
H
H
N
N
β
H
M
H
8
13
N
N
M
H
.
.
M
12
M
7
O
N
1
5
5
H
β'
.
.
O
.
1
2
13
M
V
H O
β'
.....
NH
O
M
O
......
O
H
INTRAMOLECULARLY
HYDROGEN-BONDED BILIRUBIN
BILIRUBIN RIDGE-TILE
Fig. 2 a Biogenetic sequence from heme to 4(Z),15(Z)-bilirubin
shown in a porphyrin-like shape. Rotating the bilirubin dipyrrinones
about the C(9)–C(10) and C(10)–C(11) bonds brings them into
juxtaposition with the C(8) and C(12) propionic acid groups so as to
enable intramolecular hydrogen bonding, as in planar projection (b).
When the latter is folded along the vertical dashed line, the 3D
structure of bilirubin seen in the crystal (c) emerges with a half-
opened book or ridge-tile shape with the seam along the line from the
propionic acid b and b carbons through C(10). The dotted lines in
b and c represent hydrogen bonds. V vinyl, M methyl, P propionic
acid
0
and XIIIc—all of which would have their propionic acids
located on the lactam end rings at C(2)/C(18) or C(3)/
C(17). And, like mesobilirubin IVa, neither is expected to
engage in intramolecular hydrogen bonding. Note that all
of the mesobilirubins c of Fig. 1 have propionic acid
groups located on the end rings; whereas, in the mesobi-
lirubins a they are located on the central two pyrrole rings.
Symmetric mesobilirubins with a propionic acid group
located on each lactam end ring (IIIc, XIIIc; IVc, XIIc)
had not heretofore been synthesized.
were examined as substrates for biliverdin reductases by
Prof. Timothy Mantle at Trinity College, Dublin [8]. We
also describe a new, efficient synthesis of mesobilirubin
IIIa (1), which was first prepared by Siedel and Fischer in
1933 [4] and more recently prepared by catalytic hydro-
genation of bilirubin IIIa, which had been isolated
following acid-catalyzed constitutional isomerization of
bilirubin IXa to a mixture of IIIa, IXa, and XIIIa [9–11].
In the following, we describe the syntheses and prop-
erties of two new mesobilirubins, XIIc (2) and XIIIc (3),
and their mesobiliverdins. Their syntheses provided sam-
ples for investigating the importance of hydrogen bonding
in hepatic elimination and for the preparation of mesobi-
liverdins XIIc and IIIc (4 and 5, respectively), which along
with previously prepared mesobiliverdins (XIIIa, IVa)
Results and discussion
Synthetic aspects
Two general strategies for bile pigment synthesis [12] were
considered for the preparation of the symmetrical meso-
bilirubins IIIa, XIIc, and XIIIc (1–3, Fig. 1). The syntheses
123

7
78
P. M. G. Sabido, D. A. Lightner
of 1 and 2 followed the ‘‘1 ? 2 ? 1’’ approach, where two
equivalents of a monopyrrole were condensed with one
dipyrrylmethane. The synthesis of 3 followed the ‘‘2 ? 2’’
approach where two equivalents of a dipyrrinone were self-
condensed. As indicated above, we had synthesized the
XIIIa and IVa isomers earlier [5, 6], and we considered
synthesizing mesobilirubin IIIc and IVc but our approaches
were either unsuccessful or the starting materials for 2 and
aqueous acetic acid using p-toluenesulfonic acid (p-TsOH)
as catalyst. The resultant dipyrrylmethane tetracarboxylate
ester 9 [19] was treated with trifluoroacetic acid to deprotect
the t-butyl ester groups, decarboxylate the resultant acids,
and react with trimethylorthoformate to give dialdehyde 7
[20]. Mesobilirubin IIIa (1) was obtained directly from
condensation of 7 with 6 in methanolic KOH.
The synthesis of mesobilirubin XIIc (2) also followed the
‘‘1 ? 2 ? 1’’ approach and involved making a bis(bro-
modipyrrylmethane) dihydrobromide 14 following the
general procedure of Pandey et al. [21] as outlined in
Scheme 2. Thus, the known bromopyrrole aldehyde 12 [21]
was condensed with the known symmetric dipyrrylmethane
dicarboxylic acid 13 [22] under conditions (p-TsOH) where
the latter underwent double decarboxylation to form a
reactive (bis-a-H) dipyrrylmethane intermediate. The
resulting dibromotetrapyrrole dihydrobromide 14 was then
converted to mesobiliverdin XIIc dimethyl ester (4e) in
(CH ) SO ? p-TsOH ? H O. The corresponding verdin
3
were more readily obtained.
Thus, as outlined in Scheme 1, the synthesis of 1
required pyrrolinone 6 and dipyrrylmethane-dialdehyde 7.
The former was known [13] and had been prepared pre-
viously by the Barton–Zard route [14] by first reacting
2
-acetoxy-3-nitropropane [14], obtained by acetylating the
condensation product of nitroethane with propionaldehyde,
then reacting the acetate with p-toluenesulfonylmethyliso-
cyanide, prepared by a high yield procedure [15]. The
resultant 4-ethyl-3-methyl-2-tosyl-1H-pyrrole (8) [13] was
brominated in CH Cl at the a-free carbon using N,N,N-
2
2
3 2
2
trimethylanilinium bromide perbromide to give the
diacid 4 was obtained by saponification of 4e and reduced
with NaBH to yield mesobilirubin XIIc (2).
2
-bromo-5-tosylpyrrole [16] in 86 % yield. The latter was
4
hydrolyzed to the 5-tosylpyrrolin-2-one [13] (70 % yield)
For preparation of starting monopyrrole 12, the a-CH3
of t-butyl 3,5-dimethyl-4-(2-methoxycarbonylethyl)-1H-
pyrrole-2-carboxylate (11) [18] of Scheme 1 was oxidized
to the a-CHO using ceric ammonium nitrate (CAN) to give
15 [23], and bromine was introduced to yield 12 following
the literature procedure [21].
in H O–trifluoroacetic acid and then reduced to pyrrolinone
2
6
[17] (76 %) using NaBH4.
Dialdehyde 7 was prepared in three steps from the known
monopyrrole 11 [18], first by mono-acetoxylation of the
a-CH to 10 using Pb(OAc) [18] then by solvolysis of 10 in
3
4
Scheme 1
CH O C
CO CH3
HO
2
C
CO H
2
3
2
2
1
2
) KOH/CH
) HOAc
3
OH
+
CH2
O
CH2
O
O
OHC
CHO
N
H
N
H
N
H
N
H
N
H
N
H
N
H
40%
6
7
1
(
CH
3
O)
3
CH/TFA
55%
CH O C
CO CH3
3
2
2
CH2
Ts
tBuO
2
C
CO tBu
2
N
H
N
H
N
H
8
9
p-TsOH/HOAc-H
2
O
54%
CO CH3
CO CH3
2
2
Pb(OAc)
4
t-BuO C
CH OAc
90%
tBuO C
CH3
2
2
2
N
H
N
H
1
0
11
1
23

Synthesis and properties of mesobilirubins XIIc and XIIIc
779
Scheme 2
CO CH3
CH O C
3 2
2
CO CH3
2
CH2
pTsOH/CH
2
Cl
2
-HOAC
CH2
+
RO C
2
CO R
2
Br
Br
-
N
H
N
H
N
N
H
N
H
N
+
Br
CHO
43%
-
+
N
H
Br H
H Br
1
2
13: R = H
6: R = C H CH
2
Pd(C)
89%
14
1
6
5
pTsOH/
2
5%
(
3 2 2
CH ) SO-H O
Clay 85%
CO R
RO C
2
CO CH
2
2
3
t-BuO C
CHO
CH
OAc
O
O
2
RO C
2
N
H
2
N
H
N
H
N
H
N
N
H
1
5
19: R = C H CH
4e: R = CH3
R = H
6
5
2
NaOH
5%
4
4
:
Pb(OAc)
4
81%
CAN 60%
NaBH
4
11
RO C
2
N
H
2
1
8: R = C H CH
6 5 2
6
C H
5
CH
69%
2
OH/Na
1
2
7: R = C H5
For the preparation of 13, we found it convenient to
debenzylate dibenzyl ester 16 [24], a conversion that pro-
ceeded in higher yield than saponifying the corresponding
diethyl ester, which we had converted from 17 in a similar
way. Dipyrrylmethane dibenzyl ester 16 was obtained
following solvolysis of the 5-acetoxymethyl derivative 19
air-sensitive desired product 3, but it was contaminated
with unreacted starting material and other difficult-to-
remove impurities. Attempts to purify tetrapyrrole 3 were
complicated by the facile formation of its verdin due to
adventitious air oxidation. In order to avoid problems in
isolating pure 3, dipyrrinone methyl ester 23 was self-
coupled with formaldehyde to produce the dimethyl ester
3e of mesobilirubin XIIIc, which was oxidized directly by
DDQ to the corresponding verdin dimethyl ester 5e in
62 % yield over two steps. The more easily purified verdin
dimethyl ester 5e was then saponified to verdin diacid 5,
[
22, 24] of benzyl 3,5-dimethyl-4-ethyl-1H-pyrrole-2-car-
boxylate (18) [24]. Thus, ethyl 3,5-dimethyl-4-ethyl-1H-
pyrrole-2-carboxylate (17) [5, 6, 25] was transesterified to
produce the corresponding benzyl ester 18 [26], which was
converted to the 5-acetoxymethyl derivative 19 [24] using
Pb(OAc) in acetic acid. Solvolysis [24, 27–29] of 19 on
4
and the latter was reduced to rubin 3 with NaBH . This
4
Montmorillonite clay afforded dibenzyl ester 16 [24] in
8
route to 3, similar to that developed for the synthesis of
mesobilirubin IVa (Fig. 1) [36], gave purer final product
than direct saponification of 3e.
5 % yield, which was debenzylated [28] using H /Pd(C)/
2
triethylamine in tetrahydrofuran (THF) to yield 13 in 89 %
yield.
Starting material 20 for the synthesis of 3 was prepared
from pyrrole diester 11 [18] (of Scheme 1), which was
synthesized by condensing the C-alkylation adduct of
pentane-2,4-dione and methyl acrylate [37] with the oxime
of tert-butyl acetoacetate. Elaboration of 11 to 20 pro-
ceeded via oxidation using CAN [23] to the 5-formyl
derivative 15 [18] of Scheme 2, which underwent a Bae-
yer–Villiger oxidation using m-chloroperoxybenzoic acid
to afford t-butyl 2,5-dihydro-2-hydroxy-4-(2-methoxycar-
bonylethyl)-3-methyl-5-oxo-1H-pyrrole-2-carboxylate (24).
The last compound was converted to 20 using (C H ) SiH
The synthesis of mesobilirubin XIIIc (3) followed the
‘2 ? 2’’ approach, as outlined in Scheme 3. Thus, the
‘
known pyrrolinone 20 [30, 31] was reacted with pyrrole
aldehyde 21 [23] to provide dipyrrinone diacid 22. The last
compound was converted to the a-H dipyrrinone methyl
ester 23 in 52 % yield over two steps: (1) a-decarboxyl-
ation in molten sodium acetate–potassium acetate [32, 33]
to give the mono-acid, then (2) Fischer esterification.
Direct self-coupling of a-H dipyrrinone acid of 23 using
formaldehyde plus p-TsOH or HCl [34, 35] gave the polar,
2
5 3
123

7
80
P. M. G. Sabido, D. A. Lightner
Scheme 3
CH O C
3
2
RO C
CH O C
3 2
2
1
) KOAc-NaOAc
NaOH
2%
Δ
+
4
O
R1
2) CH3OH/H2SO4
O
O
OHC
CO C H
H
N
H
N
H
2
2
5
N
H
N
H
N
H
N
H
2
0
21
22: R = H, R' = CO2H
23
TFA
Et3SiH
6%
CAN
65%
H2CO/pTsOH
7
1
7
CH O C
3
2
RO C
CO R
CH O C
CO CH
2 3
2
2
3
2
DDQ
OH
CO tBu
CH2
O
O
O
O
O
N
H
2
N
H
N
H
N
N
H
N
H
N
H
N
H
N
H
2
4
5e: R = CH3
5
6% NaOH
3e
5
:
R = H
NaBH4
7%
M-ClPBA
5%
4
5
15
3
in TFA. The overall yield from 11 was 16 %. An even
higher yield procedure to 20 (28 % overall) has been
achieved starting from acetaldehyde and methyl c-nit-
robutyrate [31]. The monopyrrole coupling partner 21 was
obtained simply by CAN oxidation [23] of the well-known
ethyl 3,5-dimethyl-4-ethyl-1H-pyrrole-2-carboxylate (17)
C(3/17)–CH3 allowed distinction between the C(2/18)–
CH3 and C(7/13)–CH . In addition to distinguishing
3
between methyl groups, the geometric structural assign-
ment, particularly the (Z)-configuration of the C(4/15)
exocyclic double bond of the dipyrrinone moieties has also
been confirmed from these experiments. Signals for the
methylene protons of the propionic acid chain often over-
lapped with the methylene signal of the ethyl group. To
distinguish one from the other, homonuclear decoupling
experiments were performed to clarify the region. The
[
25, 35].
Structural aspects and NMR
1
3
1
The constitutional structures of 1, 2, and 3 were confirmed
1
assignment of the C spectrum involved H-detected het-
1
eronuclear multiple-quantum coherence (HMQC) and H
13
by their H and C nuclear magnetic resonance (NMR)
spectra in (CD ) SO solvent. The data for these pigments
detected multiple-bond heteronuclear multiple-quantum
coherence (HMBC). The former was used to determine the
connectivity of groups in the molecule, while the latter was
used for long-range coupling.
3
2
are presented in Table 1, along with data for the well-
studied pigment mesobilirubin XIIIa [5, 6, 38]. The data
show the correct total number of protons and carbons
expected for symmetrical molecules. Clear differences are
evident among these isomers despite the fact that they all
have the same functional groups. The assignments of the
Hydrogen bonding aspects and NMR
1
1
Nuclear magnetic resonance spectroscopy, especially H
methyl groups in the H NMR are based on results from
homonuclear Overhauser effect (NOE) experiments. Thus,
in 1 a strong NOE between the hydrogens at C(5/15) and
methyls at C(7/13) distinguishes the latter from the C(3/
NMR, is also a powerful way of establishing whether a
molecule engages in conformations stabilized by
1
intramolecular hydrogen bonds. Analysis of the H NMR
1
7)–CH . Similarly, in 3 and 5 weak NOEs were found
3
chemical shifts (d) of the pyrrole and lactam N–H signals is a
direct way of verifying intramolecular hydrogen bonding.
Spectra in both (CD ) SO and CDCl were analyzed.
between C(5/15)–H and C(3/17)–CH . In 2 and 6, obser-
3
vation of a weak NOE between the C(5/15)–H CH and the
3
3 2
3
1
23

Synthesis and properties of mesobilirubins XIIc and XIIIc
781
Table 1 Carbon and proton NMR chemical shift/ppm assignments in (CD
3
)
2
SO for mesobilirubins
MBR XIIc (2)
Carbon
Atom no.
MBR XIIIa
MBR IIIa (1)
MBR XIIIc (3)
a
Carbon no.
Carbon
Proton
Carbon
Proton
Proton
Carbon
Proton
1
2
2
2
2
3
3
3
3
4
5
6
7
7
7
7
8
8
8
8
9
1
2
2
, 19
, 18
171.9
122.9
14.8
–
–
171.6
122.5
16.3
13.5
–
–
–
171.7
122.8
8.2
–
171.4
126.2
19.1
32.5
173.7
142.1
9.4
–
–
–
–
1
1
2
3
, 18
2.00(s)
2.23(q)
1.7 (s)
2.44(t)
2
, 18
–
0.99(t)
–
–
2.38(t)
3
, 18
–
–
–
–
–
12.1(bs)
, 17
1
, 17
147.1
17.2
8.1
–
140.9
9.1
–
144.2
19.4
34.3
173.5
127.3
98.3
122.3
121.8
9.1
–
–
1
2
3
2.49(q)
2.05(s)
2.75(t)
2.06(s)
2
, 17
1.08(t)
–
–
2.41(t)
–
–
3
, 17
–
–
–
–
12.2(bs)
–
–
, 16
, 15
, 14
, 13
127.8
97.7
122.4
122.0
9.2
–
129.4
98.0
121.9
119.2
9.3
–
–
130.0
98.3
121.0
114.9
17.1
61.1
–
–
5.93(s)
–
5.93(s)
6.00(s)
5.96(s)
–
–
–
–
–
–
–
1
1
2
3
, 13
1.77(s)
–
1.99(s)
2.00(s)
2.45(q)
2
, 13
–
–
–
–
–
0.99(t)
3
, 13
–
–
–
–
–
–
–
, 12
1
, 12
119.3
19.2
34.6
174.1
130.4
23.3
–
–
192.1
19.2
34.3
174.0
130.3
23.5
–
–
124.0
16.7
15.1
–
–
128.7
8.2
–
1
2
3
2.40(t)
1.99(t)
11.9(bs)
–
2.40(t)
1.95(t)
2.20(q)
0.75(t)
–
1.71(s)
–
2
, 12
–
3
, 12
11.9(bs)
–
–
–
, 11
0
130.0
23.6
–
–
130.4
23.7
–
–
3.94(s)
9.83
10.34
3.95(s)
9.75
3.92(s)
9.87
10.31
3.91(s)
9.80
10.30
1, 24
2, 23
–
–
10.32
–
–
-2
Run in (CD
a
The numbering system for the mesobilirubins may be found in 1 of Fig. 1
3
)
2
SO at approximately 2 9 10 M concentration at 25 °C. The multiplicities of the proton signals are designated in the usual way
Typically, in the former solvent, the lactam NH appears
Table 2 Comparison of N–H chemical shifts/ppm of mesobilirubins
and their dimethyl esters (e) in CDCl
3
and (CD
3 2
) SO
between 9.7 and 9.9 ppm, while the pyrrole NH appears
between approximately 10.2 and 10.4 ppm—irrespective of
whether the pigment is a diacid or diester, or a dipyrrinone
a
Mesobilirubins and
esters (e)
Chemical shift
in CDCl
Chemical shift
3
3 2
in (CD ) SO
[
39]. In contrast, in CDCl , where hydrogen bonding can
3
Lactam
N–H
Pyrrole
N–H
Lactam
N–H
Pyrrole
N–H
prevail, the lactam NH chemical shifts of rubin diacids
typically lie between approximately 10.6 and 10.8, and the
pyrrole NH resonances lie between approximately 9.2 and
IIIa (1)
XIIIa
10.59
10.58
10.85
11.72
12.12
10.54
10.47
10.64
10.26
9.16
9.15
9.74
9.77
9.82
9.87
9.80
9.74
9.78
9.88
9.84
10.31
10.31
10.36
10.31
10.30
10.40
10.32
10.30
10.30
9
.3 ppm. This pattern generally signifies intramolecular
IVa
9.65
hydrogen bonding of the type in Fig. 2. In contrast, rubin
diesters, which typically engage in dipyrrinone to dipyrri-
none intermolecular hydrogen bonding, exhibit NH
XIIc (2)
XIIIc (3)
XIIIa (e)
IVa (e)
XIIc (2e)
XIIIc (3e)
a
11.62
12.04
10.27
10.27
10.27
10.26
chemical shifts that fall between approximately 10.2 and
1
0.5 ppm. The H NMR data summarized in Table 2 show
1
little distinction between mesobilirubins and their esters in
CD ) SO but large differences in CDCl . In CDCl , the
(
3
2
3
3
dimethyl esters all exhibit similar pyrrole NH and lactam
N–H chemical shifts consistent with intermolecular hydro-
For structures, consult Fig. 1
gen bonding. The acids in CDCl , however, present a rather
3
for the pyrrole N–H chemical shifts. The data for both
compounds indicate intramolecular hydrogen bonding of the
type seen in Fig. 1c and d. Mesobilirubins IVa, XIIc (2), and
difference picture. Mesobilirubins XIIIa and IIIa (1) exhibit
very nearly identical lactam NH chemical shifts and likewise
123

7
82
P. M. G. Sabido, D. A. Lightner
XIIIc (3) differ significantly. While the predominant con-
formation of IVa might be folded, but not intramolecularly
hydrogen bonded so as to effect diamagnetic anisotropic
shielding of the pyrrole NHs each lying above the opposing
pyrrole ‘‘aromatic’’ ring, the strong deshielding of both the
lactam and pyrrole NHs of 2 and 3 suggest rather strong
intermolecular hydrogen bonding, stronger than that seen in
their dimethyl esters, and promoted perhaps by their poor
solubility in CDCl3.
These properties are in keeping with intramolecular
hydrogen-bonded structures for bilirubin IXa and meso-
bilirubins XIIIa and IIIa (1) but not mesobilirubin IVa.
Consistent with the behavior of the last compound and the
expectations from an inability of their propionic acid
groups to become involved in intramolecular hydrogen
bonding with the dipyrrinone lactam and pyrrole moieties,
mesobilirubins XIIc (2) and XIIIc (3) were expected and
found to dissolve in aq. bicarbonate and be insoluble in
CHCl3.
Solubility aspects
Chromatographic aspects
Like mesobilirubin IVa (Fig. 1), both the XIIc (2) and
XIIIc (3) pigments are extremely sensitive to light and air
oxidation. Hence, the solvents used to prepare the solutions
were saturated with nitrogen prior to use and work-up is
one under dim light. (It is due to this sensitivity to oxida-
tion that all purifications of the XIIc and XIIIc pigments
were done at the verdin ester stage.) Once the rubins were
obtained, minimum handling of the compounds was nec-
essary since the longer they remain in solution, the more
likely they are to become oxidized. The compounds once
isolated were sealed under argon and stored in the dark and
cold. Though they slowly acquire a greenish tinge, spectral
analysis (NMR) of the greenish compounds showed that
they were still quite pure. As expected, and in marked
contrast, the IIIa pigment (1) was much less air-sensitive
and behaved more like the XIIIa isomer and bilirubin IXa.
The solubility properties of bilirubin IXa and its various
analogues are known to depend considerably on the ability
of the molecule to form intramolecular hydrogen bonds
The relative polarities of bile pigments can also be assessed
by their chromatographic properties. In a silica thin-layer
chromatography (TLC) system using as eluent CHCl3–
CH OH (10:1 by vol), mesobilirubin XIIIa has a larger
3
retention factor (R = 0.9) than the more polar isomer,
f
mesobilirubin IVa (R = 0.3). Like mesobilirubin XIIIa,
f
its IIIa isomer (1) and natural bilirubin are also very fast
moving on this TLC system. In contrast, as seen in Table 3,
the R values of mesobilirubins XIIc (2) and XIIIc (3) all
f
move slower, like mesobilirubin IVa, as reflects their more
polar nature. And when converted to their less polar
dimethyl esters 2e and 3e, they move much faster.
The TLC behavior was consistent with that from the
reversed-phase high-performance liquid chromatography
(HPLC) system developed by McDonagh et al. [42]. On this
reversed-phase HPLC system, more polar pigments tend to
elute faster than the less polar. As reference HPLC standards,
intramolecularly hydrogen-bonded and thus ‘‘non-polar’’
bilirubin and mesobilirubin XIIIa exhibit approximately
18-min retention times, much longer than mesobilirubin IVa,
which because it is incapable of intramolecular hydrogen
bonding is more polar and faster moving (Table 3). In con-
trast, rubin dimethyl esters, which are also incapable of
[
38–41]. It is through engaging in such interactions that the
polar propionic acid groups of the molecule are tied up
with the dipyrrinone’s lactam and pyrrole groups (Fig. 2).
This sequestering of the polar groups renders the molecule
much less polar and more lipophilic, a property demon-
strated by the natural pigment as well as the synthetic
analogues, mesobilirubins XIIa and IIIa (1) [5, 6, 38, 42].
In contrast 2 and 3, like mesobilirubin IVa [5], with pro-
pionic acid groups located on sites removed from C(8) and
C(12), cannot participate in intramolecular hydrogen
bonding. Consequently, they are much more polar than 1,
mesobilirubin XIIIa, and bilirubin IXa. The polarity of the
pigments may be recognized by their solubility properties.
Most simple non-hydrogen-bonded carboxylic acids (e.g.,
benzoic and naphthoic acids) are soluble in weak bases,
e.g., 5 % aq. sodium bicarbonate. Thus, mesobilirubin IV
was found to be soluble in 5 % aq. sodium bicarbonate,
while bilirubin IXa and mesobilirubins XIIIa and IIIa (1)
are insoluble. In contrast, bilirubin IXa and mesobilirubins
Table 3 Comparison of TLC retention factors/R
tion times for mesobilirubins
f
and HPLC reten-
a
b
TLC
R
Mesobilirubin
HPLC
Ret. time/min
f
value
1
0
.0
.9
Bilirubin IXa
18.4
17.7
7.4
Mesobilirubin XIIIa
Mesobilirubin IVa
Mesobilirubin IIIa (1)
Mesobilirubin XIIc (2)
Mesobilirubin XIIIc (3)
2e
0.3
1.0
0.3
0.3
0.8
0.9
a
18.8
7.2
15.5
5.4
3e
7.2
-
3
XIIIa and IIIa (1) are soluble at 1 9 10 M in the non-
polar solvent chloroform; whereas, mesobilirubins IVa,
XIIc (2), and XIIIc (3) are insoluble.
TLC were run on silica gel TLC plates with CHCl
3
/CH
3
OH, 10:1
b
by vol irrigant) from [5]. Eluent: 0.1 M di-n-octylamine acetate in
(
3
3
CH OH containing 5 % water, 1.0 cm /min flow rate, as per Ref. [42]
1
23

Synthesis and properties of mesobilirubins XIIc and XIIIc
783
intramolecular hydrogen bonding, run faster (like mesobili-
rubin IVa) and in this HPLC system may be considered to be
more polar than the corresponding diacids, as has been
observed for the dimethyl esters of bilirubin and mesobili-
rubin XIIIa. As expected, the HPLC data for 1 (18.8 min)
correlates well with that of its XIIIa isomer; whereas, the
more polar XIIc isomer (2) (7.19 min) behaves like IVa and
the dimethyl esters—consistent with the absence of
intramolecular hydrogen bonding. Surprisingly, the XIIIc
isomer (3), which was predicted by TLC to behave on HPLC
like the IVa and XIIc isomers, ran slower (15.5 min). This
might suggest that while 3 is more polar than the XIIIa and
IIIa (1) isomers, as expected, it is less polar than the IVa and
XIIc (2) isomers—despite the indications from the TLC data.
It is unclear whether the divergent HPLC behavior of 2 and 3
is an artifact of this particular reversed-phase solvent system
or whether it indicates some type of intramolecular hydrogen
bonding in 3.
pigments display only a partial qualitative similarity. In
CHCl , a solvent that promotes hydrogen bonding, meso-
3
bilirubins XIIIa and IIIa exhibit an intense absorption with
max
k
intramolecularly hydrogen-bonded ridge-tile conformation
near 431 nm. These pigments are known to adopt an
(as in Fig. 1c) in CHCl [44–47]. Rather differently, 2 and
3
max
3 exhibit two well-defined k , one near 420 nm and a
max
more intense absorption at k
data for 2 and 3 are reminiscent of rubin dimethyl esters,
& 390 nm (Table 4). The
which are known to be intermolecularly hydrogen-bonded
dimers in CHCl [47] (for leading reference, see [48]). The
3
data from CD studies are even more telling.
Circular dichroism [43] is a powerful adjunct for pro-
viding insight into the conformations of bilirubin and its
analogues [44, 49]. The exciton coupling model is once
again suitable for interpreting the CD data of the bichro-
mophoric rubins. Owing to the isotropic (unpolarized)
nature of the light beam in UV–Vis spectroscopy, the two
exciton transitions exhibit the same sign. When the split-
tings are not widely separated, the substantial overlap often
gives rise to broad, non-symmetrical UV–Vis bands. With
polarized light, however, the exciton splitting leads to two
transitions with oppositely signed Cotton effects (CEs), as
seen in Table 5. According to exciton chirality theory [43,
44, 49], a negative dihedral angle formed by the electric
transition moments lying along the long axis of the dip-
yrrinone chromophores (see Fig. 3) predicts a negative first
(longer-wavelength) CE and a positive (shorter-wave-
length) second CE, i.e., a (-) exciton chirality. The signals
are predicted to be most intense when the dihedral angle is
close to 100°, as seen in folded conformations such as the
M-helical ridge-tile bilirubin shown Fig. 3. The mirror
image P-helical conformation of ridge-tile bilirubin would
be expected to express a (?) exciton chirality.
UV–Vis absorption and circular dichroism (CD)
aspects
Ultraviolet–visible spectroscopy (UV–Vis) can provide
valuable information about the conformation of bilirubin
analogues in solution and can provide insight into possible
intramolecular hydrogen bonding. Because rubin pigments
are bichromophores, formed from two dipyrrinone chro-
mophores conjoined to a CH group with minimal or no p-
2
orbital overlap, they constitute molecular excitons. Exciton
coupling theory can be a versatile adjunct for the inter-
pretation of their UV–Vis data [43, 44]. Thus, as revealed
in Table 4, where it is clear that mesobilirubins XIIIa and
IIIa (1) have nearly identical long wavelength UV–Vis
absorption characteristics across the range of solvents, and
their XIIc (2) and XIIIc (3) isomers also display charac-
teristics nearly identical to each other, the two sets of
The (-) exciton chirality of mesobilirubins XIIIa and
IIIa (1) in CHCl in the presence of quinine (Table 5) is
3
-
Table 4 Solvent dependence of UV–Vis absorption data for mesobilirubins (pigment concentration = 1.5 9 10 M)
5
Solvent
XIIIa
IIIa (1)
XIIc (2)
XIIIc (3)
max
max
max
max
max
max
max
max
k
e
k
e
k
e
k
e
Benzene
435
58,800
54,700
57,800
435
55,300
51,500
58,000
427
396
420
31,700
36,850
24,600
41,800
20,700
38,200
35,300
37,600
44,200
35,100
424
393
424
394
420
386
426
397
429
396
26,990
38,800
32,400
51,350
18,900
45,100
34,600
38,950
40,000
34,400
4
17sh
417sh
432
CHCl
3
431
3
90
CH
CH
3
CN
OH
426
56,500
46,300
50,700
43,100
57,000
49,100
424
426
54,200
55,000
418
375
431
340
432
398
3
90sh
3
426
4
01sh
(
CH
3
)
2
SO
426
426
58,150
48,250
3
97sh
395sh
max
max
3
-1
-1
k
/nm, e /dm mol cm
123

7
84
P. M. G. Sabido, D. A. Lightner
solvent ? quinine (1:200 pigment/quinine molar ratio) and in 0.1 M
CD on HSA
Table 5 Induced circular dichroism data for mesobilirubins in CHCl
phosphate buffer ? human serum albumin (1:2 pigment/HSA molar ratio)
3
Mesobilirubin
CD in CHCl
3
in quinine
k at De = 0
De (k
1
1
)
De
2
(k
2
)
De
1
(k
1
)
k at De = 0
2 2
De (k )
XIIIa
IIIa (1)
IVa
-61 (433)
-69 (431)
?8 (438)
-45 (439)
?10 (438)
-1
405
404
402
395
418
?42 (389)
?40 (386)
-7 (380)
?57 (442)
?89 (423)
?18 (439)
?105 (437)
?16 (437)
410
396
–
-62 (391
-61 (388)
?9 (365)
-65 (387)
-23 (394)
XIIc (2)
XIIIc (3)
3
?70 (371)
-15 (388)
406
416
-1
k/nm, De/dm mol cm
As in Refs. [48, 50]
interpreted in terms of intramolecularly hydrogen-bonded
conformers (Fig. 2) as M- and P-helical interconverting
conformational enantiomers (M and P) with the M-helical
predominating [44, 49]. In buffered HSA [49, 50], the signs
invert, and the major conformation is P-helical. In the
absence of chiral perturbation, the two isoenergetic con-
formations are in dynamic equilibrium, with equal amounts
of the two enantiomers present, and the pigment is thus a
racemic mixture of conformational enantiomers and does
not exhibit optical activity.
Insoluble in water, lipophilic, and protein-bound in plasma,
its hepatobiliary elimination requires hepatic acyl glucu-
ronidation by the phase 2 isozyme-specific glucuronosyl
transferase (UGT1A1), which appears to select the pig-
ment’s intramolecularly hydrogen-bonded propionic acids
for conjugation [51]. Earlier studies [7, 52] showed that
bilirubin and mesobilirubin XIIIa are glucuronidated and
eliminated in (normal) Sprague–Dawley (SD) rats, but are
not glucuronidated and eliminated intact in the mutant
(jaundiced) UGT1-deficient Gunn rat, which as in the rare,
recessive Crigler–Najjar syndrome of humans, has a con-
genital deficiency of the glucuronosyl transferase enzyme.
In contrast, mesobilirubin IVa, which is polar and not
intramolecularly hydrogen bonded, is excreted intact in
both types of rat. Consequently, we expected mesobilirubin
IIIa to require glucuronidation for transhepatic excretion
into bile; whereas, mesobilirubin XIIc and XIIIc were
expected to be excreted intact in, e.g., a Gunn rate [53]. In
rat metabolism studies conducted by the late Prof. Antony
F. McDonagh (G.I. Unit, University of California, San
Francisco Medical School) using methods described in Ref.
[51], it was shown that mesobilirubin IIIa (1) was excreted
into bile as a mixture of mono- and diglucuronides in the SD
rat but not excreted in the Gunn rat. In contrast, mesobili-
rubin XIIc (2) was excreted rapidly intact in both the SD
and Gunn rat. Similarly, the XIIIc isomer (3) was excreted
intact in the Gunn rat, along with a minor metabolite having
a verdin UV–Vis spectrum. Peculiarly, in the SD rat 3 was
excreted partly unchanged and partly as a monoglucuronide
and a trace of diglucuronide. The unexpected formation of
glucuronides of 3 in the SD rat would seem to suggest
partial intramolecular hydrogen bonding, as suggested by
the reversed-phase HPLC retention time (Table 3).
In contrast, mesobilirubin IVa, which cannot adopt the
intramolecularly hydrogen-bonded conformation of Fig. 2,
gives only a weak monosignate curve in aqueous buffered
HSA [49, 50] and a weaker (?) exciton chirality spectrum in
CHCl ? quinine (Table 5) [49]. The isomeric mesobiliru-
3
bins XIIc (2) and XIIIc (3), which likewise cannot adopt the
intramolecularly hydrogen-bonded conformations of Fig. 3,
were expected to behave similarly to IVa and exhibit weak
CD Cotton effects. Although such behavior was seen for 3, in
contrast, 2 gave an intense bisignate CD Cotton effect with
(
-) exciton chirality in CHCl ? quinine and an even more
3
intense (?) exciton chirality in aq. buffered HSA. Clearly the
data suggest that the chiral complexation action of quinine
that selects for the M-helical conformers of mesobilirubin
XIIIa and IIIa (1) also selects for the same conformation of 2
even in the absence of intramolecular hydrogen bonding due
to relocation of the propionic acids from C(8)/C(12) to C(3)/
C(17), but not when they are relocated to the ends of the
pigment at C(2)/C(18). Likewise, the binding pocket on HSA
that selects for the P-helical conformations of XIIIa and IIIa
(
1) also selects strongly for the same (P)-helicity enantiomer
of 3. Clearly, chiral complexing agents exert much less M-
vs. P-helical selectivity in IVa and XIIIc (3), but why this is
not the case for 2 is unexplained.
Hepatic metabolism
Concluding comments
Bilirubin IXa, the cytotoxic yellow pigment of jaundice, is
produced in mammals by catabolism of heme (Fig. 2).
The synthesis of mesobilirubins IIIa, XIIc, and XIIIc gave
evidence to their distinctive physical, spectroscopic, and
1
23

Synthesis and properties of mesobilirubins XIIc and XIIIc
785
-
R4
R4
+
O
R3
O
R3
H
H
O
N
O
H
NH
O
O
-
+
H
H
N
N
φ
2
φ
2
0
8
10
1
8
12
12
φ1
φ1
N
N
H
H
R2
R2
O
O........
NH
O
NH
R1
R1
O
O
H
H....... O
M
P
Fig. 3 Intramolecularly hydrogen-bonded enantiomeric conforma-
approximately 100° for /
1
& / & 60°. The double-headed arrows
2
1
3
2
tions (M and P) of bilirubin (R = R = M, R = R = V),
4
represent the approximate direction and intensity of the dipyrrinone
long-wavelength electric transition dipole moments. The relative
orientations or helicities (M minus, P plus) of those vectors are shown
(inset) for each enantiomer. The (-) dipole helicity correlates with M-
molecular chirality and the (?) helicity with P-molecular chirality
1
4
2
3
mesobilirubin IIIa 1 (R = R = E, R = R = M), and mesobili-
1
4
2
3
rubin XIIIa (R = R = M, R = R = E), folded into ridge-tile
shapes. The (M ¡ P) interconversion is accomplished by rotating the
dipyrrinones about / and / . The dipyrrinone chromophores are
1 2
planar, and the angle of intersection of the two planar dipyrrinones is
metabolic properties based on the ability to form
intramolecular hydrogen bonds. The corresponding verdins
served as substrates for distinguishing and defining struc-
tural features conducive to reduction (to the corresponding
rubins) by biliverdin IXa reductase and biliverdin IXb
reductase [8].
2 mm thick rotors of Merck silica gel PF254 with calcium
sulfate binder, preparative layer grade, using a Chromato-
tron (Harrison Research, Inc., Palo Alto, CA). All solvents
were reagent grade obtained from Fisher-Acros.
The spectral data were obtained in spectral-grade sol-
vents (Aldrich or Fisher). Quinine was from Aldrich; HSA,
fatty and free, was from Sigma; Pb(OAc)4 was from
Aldrich; CAN was from Fisher. Starting monopyrroles (8,
Experimental
11, 17, 21) were synthesized according to the literature
methods [13, 18, 23, 25, respectively].
All NMR spectra were obtained on a GE QE-300 MHz
1
1 13
H) or a Varian 500 MHz ( H) and 125 MHz ( C)
(
Mesobilirubin IIIa (1, C33H N O )
40 4 6
3
In a 10 cm round-bottom flask were placed 125 mg
spectrophotometer, respectively, in deuteriochloroform
unless otherwise indicated. Chemical shifts were reported
in ppm referenced to the residual chloroform proton signal
diformyldipyrrylmethane 7 (0.31 mmol) [20], 0.389 mg
3
pyrrolinone 6 (3.1 mmol), and 6.2 cm 4 M methanolic
potassium hydroxide solution. The solution was heated at
1
3
at 7.26 ppm and C signal at 77.23 ppm unless otherwise
noted. Melting points were taken on a Mel-Temp capillary
apparatus and are corrected. Elemental analyses, obtained
from Desert Analytics, Tucson, Arizona, were with
3
for 6 h. Then 15 cm H
reflux under N
O was added to
2
2
dilute the reaction mixture. After cooling to 0 °C, the
solution was acidified using 10 % aq. HCl. The resultant
±
0.4 % of the calculated values. High resolution deter-
precipitate was collected by filtration and triturated with
3
5 cm CH
minations of molecular ions were obtained from the
University of Nebraska Mass Spectrometry Facility. All
UV–Vis spectra were recorded on a Perkin-Elmer k-12
spectrophotometer and CD spectra were recorded on a
JASCO-600 instrument. Analytical TLC was carried out on
J.T. Baker silica gel IB-F plates (125 lm layer). Flash
column chromatography was carried out using 60–200
mesh silica gel (M. Woelm, Eschwege). For final purifi-
cation, radial chromatography was carried out on 1 or
3
OH. Purification by radial chromatography was
as the eluent. The
repeated twice, each time using CHCl
3
desired product 1 was collected in the first fraction. This
was followed by a fraction containing 23 mg (0.05 mmol)
of the monocoupled product in 16 % yield. Yield of 1:
75 mg, 0.13 mmol (40 %); m.p.: 282–284 °C (dec); IR
(KBr): mꢀ = 3,418, 3,247, 2,965, 2,915, 1,698, 1,648, 1,452,
-
1
1
13
1,250 cm ; H and C NMR in Table 1; UV–Vis in
Table 4; CD in Table 5.
123

7
86
P. M. G. Sabido, D. A. Lightner
Mesobilirubin XIIc (2, C H N O )
s, C(10)H), 6.01 (2H, s, C(5,15)H), 2.70 (4H, t,
3
3 40 4 6
The predecessor verdin ester 4e (0.15 g, 0.26 mmol) was
J = 6.3 Hz, CH CH CO H), 2.58 (4H, q, J = 6.8 Hz,
2
2
2
3
3
dissolved in 25 cm CH OH and 25 cm THF under an
CH CH ), 2.43 (4H, t, J = 6.3 Hz, CH CH ,CO H), 2.02
2 3 2 2 2
3
atmosphere of N . The mixture was cooled in an ice bath at
2
(6H, s, CH ), 1.68 (6H, s, CH ), 1.06 (6H, t, J = 6.8 Hz,
3 3
1
3
0
°C before 0.7 g sodium borohydride (18.5 mmol) was
added over a period of 30 min, after which the reaction was
CH CH ) ppm; C NMR ((CD ) SO): d = 173.5, 171.9
2
3
3 2
(C=O), 149.4, 143.4, 141.0, 139.8, 139.7, 128.8, 126.9
3
quenched with 50 cm water. The aqueous solution was
(Ar), 115.1 (C(10)), 96.3 (C(5,15)), 33.8 (CH CH CO H),
2
2
2
3
washed with CH Cl (3 9 20 cm ) to remove nonpolar
19.2 (CH CH CO H), 17.1 (CH CH ), 16.2 (CH CH ),
2 2 2 2 3 2 3
2
2
impurities before acidifying the solution with 10 % aq. HCl
to precipitate the product. Centrifugation followed by
3
washings with water (3 9 20 cm ), filtration, and drying in
9.0, 8.3 (CH3) ppm; FAB-HRMS: m/z calcd for
C H N O 586.279135, found 586.2766.
33 38 4 6
Mesobiliverdin XIIc dimethyl ester (4e, C H N O )
3
5 42 4 6
a vacuum desiccator afforded 2. Yield: 56 mg, 0.10 mmol
37 %); m.p.: 162–163 °C (dec); IR (KBr): mꢀ = 3,421,
3
In a 500 cm round-bottom flask were placed 0.62 g
(
-
,961, 1,654, 1,460 cm ; H and C NMR in Table 1;
1
1
13
2-bromo-5-formyl-4-(2-methoxycarbonylethyl)-3-methyl-
0
2
0
pyrrole (12, 1.95 mmol), 0.3 g 3,3 -diethyl-4,4 -dimethyl-
UV–Vis in Table 4; CD in Table 5. FAB-HRMS: m/z calcd
0 0
3
0 cm methanol, and 280 cm CH Cl . As soon as the
2
6
,2 -dipyrrylmethane-5,5 -dicarboxylic acid (13, 0.94 mmol),
for C H N O 588.294785, found 588.2926; calcd for
33 40 4 6
3
2
2
C H N O Na 611.284555, found 611.2841.
33 40 4 6
solutes were completely dissolved, a solution of 1.17 g
3
p-toluenesulfonic acid (6.15 mmol) in 60 cm CH OH was
Mesobilirubin XIIIc (3, C H N O )
3
3
40
4
6
3
The precursor verdin 5 (0.150 g, 0.26 mmol) was dissolved
added. The solution was stirred at room temperature
3
3
in 15 cm CH OH and 15 cm THF under an atmosphere
3
3
overnight and then washed with 570 cm H O, 150 cm
3
2
3
saturated aq. NaHCO , and again with 280 cm water. After
of N . The mixture was cooled in an ice bath at 0 °C before
2
3
0
.7 g sodium borohydride (18.5 mmol) was added over a
drying over anhyd. Na SO , the solvent was removed using
4
2
period of 30 min. As soon as the color turned to yellow-
3
brown, the reaction was quenched with 30 cm water. The
a rotary evaporator. The residue was dissolved in benzene,
which was removed under vacuum. This process was
repeated three more times before the residue was crystal-
lized from ether to give dihydrobromide salt of 14. Yield:
3
aqueous solution was washed with CH Cl (3 9 20 cm )
2
2
to remove nonpolar impurities before acidifying the
solution with 10 % aq. HCl to precipitate the product.
0.36 g, 0.4 mmol (43 %). It was used directly in the next
Centrifugation followed by washings with water
3
3 9 30 cm ), filtration, and drying in a vacuum desiccator
step.
3
In a 1 dm round-bottom flask were placed 0.36 g of the
(
3
afforded rubin 3. Yield: 71.4 mg, 0.12 mmol (47 %); m.p.:
preceding tetrapyrrole 14 (0.4 mmol) and 800 cm
1
1
76–177 °C (dec); IR (KBr): mꢀ = 3,346, 2,964, 2,921,
,671, 1,459 cm ; H and C NMR in Table 1; UV–Vis
dimethyl sulfoxide. The solution was stirred at room
temperature for 1.5 h, during which the color of the solution
changed from red to black-blue. The reaction solution was
-1 1 13
in Table 4; CD in Table 5. FAB-HRMS: m/z calcd for
3
C H N O 588.294785, found 588.2950; calcd for
3
3
40
4
6
poured into 6 dm ice cold water. The aqueous solution was
C H N O Na 611.284555, found 611.2848.
4
3
3
40
6
divided into four portions and each portion was extracted
3
with diethyl ether (3 9 150 cm ). The organic phases were
Mesobiliverdin XIIc (4, C H N O )
3
3 38 4 6
3
combined and washed with water before drying over anhyd.
Na SO . After separation, the solvent was removed by a
All solvents were purged with N before use. In a 250 cm
2
2
4
round-bottom flask were placed 60 mg verdin dimethyl
3
ester 4e (0.098 mmol), 60 cm of a 1:1 (by vol) THF–
rotary evaporator, and the black residue was purified by
three successive flash chromatography runs first using 4 %
3
CH OH solution, 40 cm 0.2 M aq. NaOH solution, 10 mg
3
(
vol) CH OH in CH Cl , then three times using increasing
3 2 2
ascorbic acid, and 10 mg EDTA. The dark blue solution
3
amounts of ethyl acetate in CH Cl (30, 50, 60 %). The dark
2 2
was stirred at 37 °C for 4 h and diluted with 100 cm
blue band was collected. Removal of the solvent and
recrystallization of the residue in CH Cl /hexane gave pure
water. The organic solvents were removed using a rotary
evaporator. The aqueous solution was cooled to 0 °C
before acidification using 10 % aq HCl. The resulting
precipitate was collected by centrifugation, washed with
2
2
verdin diester 4e. Yield: 60 mg, 0.098 mmol (25 %); m.p.:
01–202 °C; IR (KBr): mꢀ = 3,384, 2,960, 1,735, 1,690,
2
-
1 1
3
1,582, 1,212 cm ; H NMR: d = 8.20 (3H, bs, NH), 6.69
1H, s, C(10)H), 5.96 (2H, s, C(5,15)H), 3.70 (6H, s,
CO CH ), 2.82 (4H, q, J = 7.6 Hz, CH CH ), 2.59 (4H, t,
water (2 9 50 cm ), filtered, and again washed with water
(
and CH Cl . After drying in a lyophilizer, verdin acid 4
2
2
2
3
2
3
was obtained. Yield: 26 mg, 0.044 mmol (45 %); m.p.:
J = 7.6 Hz, CH CH CO CH ), 2.55 (4H, t, J = 7.46 Hz,
2
2
2
3
1
1
70–175 °C (dec); IR (KBr): mꢀ = 3,222, 2,957, 2,921,
-1 1
;
CH CH CO CH ), 2.08 (6H, s C –CH ), 1.80 (6H, s,
2 2 2 3 7 3
,697, 1,594, 1,220 cm
H
NMR ((CD ) SO):
3 2
1
C –CH ), 1.17 (6H, t, J = 7.6 Hz, CH CH ) ppm; C NMR:
3
2
3
2
3
d = 12.15 (1H, bs, CO H), 9.86 (1H, bs, NH), 6.82 (1H,
2
1
23

Synthesis and properties of mesobilirubins XIIc and XIIIc
787
d = 172.9, 172.1 (C=O), 149.9, 142.7, 141.5, 141.0, 139.2,
instantaneously. It was allowed to react for 30 min; then
3
the deep blue solution was poured into a 1 dm separatory
1
29.7, 127.3 (Ar), 114.2 (C(10)H), 96.7 (C(5,15)H), 51.7
3
3
(
CO CH ), 33.6 (CH CH CO CH ), 19.6 (CH CH CO
2
funnel containing 400 cm CH Cl and 400 cm 2 % aq.
2 2
2
3
2
2
2
3
2
2
CH ), 17.7 (CH CH ), 16.0 (CH CH ), 9.2 (CH ), 8.3
3
ascorbic acid. The aqueous layer was extracted two more
3
times with CH Cl (2 9 100 cm ). The organic fractions
3
2
3
2
3
(
CH ) ppm.
3
2
2
were combined and washed repeatedly with saturated aq.
Na CO , until the basic solution ceased to turn yellow. This
Mesobiliverdin XIIIc (5, C H N O )
3
3 38 4 6
2
3
Solvents were purged with nitrogen for 30 min before use.
3
In a 100 cm round bottom flask were placed 42.6 mg
was followed by another washing with water, drying over
anhyd. Na SO , and removal of the solvent. The black-blue
3
verdin dimethyl ester 5e (0.07 mmol), 40 cm THF/
2
4
precipitate was redissolved in the minimum amount of
CH Cl and was chromatographed to isolate the desired
3
CH OH (1:1 by vol) solution, 30 cm 0.2 M aq. NaOH
3
2
2
solution, and 10 mg ascorbic acid. The dark blue solution
blue verdin product 5e, which was recrystallized from
CH Cl /hexane. Yield: 152 mg, 0.25 mmol (62 %); m.p.:
was stirred at 40–42 °C for 4 h and was washed with
3
CH Cl (2 9 100 cm ). The aqueous layer was cooled to
2
2
2
2
2
1
28–229 °C; IR (KBr): mꢀ = 3,387, 2,909, 1,738, 1,692,
,586, 1,220 cm ; H NMR: d = 10.09 (2H, bs, lactam
0
°C before it was acidified using 10 % aq. HCl. The
-1 1
product precipitated and was collected by centrifugation
and filtration. It was washed with large amounts of water
and CH Cl and lyophilized to give verdin 5. Yield:
NH), 7.16 (1H, bs, C(10)H), 6.07 (2H, s, C(5,15)H), 3.60
(
(
(
6H, s, CO CH ), 2.59 (4H, q, J = 7.6 Hz, CH CH ), 2.47
2 3 2 3
2
2
8H, m, CH CH CO CH ), 2.36 (6H, s, C(8,12)CH ), 2.13
2
2
2
3
3
2
2.8 mg, 0.04 mmol (56 %); m.p.: 205–207 °C; IR (KBr):
6H, s, C(3,17)CH ), 1.15 (6H, t, J = 7.6 Hz, CH CH )
3
-1 1
2
3
mꢀ = 3,387, 2,909, 1,738, 1,692, 1,586, 1,225 cm
; H
1
3
ppm; C NMR: d = 173.1, 160.9 (C=O), 146.5, 143.7,
42.4, 135.7, 132.9, 131.8, 116.6 (Ar), 51.6 (CH CH CO
2
NMR: d = 10.09 (2H, bs, lactam NH), 7.16 (1H, bs,
C(10)H), 6.07 (2H, s, C(5,15)H), 3.60 (6H, s, CO CH ),
1
2
2
2
3
CH ), 31.8 (CH CH CO CH ), 19.0 (CH CH CO CH ),
3
3
2
2
2
3
2
2
2
2
.59 (4H, q, J = 7.6 Hz, CH CH ), 2.47 (8H, m,
2 3
17.7 (CH CH ), 14.8 (CH CH ), 10.2 (C(8,12)CH ), 9.8
2 3 2 3 3
CH CH CO CH ), 2.36 (6H, s, C(8,12)CH ), 2.13 (6H, s
3
2
2
2
3
(C(3,17)CH ) ppm.
3
1
3
C(3,17)CH ), 1.15 (6H, t, J = 7.6 Hz, CH CH ) ppm;
3
C
2
3
0
0
0
0
NMR: d = 173.1, 160.9 (C=O), 146.5, 143.7, 142.4, 135.7,
32.9, 131.8, 116.6 (Ar), 51.6 (CH CH CO CH ), 31.8
3,3 -Diethyl-4,4 -dimethyl-2,2 -dipyrrylmethane-5,5 -
1
dicarboxylic acid (13, C H N O )
17 22 4 4
2
2
2
3
(
CH CH CO CH ), 19.0 (CH CH CO CH ), 17.7 (CH
2
To 5 g benzyl 5-acetoxymethyl-4-ethyl-3-methyl-1H-pyr-
3
2
2
2
3
2
2
2
3
CH ), 14.8 (CH CH ), 10.2 (C(8,12)CH ), 9.8 (C(3,17)
3
role-2-carboxylate (19, 15.87 mmol) [24] in 110 cm
CH Cl , 22 g Montmorillonite K-10 clay was added, and
2
3
3
CH ) ppm; FAB-HRMS: m/z calcd. for C H N O
3
33 38
4
6
2
2
5
86.279135, found 586.2778.
the mixture was allowed to react for 30 min at room
temperature. When TLC indicated completion of the
reaction, the clay was removed by filtration, followed by
Mesobiliverdin XIIIc dimethyl ester (5e, C H N O )
3
5 42 4 6
3
In a 25 cm round bottom flask equipped with a magnetic
3
washings with 100 cm CH Cl . Evaporation of the solvent
2
3
stir bar were placed 15 cm CH Cl , 50 mg paraformalde-
2
2
2
on a rotary evaporator gave a cream-colored residue. Flash
hyde (1.67 mmol), and 40 mg p-toluenesulfonic acid
0.21 mmol). Note: Paraformaldehyde is not very soluble
in CH Cl ; it was allowed to stir in the solvent with
chromatography and recrystallization from CH OH gave
3
(
1
6. Yield: 3.37 g, 6.77 mmol (85 %); m.p.: 125–127 °C
2
2
(Ref. [21] 126–127 °C).
p-toluenesulfonic acid before adding in 23. Thus, the
mixture was allowed to stir for 1.5 h. Then, 400 mg a-H
dipyrrinone methyl ester 23 (1.32 mmol) was added. The
mixture was allowed to stir at room temperature in the
absence of light for 16 h and an atmosphere of N2.
The course of the reaction was followed by TLC. Upon
Dibenzyl ester 16 (5 g, 10.04 mmol) was placed in a
3
dm round-bottom flask together with 1 dm THF,
3
2
0
3
.28 cm diethylamine, and 1.11 g 10 % palladium on
carbon. The suspension was hydrogenated at room tem-
perature and one atmosphere overnight while following the
reaction by TLC. The catalyst was removed by filtration
through a bed of Celite. The solvent was removed, and the
reddish residue was recrystallized from THF–hexane.
completion of the reaction, the mixture was diluted with
3
00 cm CH Cl . Then it was washed successively with
1
2
2
3
water (2 9 100 cm ), saturated aq. NaHCO (2 9 100 cm ),
3
3
Yield: 2.85 g, 8.96 mmol (89.3 %); m.p.: 126–128 °C
3
and again water (1 9 100 cm ). After drying over anhyd.
1
(
dec); H NMR ((CD ) SO): d = 11.0 (2H, s, NH), 3.73
3
2
Na SO , the solvent was removed. The residue of 3e was
2
4
(
2H, s CH ), 2.33 (4H, q, J = 7.3 Hz, CH CH ), 2.14 (6H,
2
2
3
dried in a vacuum desiccator and was used in the next step
without purification.
1
s, CH ), 0.87 (6H, t, J = 7.3 Hz, CH CH ) ppm; C NMR
3
3
2
3
(
(
(
(CD ) SO): d = 162.4 (C=O), 129.9, 124.7, 122.1, 117.3
3
In a 250 cm round bottom flask were placed 246.4 mg
3 2
Ar), 21.7 (CH ), 16.6 (CH CH ), 15.2 (CH CH ), 10.0
2
3
of the crude 3e (0.4 mmol) from above, 120 cm dry THF,
and 90.8 mg DDQ (0.4 mmol). The solution turned blue
2
3
2
3
CH ) ppm.
3
123

7
88
P. M. G. Sabido, D. A. Lightner
3
CH OH, and 7 cm 2 N sulfuric acid. The mixture was
1
,10-Dihydro-2-(carboxyethyl)-7-ethyl-3,8-dimethyl-1-
3
oxodipyrrin-9-carboxylic acid (22, C H N O )
7 20 2 5
heated to reflux temperature under an atmosphere of
nitrogen. After 30 min CH Cl was added and the acid was
1
3
In a 50 cm round-bottom flask equipped with a magnetic
2
2
stir bar were placed 0.6862 g pyrrolinone 20 (3.75 mmol)
[
removed by washing the organic solution with saturated aq.
30, 31], 0.5225 g formylpyrrole 21 (2.5 mmol) [23],
NaHCO . The solvent was removed using a rotary
3
3
.21 g KOH (21.6 mmol), and 9 cm CH OH. The reaction
1
evaporator, and the residue was purified by flash chroma-
tography using 5–8 % CH OH in CH Cl by vol as an
3
mixture was stirred at room temperature overnight under an
3
2
2
atmosphere of N . The solvent was then removed on a
2
eluent. Recrystallization from CH OH–H O gave methyl
3 2
rotary evaporator, and the residue was redissolved in
3
ester 23. Yield: 435 mg, 1.44 mmol (52 %); m.p.:
188–189 °C; IR (KBr): mꢀ = 3,387, 3,000, 1,730, 1,672,
3
1
0 cm water and 1 cm CH OH and then heated to reflux
3
-
1
max
for 1 h. A small amount of solid remained undissolved; so,
the solution was filtered before it was cooled to 0 °C then
acidified with glacial acetic acid. The precipitate was
1,176 cm
;
UV–Vis:
k
(e) = 392 (32,400) nm
-
1
3
-1
1
(mol dm cm ); H NMR: d = 11.2, 10.38 (1H, bs,
NH), 6.84 (1H, d, J = 2.4 Hz, Ar–H), 6.18 (1H, s, C(5)H),
3.69 (3H, s, CO CH ), 2.66 (4H, m, CH CH CH ), 2.57
collected and triturated using CH OH to give the desired
3
2
3
2
2
3
yellow dipyrrinone 22. Yield: 0.347 g, 1.05 mmol (42 %);
1
(2H, q, J = 7.6 Hz, CH CH ), 2.18, 2.06 (3H, s, CH ),
2 3 3
13
m.p.: 211–212 °C; H NMR ((CD ) SO): d = 12.17 (2H,
1.13 (3H, t, J = 7.6 Hz, CH CH ) ppm; C NMR:
2 3
3
2
bs, CO H), 10.93 (1H, s, NH), 10.56 (1H, s, NH), 5.89 (1H,
d = 173.5, 173.4 (C=O), 143.4, 131.6, 129.1, 125.8,
123.4, 122.1, 118.8 (Ar), 102.2 (C(5)H), 51.6 (CO CH ),
2
s, C(5)H), 2.48 (4H, m CH CH CO CH ), 2.38 (2H, q,
3
2
2
2
2
3
J = 7.1 Hz, CH CH ), 2.17 (3H, s, CH ), 2.05 (3H, s,
2
32.7 (CH CH CO CH ), 19.5 (CH CH CO CH ), 17.8
2 2 2 3 2 2 2 3
3
3
1
CH ), 0.98 (3H, t, J = 7.1 Hz, CH CH ) ppm; C NMR
3
(CH CH ), 16.1 (CH CH ), 10.0, 9.8 (CH ) ppm; FAB-
2 3 2 3 3
3
2
3
(
(CD ) SO): d = 173.3, 171.9, 162.0 (PyrC=O), 142.4,
MS: m/z calcd for C H N O 302.4, found 302.4.
17 22 2 3
3
2
1
3
33.7, 129.4, 128.7, 126.4, 125.0, 121.7 (Ar), 96.0 (C(5)H),
Acknowledgments PMS was an R.C. Fuson graduate fellow. Partial
support of this work came from the U.S. National Institutes of Health
2.2 (CH CH CO CH ), 18.9 (CH CH CO CH ), 16.6
2
2
2
2
3
2
2
3
(
CH CH ), 15.4 (CH CH ), 9.8 (CH ), 9.1 (CH ) ppm.
2 3 2 3 3 3
(
HD 17779). We thank the late Dr. A.F. McDonagh, GI Unit,
Department of Medicine, University of California, San Francisco for
conducting the metabolism studies of this work.
1
,10-Dihydro-7-ethyl-3,8-dimethyl-2-
(
methoxycarbonylethyl)-1-oxodipyrrin
(
23, C H N O )
1
7 22 2 3
3
References
In a 5 cm round-bottom flask were placed a mixture of
.147 g dipyrrylmethane diacid 22 (0.443 mmol), 0.658 g
0
1
2
3
. Fischer H, Stangler G (1927) Justus Liebigs Ann Chem 459:53
. Fischer H, Zeile K (1929) Liebig’s Annalen 468:98
. Fischer H, Plieninger H (1942) Hoppe-Seyler’s Z Physiol Chem
274:231
potassium acetate, and 0.614 g sodium acetate. The
mixture was placed in an oil bath heated to 120 °C; then
the bath temperature was increased further until bubbles
were observed in the reaction flask at 130 °C. The reaction
was allowed to proceed for 20 min more until the
temperature reached 180 °C. After allowing the mixture
4
5
. Siedel W, Fischer H (1933) Hoppe-Seyler’s Z Physiol Chem
2
14:145
. Trull FR, Franklin RW, Lightner DA (1987) J Heterocycl Chem
4:1573
6. Shrout DP, Puzicha G, Lightner DA (1992) Synthesis 328
2
3
to cool to room temperature, 10 cm water was added to
7
. McDonagh AF, Lightner DA (1991) The importance of molecular
structure in bilirubin metabolism and excretion. In: Bock KW,
Gerok W, Gerok W, Gerok W, Matern S, Matern S (eds) Hepatic
metabolism and disposition of endo- and xenobiotics: Falk
Symposium no. 57. Kluwer, Dordrecht, p 47–60
the yellow residue. The aqueous mixture was acidified with
concentrated hydrochloric acid to pH 2. The greenish-
yellow precipitate of the propionic acid of 23 was collected
by filtration, washed with large amounts of cold water, and
1
8
9
. Cunningham O, Dunne A, Sabido PM, Lightner DA, Mantle TJ
(
used in the next step without further purification. H NMR
2000) J Biol Chem 275:19009
(
(CD ) SO): d = 12.19 (1H, bs, CO H), 10.50 (1H, bs,
3
2
2
. McDonagh AF (1979) Bile pigments: bilatrienes and 5,15-bila-
dienes. In: Dolphin D (ed) The porphyrins, vol VI. Academic,
New York
NH), 9.79 (1H, bs, NH), 6.75 (1H, s, Ar–H), 5.98 (1H, s,
C(5)H), 2.49 (4H, m, CH CH CH ), 2.42 (2H, q,
3
2
2
1
0. Dicesare JL, Vandemark FL (1981) Chromatogr Newsl 9:7
11. Ma J-S, Lightner DA (1984) J Heterocycl Chem 21:1005
2. Boiadjiev SE, Lightner DA (1994) Synlett 777
J = 7.1 Hz, CH CH ), 2.09 (3H, s, C(3)CH ), 1.97 (3H,
2
3
3
1
3
s, C(8)CH ), 1.03 (3H, t, J = 7.1 Hz, CH CH ) ppm;
3
C
3
2
1
NMR ((CD ) SO): d = 173.6, 171.3 (C=O), 142.1, 129.6,
3
2
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2
2
2
3
2
2
2
3
CH CH ), 15.9 (CH CH ), 9.7, 9.3 (CH ) ppm.
3
2
3
2
3
(
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3
In a 500 cm round bottom-flask were placed 800 mg of
1
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3
the propionic acid of dipyrrinone 23 (2.78 mmol), 250 cm
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3
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123