Converting 9-methyldipyrrinones to 9-H and 9-CHO dipyrrinones

Article abstract of DOI:10.1016/j.tet.2007.06.009

Yellow 9-methyldipyrrinones can be converted readily and in high yields to symmetric linear tetrapyrroles, blue biliverdinoids, which are cleaved in half, smoothly at room temperature to afford yellow 9-H dipyrrinones, and 9-CHO dipyrrinones as their viol

Full text of DOI:10.1016/j.tet.2007.06.009

Tetrahedron 63 (2007) 8962–8976
Converting 9-methyldipyrrinones to 9-H and 9-CHO dipyrrinones
*
Stefan E. Boiadjiev and David A. Lightner
Chemistry Department, University of Nevada, Reno, NV 89557-0020, USA
Received 25 April 2007; revised 31 May 2007; accepted 2 June 2007
Available online 9 June 2007
Abstract—Yellow 9-methyldipyrrinones can be converted readily and in high yields to symmetric linear tetrapyrroles, blue biliverdinoids,
which are cleaved in half, smoothly at room temperature to afford yellow 9-H dipyrrinones, and 9-CHO dipyrrinones as their violet to orange
colored adducts with the carbon acid used for the scission: thiobarbituric acid (TBA), N,N0-diethylthiobarbituric acid, barbituric acid, N,N0-
€
dimethylbarbituric acid, and Meldrum’s acid. The adducts, usually only of passing interest, are formally Knovenagel condensation products of
a 9-CHO dipyrrinone with TBA and other carbon acids of this work, and a reverse Knovenagel reaction of such adducts leads to 9-CHO
€
dipyrrinones. Under a set of improved reaction conditions the sequence thus efficiently converts 9-CH3 dipyrrinones to 9-H and 9-CHO
dipyrrinones.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
group.3 The ‘other half’ of the tetrapyrrole, with the exo-
vinyl group, was not recovered—an observation that led
Fischer to first assume a symmetrically-substituted linear
tetrapyrrole structure for bilirubin (which he subsequently
disproved). In contrast, mesobilirubin cleaves to a good
yield of a mixture of neo- and iso-neoxanthobilirubinic acids
(Scheme 1) that proved difficult to separate at the time.
Dipyrrinones1 are the chromophores of bilirubin (Scheme
1), the yellow-orange pigment of mammalian bile and of
jaundice, and they also constitute the two halves of biliverdin
(Scheme 1), the blue-green biological precursor of bilirubin
and the pigment of non-mammalian bile.2 In both bilirubin
and biliverdin, the dipyrrinone units are connected by a sin-
gle carbon, C(10). Although these pigments are not biosyn-
thesized in nature by conjoining two dipyrrinones, bilirubin
and biliverdin analogs have been prepared synthetically by
coupling two 9-H dipyrrinones with formaldehyde or its
equivalent, or by coupling a 9-formyldipyrrinone with a
9-H dipyrrinone or even by oxidative coupling of 9-methyl-
dipyrrinones.1 The 9-H dipyrrinone precursors, as well as
9-formyldipyrrinones have been prepared by synthesis, typ-
ically from monopyrroles. 9-Methyldipyrrinones are like-
wise synthesized from monopyrroles, but direct conversion
of these synthetically more accessible pigments to syntheti-
cally useful 9-H dipyrrinones has not been achieved.
Some 50 years later, Manitto and Monti4 demonstrated
a novel, less vigorous, and high yield fragmentation of bili-
verdin and its symmetric analog, biliverdin-XIIIa dimethyl
ester, a biliverdin analog with two endo-vinyl groups, to
afford 9-H dipyrrinones from reaction with 1.5 equiv of thio-
barbituric acid (TBA) (Scheme 2) in methyl acetate at room
temperature. The green-blue color of the verdin changed
gradually over 6 h to purple; and poorly-soluble, magenta-
colored TBA adducts of dipyrrinones were precipitated
from chloroform/hexane in w80% yield. The pale yellow
filtrates yielded 9-H dipyrrinones. The reaction has several
advantages, the most significant are its simplicity and high
yields, and the fact that the vinyl groups remain intact. Al-
though unprecipitated TBA adducts render chromatographic
separation of the 9-H dipyrrinones difficult, the cleavage
reaction of biliverdin is probably the most convenient alter-
native to prepare not readily accessible 9-H dipyrrinones
possessing vinyl groups, and it offers a convenient way to
make other 9-H dipyrrinones,5–7 given the verdin.
In the mid 1920s Hans Fischer renewed his investigations of
the constitutional structure of bilirubin and learned subse-
quently that bilirubins and biliverdins are cleaved to 9-H di-
pyrrinones in boiling resorcinol. Under brief reaction,
bilirubin, its dimethyl ester and biliverdin dimethyl ester af-
forded only low yields of vinyl-neoxanthobilirubinic acid
(Scheme 1) or its methyl ester—all with an endo-vinyl
When a symmetrical verdin such as biliverdin-XIIIa di-
methyl ester is treated with TBA, only one 9-H dipyrrinone
product is possible: vinyl-neoxanthobilirubinic acid methyl
ester. In this case, as with biliverdin, one half of the verdin
is ‘lost’ to the TBA adduct, which might be viewed formally
€
Keywords: Pyrroles; Biliverdinoids; Retro-Knovenagel reaction; Carbon
Acids.
* Corresponding author. Tel.: +1 775 784 4980; fax: +1 775 784 6804;
e-mail: lightner@scs.unr.edu
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.06.009

S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
8963
C
A
CO2H
10
CO2H
CO2H
10
CO2H
endo
exo
H2
1
19
O
1
19
O
CH2
CH2
O
O
N
H
N
H
N
H
N
H
N
N
H
cat.
N
H
N
H
H
Dipyrrinone
Dipyrrinone
MESOBILIRUBIN
boiling resorcinol
BILIRUBIN
boiling resorcinol
5
CO2HHO2C
CO2H HO2C
N
N
N
N
N
N
N
H H
N
H H
HH
HH
1
1
9
9
O
O
O
O
H
H
H
H
NOT FOUND
Vinyl-neoxantho-
bilirubinic Acid
Vinyl-isoneoxantho-
bilirubinic Acid
Neoxantho-
Iso-neoxantho-
bilirubinic Acid
bilirubinic Acid
boiling resorcinol
B
CO2H
CO2H
exo
endo
10
1
19
O
CH
O
N
N
H
N
N
H
H
BILIVERDIN
Scheme 1. Linear representations of (A) bilirubin, (B) biliverdin and (C) mesobilirubin and their 9-H dipyrrinone products isolated from molten resorcinol.
CO2H
€
as the Knovenagel condensation product between a 9-CHO
dipyrrinone and TBA. There is no evidence that a reverse
CO2H
N
€
Knovenagel reaction (see overview in Section 2.5) has
10
CH
O
O
been carried out with the adducts of Scheme 2; yet, one
might imagine the adducts to be a useful source of 9-CHO
dipyrrinones. In this case the TBA cleavage reaction would
ultimately yield (theoretically) 1 equiv of 9-H dipyrrinone
and 1 equiv of 9-CHO dipyrrinone, which is structurally
the reverse of the verdin-forming acid-catalyzed condensa-
tion of 9-H and 9-CHO dipyrrinones. It also suggests
a way for converting 9-CH3 dipyrrinones to 9-H because
N
H
N
H
N
H
BILIVERDIN
O
H
O
Thiobarbituric
Acid (TBA)
N
N
H
S
CO2H
CO2H
10
CH
O
O
A
N
N
N
H
N
H
R
R
OH
H
H
H
O
N
N
CH
H
O
O
N
H
N
H
N
N
H
S
H+
1: R = CH2CH3
2: R = (CH2)5CO2CH3
endo
exo
B
CO2H
HO2C
N
N
N
N
H H
O
O
9
H
H
+
pKa
9
1
R
O
O
H
O
3.75a
10
H (Thiobarbituric Acid)
CH2CH3
N
N
N
O
R
R
Vinyl-neoxantho-
5'
NH
3'
bilirubinic Acid
O
S
N
1'
S
H
O
O
H (Barbituric Acid)
CH3
3.99b
4.68c
N
R
R
CO2H
HO2C
N
N
O
N
N
H H
O
H H
9
+
9
1
O
O
O
H
O
10
Meldrum’s Acid
4.83d
5'
H
Vinyl-iso-neoxantho-
bilirubinic Acid
N
O
3'
O
N
S
1'
H
Figure 1. (A) The symmetric verdins and (B) the C–H acids of this work.
The C–H acid pKa values may be found in the following: aRef. 10a,
bRef. 10b, cRef. 10c, dRef. 10d.
Scheme 2. Cleavage of biliverdin into its component 9-H dipyrrinones and
the complementary 9-CHO dipyrrinone thiobarbituric acid adducts (Ref. 4).

8964
S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
9-CH3 dipyrrinones are converted smoothly and in good
yields to (typically symmetrically-substituted) biliverdins.
A
N
N
H H
1
9
Kryptopyrro-
methenone
O
10
We have reinvestigated the Manitto–Monti reaction with an
eye toward (1) improving reaction conditions and product
separation, (2) investigating whether the cleavage might
also occur from the action of carbon acids other than TBA,
and (3) converting the TBA adducts to 9-formyldipyrri-
nones. The verdins used in the current work are symmetric:
for simplicity, etiobiliverdin-IVg;8 and for improved verdin
and product solubility, an analog of mesobiliverdin-XIIIa
with hexanoic acids replacing propionic (Fig. 1).8b The C–
H acids used include TBA, its N,N0-diethyl derivative,
barbituric acid (BA), its N,N0-dimethyl derivative, and
Meldrum’s acid, which exhibit acidity varying between
pKa 3.75 and 4.83.
ꢀ
CH2Cl2 /
chloranil HCO2H
10
O
N
O
N
H
N
H
N
H
1
O
7
X = S (TBA)
X = O (BA)
CH3OH, 25 °C
O
N
R'
N
R'
X
32
31
3
71
32
R
31
3
5
5
4
6
7
4
6
8
R
+
R
8
2
2
N
N
N
N
H H
H H
O
9
1
9
1
O
H
O
10
3 or 5
3'
5'
R'
N
O
4, 7-9
N
2. Results and discussion
X
1'
R'
R
R
X
S
S
O
O
R
H
2.1. Synthesis: 9-CH3 to 9-H dipyrrinones
3: CH2CH3
4: CH2CH3
CH2CH3
CH2CH3
7:
8:
CH2CH3
H
CH3
Etiobiliverdin-IVg8 (1) and mesobiliverdin-XIIIa 8,12-bis-
hexanoic acid dimethyl ester (2)8b were chosen as standard
biliverdinoid reaction substrates that are readily synthesized
by p-chloranil-promoted self-coupling of easily available
9-CH3 dipyrrinones, kryptopyrromethenone,8c,9 and xantho-
bilirubinic-8-hexanoic acid methyl ester8b (Scheme 3). Ver-
din 1 has an all-alkyl pyrrole b-periphery and possesses
sufficient solubility in common organic solvents for cleavage
reactions in a homogenous phase. As described originally
by Manitto and Monti,4 reaction of biliverdins with TBA sug-
gested very low concentrations in methyl acetate (w0.6 mM),
and long reaction times. In 2001, Sawamoto and Inomata6
noted greater reactant solubility and significant reaction
rate acceleration by replacing methyl acetate with methanol,
which prompted us to use methanol solvent for reactions of
verdin 1, examined in detail with TBA and with other C–H
acids as projected in Scheme 3.
9: CH2CH3
5: (CH2)5CO2CH3
6
(CH2)5CO2CH3
H
S
O
B
O
CH3OH, 25 °C
CO2CH3
N
H
N
H
S
CH3O2C
(CH2)5(CH2)5
10
O
O
N
H
N
H
N
N
H
2
ꢀ
chloranil
CH2Cl2 /
HCO2H
8
(CH2)5CO2CH3
N
N
H H
9
1
Xanthobilirubinic-8-
O
hexanoic Acid Methyl Ester
10
The reaction mechanism postulated by Manitto and Monti
(Scheme 2) involves nucleophilic addition of the TBA anion
at C(10) of the verdin followed by protonation of the erst-
while verdin isopyrrole nitrogen to give a C(10)-TBA
substituted bilirubin intermediate that undergoes acid-
catalyzed tetrapyrrole scission, formally a retro-Michael
reaction. Since we did not expect complications from any
second bimolecular process that might interfere with the
spontaneous scission step, the reactant concentrations were
increased 7-fold (to 240 mL mmolꢀ1). Care was exercised
to obtain homogenous solutions of both the verdin and the
C–H acid (2.4 equiv), which required brief warming in
some cases, then the solutions were mixed at 25 ꢁC. Optimi-
zation of the reaction time was easily achieved by following
the disappearance of intense blue color of 1 or 2 by TLC.
Thus, the reaction time did not exceed 2 h, but slightly lon-
ger times did not decrease the product yields. (The products
3 and 4 are stable in EtOAc for 20 h, and all reactions in
CH3OH were worked up after ꢂ2 h when TLC showed ver-
din disappearance.) Separation of the magenta-colored TBA
adduct 4 (in 87% purified yield) from 9-H dipyrrinone ana-
log (3) of kryptopyrromethenone (in 76% purified yield) was
achieved by radial chromatography, but only on a small scale
Scheme 3. (A) Conversion of kryptopyrromethenone to etiobiliverdin-IVg
(1) and 1 to 9-H dipyrrinone 3 and adducts 4 and 7–9. (B) Conversion of xan-
thobilirubinic-8-hexanoic acid methyl ester to 9-H dipyrrinone 5 and TBA
adduct 6.
(w0.1 mmol) due to the rather low solubility of 4 in nonpolar
solvents. Adduct 4 formed saturated solution in chloroform
of onlyw0.7 mg mLꢀ1, and its low solubility rendered diffi-
cult its complete characterization in CDCl3 using 2D NMR
techniques. The literature4–7 suggested that manipulations
involving precipitation were better for working up the reac-
tion of 1 with TBA rather than chromatography of the total
crude mixture if pure 4 is not needed. This afforded a 79%
yield of 3.
To overcome, at least partially, the insolubility issues
encountered with 4 and to secure reliable NMR data of
a TBA adduct in CDCl3, we cleaved 2, a dimethyl bis-
hexanoate analog8b of mesobiliverdin-XIIIa (Fig. 1). The
reaction proceeded verywell and, after chromatographic sep-
aration, TBA adduct 6 was isolated in 95% yield (recrystalli-
zation from ethyl acetate/hexane) and the 9-H dipyrrinone
5 in 76% yield (lowered by losses occurring in the last

S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
8965
recrystallization step from CH3OH). Adduct 6, also magenta-
colored, in fact exhibited enhanced solubility in CDCl3
although longer NMR acquisitions were performed at an el-
evated temperature in order to preventsample crystallization.
precipitated directly from the methanol solvent, thus indi-
cating their increased solubility versus adduct 8, probably
due to the presence of the propionic ester group. The filtrate
contained a 50:50 mixture of the methyl esters of vinyl-
neoxanthobilirubinic acid and vinyl-iso-neoxanthobiliru-
binic acid (Scheme 2). These 9-H dipyrrinones are easier
to obtain by this process than by the lengthy synthetic
method and may be separated chromatographically.
The available literature5–7 on the Manitto–Monti4 verdin
cleavage reaction focuses only on TBA as the reagent. To ex-
plore the generality of the verdin cleavage we first examined
the effect of N-substitution on TBA by using 1,3-diethyl-2-
thiobarbituric acid (1,3-diethyl-2-thioxo-(1H,3H,5H)pyri-
midine-4,6-dione) and found that it cleaved verdin 1 to 3
and 7, quantitatively, with adduct 7 being isolated in 92%
yield (Scheme 3). A practical advantage of using the dialky-
lated TBAwas noticed immediately: it is much more soluble
than TBA in CH3OH and reacts in a somewhat shorter reac-
tion time. Consequently, the concentrations of the reactants
could be increased for convenient, large-scale preparations
of dipyrrinones. The work-up and product separation by ra-
dial chromatography are also easier than in the reaction of 1
with TBA. Radial chromatographic separation of neokrypto-
pyrromethenone 3 from the magenta-colored adduct 7 is
rather easier than the separation of 3 from 4. The polarity or-
der on silica gel is reversed relative to that observed for 3 and
4, i.e., 7 is less polar than 3 giving cleaner concentrated frac-
tions from the chromatographic separation. Final recrystalli-
zation of these fractions yielded 92% of 7. From several
experiments using diethyl TBA, a pure sample of 3 (82%)
was also isolated. From the accelerated scission of 1 using
diethyl TBA, one may conclude that the acidic NH protons
on TBA are not involved in any crucial way in acid–base
or other proton transfer reactions necessary for a successful
reaction.
In order to assess further the generality of the verdin scis-
sion reaction using related carbon acids, and taking note
of the similar pKas of TBA (pKa 3.75)10a and BA (pKa
3.99),10b and the higher value (pKa 4.68)10c of N,N0-
dimethyl BA (the pKa value of N,N0-diethyl TBA was not
available), we learned that all seem to cleave the verdins
of this study under the same reaction conditions: CH3OH,
25 ꢁC, 2 h. Meldrum’s acid, which has an even slightly
higher pKa (4.83),10d was also found to react with verdins.
However, uncatalyzed spontaneous scission of the verdin
(1) did not occur in methanol at 25 ꢁC, even after 4 days.
The literature indicates that Meldrum’s acid, surprisingly,
does not enolize, which we link to its failure to induce
spontaneous verdin scission. In order to promote reaction,
triethylamine was added to remove the acidic proton at
C(50) of Meldrum’s acid, and after 3 days at room temper-
ature, verdin 1 was converted to give a complex mixture,
from which an orange, nonpolar and easily crystallizable
compound was isolated in 6% yield whose 1H and 13C
NMR spectra corresponded to the expected conjugate 10
(Scheme 4).
1
O
O
This investigationthen led to questions as towhether a related
carbon acid, barbituric acid (BA), and its N,N0-dimethyl de-
rivative might replace TBA as potential cleavage reagents.
Verdin cleavage reactions using these two carbon acids
were examined in parallel. Both C–H acids reacted rapidly
with etiobiliverdin-IVg (1) under standard conditions: ho-
mogenous solution in methanol (240 mL mmolꢀ1) at 25 ꢁC
for 2.5 h (Scheme 3). A red-colored product (8) was precip-
itated in 94% of the theoretical yield in >95% purity, and the
filtrate afforded (after an undemanding chromatography and
recrystallization) 83% yield of neokryptopyrromethenone 3.
Adduct 8 is insoluble in CDCl3; therefore, its NMR spectra
were acquired in only (CD3)2SO.
DBU, DMSO, 65°
O
O
H
C
O
O
N
H
N
N
H
N
H
22
23
H
O
O
O
O
N
12
Reaction of 1 with dimethyl BA (Scheme 3) was also suc-
cessful, but here no products precipitated, and the total crude
mixture, after work-up, was separated by radial chromato-
graphy on silica gel. In contrast to diethyl TBA adduct 7,
dimethyl BA adduct 9 is more polar on silica gel than neo-
kryptopyrromethenone 3. After recrystallization, pure 9
was obtained in 88% yield as a purple solid.
N
H H
O
+
+
3
O
5'
O
10
O
O
O
H
N
N
O
O
N
H
N
H
Encouraged by the easy separation of 8 from 3, and by
predicting a further advantage in separability based on the
very low solubility of BA adduct 8 in the methanol reaction
medium that would facilitate the isolation of various 9-H
dipyrrinones, we reacted BA with an unsymmetrical verdin,
the dimethyl ester of biliverdin-IXa (Scheme 2),2 derived
from natural bilirubin. The reaction proceeded rapidly,
and 80–85% of the expected mixture of adducts (X¼O)
11
Scheme 4. Reaction of etiobiliverdin-IVg (1) with Meldrum’s acid (Fig. 1)
to give 9-H dipyrrinone 3 (Scheme 3), adduct 10 (16% yield), and tetrapyr-
role 11, with the last presumably arising from an initially-formed tetrapyr-
role adduct (12). A higher yield of 10 (46%) was obtained from refluxing
methanol solvent.

8966
S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
Table 1. 1H and 13C NMR chemical shiftsa and assignments of 9-H dipyrri-
nones 3 and 5
Because dimethyl sulfoxide solvent was found to have
a greater accelerating effect than methanol on the reaction
rate of 1 with diethyl TBA, 1 was reacted with Meldrum’s
acid in DMSO and in the presence of DBU at 65 ꢁC (reflux
temperature of methanol). An orange-magenta colored pig-
ment (characterized as 10) was isolated in 16% yield from
this reaction. However, most of the verdin was consumed
by conversion to a very polar yellow product, which was pu-
rified by chromatography. It is insoluble in CHCl3, sparingly
soluble in CH3OH, and soluble in (CH3)2SO, and its NMR
spectra in (CD3)2SO are consistent with structure 11
(Scheme 4). Transformation of 1 into 11 is feasible from
an initially-formed tetrapyrrole adduct (12) that is deproto-
nated at N(22) or N(23), with the resulting anion attacking
one of the Meldrum’s acid carbonyls. Base-catalyzed release
of CO2 and (CH3)2CO from the Meldrum’s acid moiety
would lead then to 11. The best results on a preparative scale
for cleaving verdin 1: Meldrum’s acid (3 equiv) and DBU
(3 equiv) in refluxing methanol, gave a 46% yield of 10.
2
3
1
1
7
3
5
7
9
3
4
6
1
3: R = H
1
8
8
2
2 R
2
N
10 11
N
H H
5: R = (CH2)3CO2CH3
1
8
O
H
Carbon
13C NMR
1H NMR
3
5b
3
5c
1 C]O
2 ]C–
21 CH3
3 ]C–
31 CH2
32 CH3
4 ]C–
5 ]CH–
6 ]C–
7 ]C–
71 CH3
8 ]C–
81 CH2
174.3
123.1
8.0
174.3
123.2
8.1
—
—
1.96 (s)
—
2.56 (q, J¼7.6)
1.19 (t, J¼7.6)
—
6.17 (s)
—
—
2.17 (s)
—
2.46 (t, J¼7.6)
1.21 (t, J¼7.6)
6.84 (d, J¼2.8)
11.06 (br s)
10.46 (br s)
—
—
1.96 (s)
—
2.55 (t, J¼7.6)
1.18 (t, J¼7.6)
—
6.16 (s)
—
—
2.13 (s)
—
2.43 (t, J¼7.6)
1.58 (m)
6.82 (d, J¼2.8)
11.04 (br s)
10.47 (br s)
148.4
18.0
14.9
128.3
101.3
124.4
123.4
9.4
126.4
18.5
14.6
120.2
—
148.4
18.0
14.9
128.3
101.3
124.3
123.6
9.5
124.5
25.1
30.0
120.8
—
2.2. Constitutional structures from NMR
82 CH2/CH3
9 ]C–
10 NH
Dipyrrinones are generally well-known in the chemical liter-
ature.1 Neokryptopyrromethenone 3, however, has been de-
scribed only once—in 1932 by Fischer,11 and of course no
modern spectroscopic methods could have been applied.
Its structure can be deduced by the method of preparation,
according to the Manitto–Monti cleavage4 of the known ver-
din and a melting point that corresponds to that reported by
Fischer.11 It was reconfirmed by its 13C NMR spectrum (Ta-
ble 1), which correlates well with other known 9-H dipyrri-
nones, such as the tetramethyl and tetraethyl analogs.12 (The
13C (and 1H) NMR assignments of 3 were firmly established
11 NH
—
—
Chemical shifts (d, ppm) downfield from (CH3)4Si at 25 ꢁC in 3ꢃ10ꢀ3 M
for 1H NMR and 2ꢃ10ꢀ2 M for 13C NMR solutions in CDCl3. J values are
in hertz.
a
24.9 ppm (84-CH2), 29.0 ppm (83-CH2), 34.1 ppm (85-CH2), 51.4 ppm
(OCH3), 174.3 ppm (86-CO).
b
c
1.40 ppm (m, 83-CH2), 1.68 ppm (m, 84-CH2), 2.33 (t, J¼7.5, 85-CH2),
3.67 ppm (OCH3).
1
by a combination of data from 2D gHMBC and H{1H}
signals of 3 and 5, which can later be correlated with those of
adducts 4, 6–9. The NMR chemical shift assignments of 3
(Table 1) are in agreement with previous extensive studies
on dipyrrinones by NMR.1a,7,13,14 In CDCl3 solvent, which
NOE experiments in CDCl3.) Although the hexanoic ester
analog (5) of neoxanthobilirubinic acid (Scheme 1) was un-
known, its structure, too, was deduced from the known struc-
ture of the verdin precursor and the mechanism of the
cleavage reaction, and was confirmed by 13C NMR.
1
promotes hydrogen bonding, the most downfield H NMR
signal of 3 at 11.06 ppm is attributed to the lactam NH and
the more shielded pyrrole NH resonates at 10.46 ppm—
both values are in the usual reported range for intermolecu-
larly hydrogen-bonded dimers.7 The observed doublet at
6.84 ppm is assigned to C(9)–H proton, spin-coupled
(3J¼2.8 Hz) to the pyrrole NH whose three-bond proximity
is confirmed by gHMBC. The singlet at 6.17 ppm belongs to
C(5)–H methine hydrogen (for the numbering scheme, see
Table 1), which is correlated by NOEs to the C(3)-ethyl
and C(7)-methyl and thus supports a syn-(Z)-configuration
of the exocyclic C(4)–C(5) double bond of 3, as confirmed
by X-ray crystallography of similar dipyrrinones.15
In contrast to 3, the magenta-colored TBA adduct 4 has very
low solubility in CDCl3 but it is sufficiently soluble in
1
(CD3)2SO for NMR studies. As for 3, the 13C (and H)
NMR assignments were firmly established in 4 by a combina-
tionof2DgHMBCand1H{1H}NOEexperiments. Likethose
of 3 and 5, the 13C NMR spectra of 4 and 6 in (CD3)2SO
correlate well (Table 2) with their structures assigned on the
basis of the verdin cleavage mechanism of Manitto and
Monti.4 One can readily detect the characteristic dipyrrinone
carbon resonances, that of the C(10)-methine (originally
C(10) in the verdin), and the carbons of the TBA. The di-
pyrrinoneresonancesareshifted, relativeto9-Hdipyrrinones,
especially those of the pyrrole ring and C(4), due to field
effects and conjugation across the C(10) methine between
the TBA and the dipyrrinone. Remarkably, excellent cor-
relation with only small differences in chemical shifts is
found across a wide range of adducts produced in this work,
from N,N0-diethyl TBA, BA, N,N0-dimethyl BA, and
Meldrum’s acid (7–10, respectively), whose formation is
discussed below.
It is now well-known that DMSO-d6 solvent disrupts the hy-
drogen-bonding network that stabilizes dimeric structures of
dipyrrinones, which may be recognized by NH signals that
undergo a cross-over resulting in more deshielded pyrrole
NH from hydrogen bonding to solvent.1,14 Accordingly, in
DMSO-d6 3 showed a pyrrole NH signal at 10.46 ppm, lac-
tam NH at 9.69 ppm, C(9)–H at 6.71 ppm, and C(5)–CH at
5.95 ppm. The almost equal rate of deuterium exchange of
the lactam and pyrrole NHs when D2O was added to
a DMSO-d6 solution of 3 is much faster than in CDCl3, in
which the pyrrole NH exchanges much more slowly than
the lactam.16
In the 1H NMR spectra, of particular relevance to the current
study is firm assignment of the most deshielded proton NMR

S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
8967
Table 2. 13C NMR chemical shifts (d)a and assignments of adducts 4 and
6–10 in (CD3)2SO
Table 3. Comparison of 1H NMR chemical shiftsa for adducts 4, 6–10, 14,
and 15
2
1
R
X
Y
3
3
Compound
Pyrrole NH
Lactam NH
10-H
TBA NH
5
6
4
3
4: CH2CH3
6: (CH2)5CO2CH3 NH
7: CH2CH3
8: CH2CH3
9: CH2CH3
10: CH2CH3
NH
S
S
S
O
O
(CH3)2
7
9
1
4
14.08
13.45
8.37
9.93
8.00
7.92
9.60, 9.65
12.23, 12.36
2
R
8
2
N
N
H H
O
NCH2CH3
NH
NCH3
O
1
H
O
10
6
7
13.89
13.46
8.95
9.93
7.90
7.91
10.24, 10.59
12.23, 12.36
3'
5'
X
O
X
14.35
13.36
8.15
9.92
8.24
8.02
—
—
Y
1'
8
9
13.45
9.81
7.96
11.09, 11.25
Carbon
4
6b
7c
8
9d
10e
14.13
13.30
8.06
9.84
8.24
8.04
—
—
1 C]O
2 ]C–
21 CH3
3 ]C–
31 CH2
32 CH3
4 ]C–
5 ]CH–
6 ]C–
7 ]C–
77 CH3
8 ]C–
81 CH2
82 CH3
9 ]C–
10 ]CH–
173.2
129.1
8.3
147.5
17.0
14.4
141.8
94.3
140.4
125.6
8.9
144.2
17.4
16.1
129.2
132.7
176.8
162.7
103.6
162.9
173.2
129.1
8.3
147.5
17.0
14.4
141.7
94.3
140.3
125.9
9.0
142.6
24.1
—
129.7
132.8
176.8
162.7
103.6
162.9
173.2
129.4
8.3
147.6
17.1
14.4
142.5
94.1
141.4
125.9
8.9
145.1
17.5
16.1
129.7
133.8
177.4
160.9
103.3
161.1
173.2
128.1
8.3
147.5
17.0
14.4
140.7
94.6
138.4
124.6
8.8
143.1
17.4
16.1
128.6
133.2
150.1
164.5
103.3
165.3
173.2
128.1
8.2
147.5
17.0
14.4
141.0
94.5
138.8
124.7
8.8
143.6
17.4
16.1
128.8
133.8
151.0
162.9
103.0
163.3
173.3
128.9
8.2
147.6
17.0
14.3
141.2
94.2
139.5
124.6
8.7
144.2
17.3
16.0
127.3
134.3
—
163.9
96.9
164.0
10
13.33
12.57
7.56
9.93
8.15
7.94
—
—
14a
14b
15a
13.45
13.43
—
—
8.06
7.88
8.89, 9.22
12.16, 12.20
13.54
13.22
—
—
8.20
8.03
—
—
8.53
11.94
—
—
8.44
8.19
8.77, 8.83
11.87, 11.99
15b
8.61
12.06
—
—
8.50
8.32
—
—
d, parts per million downfield from (CH3)4Si for 3ꢃ10ꢀ3 M solutions in
CDCl3 and (CD3)2SO at 25 ꢁC. The data from (CD3)2SO are shown in
italics.
a
20 C]S(O)
40 C]O
50 ]C–
60 C]O
a close proximity of a TBA C]O to the pyrrole NH, and
with the expectation of a strong hydrogen bond between
them, one can expect an unusually strong deshielding of
the pyrrole NH.
In parts per million downfield from (CH3)4Si for 2ꢃ10ꢀ2 M solutions in
a
(CD3)2SO at 25 ꢁC.
b
c
30.7 ppm (d-CH2), 28.1 ppm (g-CH2), 23.8 ppm (b-CH2), 33.1 ppm
(a-CH2), 173.2 ppm (a-CO2CH3), 51.1 ppm (a-CO2CH3).
12.1 ppm (10-CH2CH3), 43.1 ppm (10-CH2CH3), 12.2 ppm (30-CH2CH3),
43.2 ppm (30-CH2CH3).
28.1 ppm (10-CH3), 28.4 ppm (30-CH3).
26.6 ppm (20-CH3), 103.5 ppm (20-C).
d
e
A further examination of the distinction between the lactam
and pyrrole NHs, and their assignments, was accomplished
with 4 in CDCl3 by deuterium exchange with added D2O.
A fast exchange was clearly indicated for the more shielded
NH (lactam) versus the more deshielded NH (pyrrole), with
extremely slow exchange attributed to intramolecular hydro-
gen bonding (Fig. 3). Quantitatively similar results were
found with 6 in CD2Cl2 and CDCl3. Thus, the most de-
shielded signal in CD2Cl2 did not exchange considerably
with deuterium, confirming not only that the signal belongs
to pyrrole NH but also the persistence of intramolecular hy-
drogen bonding in CD2Cl2. Even in (CD3)2SO solvent, ex-
change of the more deshielded NH was very slow, whereas
that of the more shielded NH was fast—a behavior without
parallel in simple dipyrrinones like 3.14
In the 1H NMR spectra of adducts 4 and 6, the NH dipyrri-
none chemical shifts are especially interesting (Table 3). In
CDCl3 a very strongly deshielded nucleus exhibits a signal
near 14 ppm that we assign to the pyrrole NH, and a more
upfield signal near 8 ppm that we assign to the lactam NH.
As will be explained later in this work, the assignments of
these signals were confirmed as belonging to the dipyrrinone
unit by 1H{1H} NOE measurements and their rates of N–H
to N–D exchange with D2O.16 (The various assignments of
the 1H NMR signals of the adducts contributed to the assign-
ments of their 13C NMR chemical shifts by a combination of
1D and 2D experiments.) Adduct 4 is not very soluble in
CDCl3; adduct 6, prepared by reaction of TBA with verdin
The D2O exchange experiments showed, however, a subtle
difference between 4 and 7 in (CD3)2SO only. Upon adding
1
2, is significantly more soluble. Their H NMR spectra in
CDCl3 were obtained with difficulty from non-homogenous
solutions, but with somewhat improved solubility in CD2Cl2
32
H
1
(and (CD3)2SO), one finds the H{1H} NOE correlations
31
3
R
H
X
S
S
S
O
71
8
R
H
H
5
7
81
83
shown in Figure 2. These data show that the dipyrrinone
component of 4 (as well as 6) adopts a syn-Z conformation
found in typical dipyrrinones; viz. NOE correlations show
that the lactam and pyrrole NHs lie close to one another
and that the C(5)–H lies proximal to the C(3)-ethyl and
C(7)-methyl. The conformation about the C(9)–C(10)
bond connecting the dipyrrinone and TBA is one where
the C(10)–H lies close to the dipyrrinone C(8)-ethyl in 4.
The conformation defined by the NOEs is thus one with
4:
2
N
N
6: (CH2)3CO2CH3
HH
O
R
1
O
9
10
CH2CH3
H
CH3
7:
8:
9:
H
H
H
H
O
R'N
X
O
N
R'
Figure 2. Nuclear Overhauser Effect (NOE) enhancements observed for ad-
ducts 4 and 6–9 and represented by curved arrows. A dashed arrow indicates
a weak NOE.

8968
S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
5
confirmed the syn-Z configuration at C(4)]C(5) double
3
bond of the dipyrrinone portion and a conformation around
C(9)–C(10) single bond rendering the C(10)-methine hydro-
gen proximal to C(8)–CH2CH3 (as shown in Fig. 2). Taken
collectively, the NMR properties of BA adduct 8 are very
similar (except solubility in CHCl3) to the TBA adducts,
and clearly the pigment exists in a conformation supporting
strong intramolecular hydrogen bonding involving the
pyrrole NH.
2
8
N
N
HH
O
9
O
H
10
H
N
4
O
N
S
H
The NMR characteristics of 1,3-dimethyl BA analog 9 are
identical to those of 8, with additional data being available
in CDCl3 solvent. The pyrrole NH of 9 is even more de-
shielded in CDCl3 to 14.13 versus 13.30 ppm in (CD3)2SO;
whereas, the lactam NH of 9 is more shielded in CDCl3 to
8.06 versus 9.84 ppm in (CD3)2SO. The NOE correlations
of 9 in CDCl3 are identical to those found in (CD3)2SO for
8 (Fig. 2). The rate of deuterium exchange in 9 is in line
with those found in TBA derivatives like 4, 6, and 7, thus re-
affirming that the pyrrole NH is the most deshielded proton,
which becomes strongly electron-deficient from participa-
tion in strong intramolecular hydrogen bonding.
The strongly deshielded NH signal at 13.33 ppm of 10 in
CDCl3 remained intact following the addition of D2O (as
it did in (CD3)2SO); whereas, the signal at 7.56 ppm was
diminished significantly within 4 min, thereby suggesting
that the latter is the lactam NH and the former is the pyrrole
NH. Consequently, the conformation in the Meldrum’s acid
conjugate (10) corresponds to that found in 4 and 8 in both
polar and nonpolar solvents.
2.3. Reaction mechanism
The mechanism and kinetics of the cleavage reaction were
studied by 1H NMR. On the basis of its favorable solubiltiy,
1,3-diethyl TBAwas judged to be an excellent candidate for
examining the cleavage reaction kinetics. Perdeuterated
methanol solvent was used in the initial study. The reaction
concentration for NMR experiments was kept rather low and
equal to the concentration used for preparative syntheses
(w4.2 mmol mLꢀ1), and diethyl TBA was decreased from
2.5 to 1.2 equiv in order to slow the transformation. Separate
solutions of diethyl TBA and verdin 1 were prepared, and
Figure 3. Timewise progression of deuterium exchange of the NHs of
3.5 mM 4 in CD2Cl2 solution at 25 ꢁC.
D2O to a solution of 7 in CDCl3, the lactam NH signal at
8.15 ppm decreased in intensity tow10% of its initial value;
whereas, the C(10)–H (as a reference point at 8.24 ppm) and
the pyrrole NH signal at 14.35 ppm remained unperturbed.
In contrast, in (CD3)2SO solvent both NHs (pyrrole at
13.36 ppm and lactam at 9.92 ppm) of 7 were replaced by
deuterium within 4 min. This rapid exchange of the pyrrole
NH of 7 contrasts with the much slower rate of exchange
found in 4 where the pyrrole NH is not as mobile, even in
(CD3)2SO.
1
their H NMR spectra obtained (Fig. 4). Verdin 1, as ex-
pected, did not exhibit any NH signals because the protons
were exchanged with deuterium from CD3OD solvent. The
1H NMR chemical shifts followed during the experiment
were from protons at C(5) and C(15) at 6.09 ppm and from
the C(10)–H at 6.86 ppm, which was remarkably quite broad
in CD3OD. (An explanation for such broadening might in-
voke spin–spin coupling to ND deuterium with a small
Diethyl TBA adduct 7 was characterized by the usual NMR
experiments in CDCl3 and (CD3)2SO solvents. The carbon
signal assignments (Table 2) followed those made earlier
by 2D NMR spectroscopy on 4 and 6. In both solvents, 7
showed identical NOE enhancements (Fig. 2), indicating
a conformational preference similar to that in 4 (and 6).
1
3JH–D.) The H NMR of diethyl TBA in CD3OD was even
more interesting because the TBA methylene C(50) protons
did not appear at all, and two sets of N-ethyl groups were
found in a 60:40 ratio. Such data can be explained by enoli-
zation that leads to rapid and total deuterium exchange at
C(50) and the significant presence of the enol form in
CD3OD. In contrast, diethyl TBA in CD2Cl2 shows only
one species in solution, with the C(50)–CH2 at 3.70 ppm
and a correct integral for two protons, and with one set of
ethyl group signals. In (CD3)2CO solvent, diethyl TBA
also exhibited the presence of two species (ratio 80:20)
In general agreement with these findings, BA adduct 8 ex-
hibited a resonance at the lower field (13.45 ppm) that did
not exchange with D2O, but the other three NHs were com-
pletely exchanged with deuterium within 4 min in (CD3)2SO
solvent. Nuclear Overhauser effect experiments on 8

S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
8969
C(5)–H of dipyrrinone 3, respectively. The rapidly increas-
ing, weak signal at 6.75 ppm is that of C(9)–H from 3. The
data show that the broadened C(10)–H signal of 1 moves
quickly from 6.86 ppm to about 6.4 ppm, then slowly disap-
pears. The initially sharp C(5,15)–H singlet of 1 found at
w6.2 ppm after mixing with diethyl TBA also slowly de-
creases in intensity at the base of emerging peaks at 6.18
and 6.21 ppm. Of particular interest is the range of chemical
shifts, which is shown expanded in Figure 4. A new signal
appears at 5.96 ppm, immediately after mixing the compo-
nent solutions, grows in intensity, reaches maximum at about
15 min and then its intensity slowly decreases. This signal is
consistent with accumulation of an intermediate of rubin-
type 13 (Scheme 5) according to the previously proposed
mechanism.4 Presumably, in addition to the C(10)–H there
should have been another emergent signal for the C(50)–H
of diethyl TBA, which is absent because it had been ex-
changed with deuterium (from CD3OD solvent) even before
the reaction started, as pointed out above. Moreover, if
a C(50)–H was present instead of D in 13, then the C(10)–
H would be spin–spin-coupled (3J) to the erstwhile C(50)–
H, which is not observed.
H
Figure 4. Timewise 1H NMR scans in the expanded region of the C(5)–H
methine hydrogens of (6.18 ppm), (6.21 ppm), and C(10)–CH
(5.96 ppm) of transient 13 during reaction between etiobiliverdin-IVg (1)
and 1,3-diethyl TBA in CD3OD at 20 ꢁC (Scheme 5). N,N0-Diethyl-TBA
does not exhibit NMR signals in the region shown.
10
3
7
O
O
N
H
N
H
N
H
N
1
D
D
O
O
CD3OD
N
N
Et
Et
S
and a minor broad feature at 3.85 ppm. These observations
suggest different amounts of the enol form of diethyl TBA
are present in different solvents and a possible link to the
faster reaction of diethyl TBA observed in more polar sol-
vents, presumably via the enol. The verdin scission rate is
even faster in (CD3)2SO than in CD3OD, and much slower
in ethyl acetate.
H
10
C
O
O
N
D
N
N
N
D
N
5'
D
D
O
D
O
N
Et
Et
S
13
The two separate solutions of 1 and diethyl TBA in CD3OD
were mixed in an NMR tube, and the H NMR spectra
1
(Fig. 4) of the resulting mixture were acquired in 5 min in-
tervals at 20 ꢁC. Of course, each resulting spectrum is an av-
erage for the acquisition time of 5 min. After only 25–30
spectra, the reaction was complete. In the final spectrum, ob-
tained after 125 min, the appearance of the expected 1:1
mixture of 3 and 7 (Scheme 3) is rather clean, and all signals
can be accounted for, including some from unreacted diethyl
TBA and those from wet (HDO)CD3OD. Conspicuously
missing, however, is the C(9)–H signal of the neokryptopyr-
romethenone (3) product at 6.75 ppm whose integral is only
9% of each integral for C(10)–H at 8.18 ppm and C(5)–H at
6.21 ppm for adduct 7 or at 6.18 ppm for 3. This unexpected
result clearly shows that a deuteration step occurs during the
verdin fragmentation. In controlled experiments a purified
sample of 3 in contact with CD3OD and sonicated for 1 h ex-
hibited the correct integral for exactly one proton at C(9).
Similarly, exchange at C(9) by deuterium in 3 did not happen
when CD2Cl2 solvent was used for measuring the kinetics of
the scission of verdin 1. These data indicate that the 9-H or
9-D of 3 is incorporated at a final step in the mechanism.
+
N
N
N
N
D D
D D
O
9
D
1
10
O
O
H
d3-3
5'
NEt
3'
O
N
1'
O
Et
d2-7
Scheme 5. Mechanism of verdin cleavage by diethyl TBA in CD3OD.
After these results had been interpreted, it seemed worth-
while examining the reaction in several different solvents
of varying polarity (and thus different enol content in the di-
ethyl TBA) where the reaction might be slower and accumu-
lation of a rubin-type intermediate akin to 13 was even more
detectable. Reactions in non-deuterated solvents on the mi-
croscale were run with similar stoichiometry and concentra-
tions as in CD3OD. Strikingly, the reaction between 1 and
diethyl TBA in (CH3)2SO was complete in less than
15 min. In CH2Cl2, the reaction was sluggish but was com-
plete after 2.5 h at 45 ꢁC (at reflux). In an NMR experiment
conducted at 20 ꢁC in CD2Cl2, the appearance of the final
Certain signals grew fast over time: 8.18, 6.21, and 6.18 ppm
belonging to the C(10)–H, C(5)–H of adduct 7, and to the

8970
S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
spectrum after 3 h was very clean, and all signals were easily
assigned. However, the initial spectra are rather complex,
suggesting multiple species/equilibria. Although no signals
for intermediate 13 could be firmly assigned in CD2Cl2,
the experiment allowed one to conclude that deuterium is
not incorporated at C(9) in 3 (the signal at 6.83 ppm is as in-
tense as those at 5.98 and 6.16 ppm, but is with lower height
due to coupling to the pyrrole NH).
3
2
4
1
5'
H
5'
5
2
O
O
O
N
H
2
H
+
N
N
R
R
NR
S
N
H
O
CH3CH2OH
55 °C, 3 h
O
N
S
R
R = H or CH2CH3
14a: R =H
14b: R = CH2CH3
S
R
N
O
RN
O
5'
Acetone-d6 was also used as a solvent in the cleavage reac-
tion, and in this solvent diethyl TBA alone gave an indication
for two species in equilibrium. Etiobiliverdin 1 did not show
the lactam NH, lost presumably by exchange with HDO
present in the solvent. Similarly, in the final spectrum, the
acidic ‘protons’ of products 3 and 7 are barely visible. Al-
though the reaction in acetone seemed slower than in meth-
anol or DMSO on microscale, it was complete after 3 h at
20 ꢁC. Again, the C(9)–H signal of 3 at 6.78 ppm showed
a decreased intensity due to D exchange. Immediately after
mixing, two new intense peaks appeared at 6.0–6.1 ppm,
then their intensity gradually decreased. They might be at-
tributed to the C(10)–H as in 13, and if correct, the interme-
diate is apparently formed very quickly (there is no intensity
increase similar to that found in CD3OD), followed by
a slower fragmentation step.
O
O
H
31
5'
+
+
3
O
4
H
N
N
R
R
CH3CH2OH
55 °C, 3 h
3
5
2
N
H
S
N
H
R = H or CH2CH3
15a: R = H
15b: R = CH2CH3
O
H
H
O
O
R
AcOH/Ac2O
115 °C, 1.5h
N
O
N
N
R
R
N
R
S
O
S
16a: R = H
R = H or CH2CH3
16b: R = CH2CH3
€
Scheme 6. Knovenagel condensation of monopyrrole aldehydes and
o-tolualdehyde with TBA and diethyl TBA.
2.4. Synthesis and characterization of model monopyr-
role conjugates of thiobarbituric acid
(CD3)2SO: pyrrole NH at 11.94 ppm and TBA NHs at
11.87 and 11.99 ppm (differentiated by larger broadening
of the pyrrole NH). In (CD3)2SO, however, the a-derivative
(14a), showed a pyrrole NH at 13.43 ppm, deshielded by
1.2 ppm relative to the TBA NHs at 12.16 and 12.20 ppm.
This difference is even more pronounced in CDCl3 solvent,
for non-homogenous solutions. The b-derivative 15a showed
a pyrrole NH signal at 8.53 ppm and TBA NHs at 8.77 and
8.83 ppm; whereas, the hydrogen-bonded pyrrole NH of a-
derivative 14a is at 13.45 ppm (very similar to that in 4 and
6) and the TBA NHs are at 8.89 and 9.22 ppm. The pyrrole
NH signal of 14a in both (CD3)2SO and CDCl3 is unex-
changeable with deuterium (D2O) over 15–20 min but the
TBA protons are completely exchanged within 5 min. In con-
trast, all of the NHs of 15a exchanged rapidly with deuterium
in (CD3)2SO. The significantly more soluble diethyl TBA
derivatives 14b and 15b exhibited similar trends.
1
The strongly deshielded pyrrole NH observed in the H
NMR of dipyrrinone–TBA conjugates such as 4 and 6 (Table
3), and the failure of their pyrrole NH signals to cross that of
the lactam NH in (CD3)2SO (the pyrrole NH of 4 appears in
CDCl3 at d¼14.08 and moves to 13.45 ppm in (CD3)2SO)
suggested building simpler model compounds with but one
pyrrole ring. In one model compound, the conjugate is at
the a-position of the pyrrole in order to mimic the arrange-
ment in 4 and be able to participate in intramolecular hydro-
gen bonding. In a second, it would be attached at the pyrrole
b-position, thereby excluding such hydrogen bonding. The
syntheses (Scheme 6) of two such model compounds (14
€
and 15) relied on a Knovenagel condensation between
TBA or diethyl TBA with 3,4-diethyl-5-methylpyrrole-2-
carbaldehyde17 and with 2,5-dimethylpyrrole-3-carbalde-
hyde,18 where the latter was synthesized by a Vilsmeier
reaction of 2,5-dimethylpyrrole. Both aldehydes reacted
smoothly with TBA (1.1 equiv) in ethanol at 55 ꢁC during
3 h without the need of base catalysis.19 The precipitated in-
dividual products were separated by filtration and, rather un-
expectedly, both TBA derivatives 14a and 15a were found to
exhibit extremely low solubility in CHCl3 and CH3OH,
which excluded chromatographic purification. Therefore,
the syntheses were repeated with diethyl TBA to yield
adducts (14b and 15b) with more favorable solubility.
1
Taken collectively, the H NMR chemical shifts and ex-
change rates indicate that the pyrrole NH of 14 is engaged
in strong intramolecular hydrogen bonding to TBA carbonyl
oxygen, in agreement with the observations on dipyrrinone
adducts. However, an alternativerationalizationforthestrong
deshielding of the pyrrole NH might come simply from lying
in an anisotropic deshielding region of a TBA C]O. In order
€
to disprove this possibility, the Knovenagel products (16) of
o-tolualdehyde and TBA or diethyl TBA were prepared
1
(Scheme 6), and their H NMR spectra were measured. In
16 the benzene o-H, which lies in a C]O deshielding cone,
was observed to be a maximum of only 0.4–0.5 ppm more
deshielded (16b in CDCl3) than the m or p-Hs.
These conjugates were characterized by NMR in (CD3)2SO.
It is noteworthy that the carbon chemical shifts of the TBA
carbonyls at C(40) and C(60) are very close (162.90 and
162.94 ppm) in 14a but are rather different in 15a (160.42
Nuclear Overhauser effect spectra of 15 in (CD3)2SO indi-
cated that the C(30)–H bridging methine hydrogen is ori-
ented preferentially toward pyrrole C(2)–CH3 methyl
group (as drawn in Scheme 6) and not toward pyrrole C(4)
position, in which conformation the C(2)–CH3 would
1
and 163.29 ppm). The H NMR chemical shifts of interest
confirmed all expectations. The b-derivative (15a), which
is incapable of intramolecular hydrogen bonding exhibited
all NH signals of almost identical chemical shifts in

S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
8971
buttress a carbonyl. In accord with the conclusion above, the
NOE in 14 indicated a spatial proximity between the C(21)–
H and C(3)–CH2 (Scheme 6).
arylidene barbituric acids has been reported and their kinetics
examined spectrophotometrically, without isolation of the re-
sulting benzaldehydes.24 Inspired by the removal of the ma-
lononitrile protecting group25 from 2-formyl and 3-formyl
€
2.5. Converting adducts to 9-CHO dipyrrinones
pyrroles with strong aqueous alkali, formally a retro-Knove-
nagel reaction, we anticipated that high temperature and con-
centrated aqueous hydroxidewould be a prerequisite for such
energy unfavorable process.
Most of the chemistry of TBA derivatives is concerned with
their rich functionality that is often used for further building
of more complex heterocyclic systems.20 Very limited infor-
mation is scattered about on the possibility of breaking the
50-exocyclic double bond, e.g., in Scheme 7. Addition of
nucleophiles, such as carbanions derived from malononitrile,
ethyl methyl ketone, cyclopentanone, and camphor, to this
type of double bond, has been described,21 as well as addition
of secondary amines,21b and even addition of water in the
special case of an electron-withdrawing p-nitrophenyl sub-
stituent on the double bond (the condensation product of
TBAwith p-nitrobenzaldehyde)21c—in all cases without fur-
ther cleavage or degradation. TBA adducts such as 4, 6–9,
and 17 (and their analogs of Scheme 2) could be viewed as
CO2CH3
CO2CH3
N
N
N
N
HH
O
H
H
a, b, c
10
O
O
O
H
O
5'
H
N
17
18
N
S
H
N
H
N
N
N
H
H
H
H
a,b
d
Knovenagel condensation products19,22 between an aromatic
O
10
O
O
€
aldehyde and C–H acidic b-dicarbonyl component. Surpris-
O
H
O
5'
N
4
19
ingly, the term ‘retro-Knovenagel reaction’23 has not been of-
ten used in the literature, although a mild hydrolytic cleavage
€
N
S
H
€
of barbituric acid derivatives (retro-Knovenagel) has been
demonstrated in a monolayer with 2,4,6-triaminopyrimidine
€
Scheme 7. Retro-Knovenagel reactions leading to aldehydes 18 and 19.
Reagents and conditions: (a) aq NaOH/D, (b) HCl, (c), CH2N2, (d) TBA,
piperidine.
inserted from the aqueous phase.23a Alkaline solvolysis of
Table 4. Comparison of UV–vis spectral data of adducts 4, 6–10, 14, and 15a
Pigment
3max (lmax, nm)
C6H6
CHCl3
CH3CN
CH3OH
(CH3)2SO
4
62,100 (564)
36,600 (527)
44,000 (326)
67,000 (563)
38,700 (526)
52,900 (324)
55,300 (552)
41,400 (518)sh
47,900 (321)
58,500 (552)
42,600 (518)sh
47,000 (321)
53,700 (558)
41,300 (526)sh
41,000 (326)
6
7
63,800 (564)
36,800 (527)
44,300 (326)
64,000 (563)
39,000 (525)
52,500 (324)
57,700 (552)
42,500 (519)sh
50,400 (321)
60,400 (552)
43,800 (521)sh
48,800 (321)
55,300 (558)
42,300 (526)sh
41,100 (326)
62,200 (567)
38,800 (531)sh
39,400 (328)
68,500 (566)
40,400 (530)sh
44,000 (327)
60,700 (557)
41,600 (522)sh
42,100 (323)
62,300 (555)
45,100 (522)sh
42,600 (323)
58,800 (564)
42,300 (528)sh
40,200 (329)
8b
44,700 (535)
34,300 (503)sh
38,200 (317)
48,500 (537)
34,000 (505)
45,900 (315)
42,500 (526)
35,300 (497)sh
43,100 (311)
45,100 (527)
36,900 (498)sh
43,300 (313)
41,400 (531)
35,200 (501)
37,900 (316)
9
48,700 (539)
34,700 (508)sh
39,900 (317)
52,300 (539)
35,700 (505)sh
46,500 (316)
46,800 (528)
38,300 (499)sh
44,500 (312)
48,300 (528)
39,800 (500)sh
44,300 (313)
43,200 (534)
37,000 (505)sh
38,600 (316)
10
41,500 (523)
32,600 (494)
40,600 (311)
42,600 (521)
33,800 (490)sh
47,600 (310)
41,200 (513)
34,800 (487)sh
46,800 (307)
41,500 (512)
36,900 (486)sh
45,700 (308)
40,200 (519)
34,200 (491)sh
40,300 (311)
14ab
15ab
14b
118,700 (460)
43,800 (439)sh
6100 (324)
126,300 (460)
47,500 (439)
5900 (312)
122,300 (455)
47,500 (434)sh
4300 (312)
130,200 (456)
50,000 (436)sh
5700 (316)
121,800 (461)
43,400 (439)sh
7700 (306)sh
43,200 (427)
11,900 (357)
5900 (311)sh
39,900 (431)
11,900 (360)
5900 (308)sh
42,200 (425)
12,300 (353)
6500 (310)sh
44,000 (428)
12,500 (360)
5700 (312)sh
43,400 (431)
11,700 (360)
6400 (313)sh
125,000 (464)
41,000 (440)sh
6100 (328)
130,000 (464)
45,600 (442)sh
5700 (328)
126,600 (459)
48,700 (439)sh
5600 (309)
136,300 (459)
50,500 (439)sh
5000 (312)
124,200 (464)
44,900 (442)sh
5600 (313)
15b
42,900 (428)
10,900 (357)
6600 (314)sh
42,700 (430)
11,300 (358)
6300 (309)sh
42,400 (428)
11,100 (357)
6700 (308)
44,100 (423)
11,700 (363)
5900 (305)
42,200 (437)
11,000 (364)
6400 (305)
Concentration range 8.1ꢃ10ꢀ6–3.6ꢃ10ꢀ5 of solutions containing 2% v/v CHCl3.
a
b
Solutions containing 2% v/v (CH3)2SO.

8972
S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
Preliminary experiments on the TBA adduct 17 of 9-formyl-
neoxanthobilirubinic acid (18, Scheme 7), available from
TBA cleavage of mesobiliverdin-XIIIa dimethyl ester, indi-
cated that ethanol co-solvent was necessary for solution ho-
mogeneity; however, the lower (w70 ꢁC) reflux temperature
of aqueous ethanolic sodium hydroxide did not give satisfac-
tory results. Evaporation of the ethanol from the reaction
mixture by heating led to an increased reaction temperature
(to w90 ꢁC) and to the cleavage of the C(50)–C(10) double
bond in 17. The pale yellow aldehyde 18 product was iso-
lated in 26% yield after acidification, followed by separation
of the crude dark acid by filtration and esterification with di-
azomethane. The necessity to re-esterify the propionic acid
side chain might have had a deleterious effect on the overall
yields for aldehydes 18 and 19, which can also be
synthesized efficiently by a one-step ester deprotection–
formylation procedure from the corresponding 9-carbo-
tert-butoxy esters,27 their recovery from pigments 4, 8, and
17 proved the concept that both the 9-H and 9-CHO dipyrri-
nones of biliverdins can be recovered.
2.6. UV–visible spectra of adducts
Magenta-colored TBA adducts 4 and 6 show a strong ab-
sorption (3w60,000) near 560–550 nm, with weaker absorp-
tion (3w40,000) near 525 (shoulder) and 325 nm (Table 4).
The influence of ethyl groups (spectra from diethyl TBA ad-
duct 7) is only small. However, the spectra were found to be
sensitive for replacing the C]S group of TBA. The BA and
dimethyl BA adducts (8 and 9, respectively) showed an ap-
proximately 30 nm hypsochromic shift in the longest wave-
length band and a hypochromic shift of approximately
15,000–20,000 3 units. A similar hypsochromic shift was
found for the 520 nm band, but with a much smaller drop
in 3. Similarly, the band near 325 nm was shifted to
w315 nm and 3 dropped byw10,000 units. The trend toward
hypsochromic wavelength shifts is seen further in Mel-
drum’s acid derivative 10. The influence of solvent polarity
and protic versus aprotic on the lmax and 3 values is not large.
€
yield; therefore, the retro-Knovenagel reaction was also per-
formed on adduct 4, and this cleanly afforded aldehyde 19 in
44% yield. Further improvement (to 61% yield of 19) was
achieved when diethylene glycol co-solvent (8% v/v) in re-
fluxing aqueous NaOH was used. Similarly, cleavage of the
BA residue of 8 was accomplished in an initial mixture of
ethanol/diethylene glycol/aq NaOH and after evaporation
of all ethanol at reflux, the reaction provided 19 in 66%
yield. The structure of known26 aldehyde 19 was confirmed
by its NMR spectra and, more importantly, it was converted
back to magenta-colored adduct 4 (77% yield, identical in all
aspects to that isolated from verdin cleavage) by a typical
€
Knovenagel condensation with TBA in refluxing chloroform
in the presence of piperidine catalyst. Despite the lower
The data may be compared with those from adducts 14 and
15, which possess only one pyrrole ring. Solutions of both 14
A
B
ε x 10-3 / L.mol-1.cm-1
ε x 10-3 / L.mol-1.cm-1
120
100
80
60
40
20
0
120
100
80
60
40
20
0
300
400
500
600
15
20
25
30
λ / nm
ν x 10-3 / cm-1
ν1-ν2 (cm-1
)
Area x 10-8
1.57
λ1-λ2 (nm)
X
20408
16234
19802
20000
31056
24155
26738
26455
(490-322)
(616-414)
(505-374)
(500-378)
4
1.54
15
14
1.43
1.79
Figure 5. (A) UV–visible absorption spectra of dipyrrinone TBA adduct 4 (magenta), BA adduct 8 (red), Meldrum’s acid adduct 10 (blue) and monopyrrole
TBA adducts 14a (green) and 15a (yellow) in CHCl3 atw10ꢀ5 M concentrations. (B) Comparison of UV–visible absorption spectra of monopyrrole–TBA con-
jugates 14a (green) and 15a (yellow), dipyrrinone–TBA adduct 4 (magenta) and methyl xanthobilirubinate (X, an analog of 5 (yellow-green)) in methanol. The
R
integrated absorption coefficients in L molꢀ1 cmꢀ2, defined by v2 3 dv, are shown below the graph B.
v1

S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
8973
and 15 in (CH3)2SO are yellow (somewhat deeper yellow
than that found in a typical dipyrrinone), but 15 is a dark,
red-orange solid and 14 is a yellow solid.
A combination of heteronuclear multiple bond correlation
(HMBC) spectra and 1H{1H} NOE data were used to assign
1H and 13C NMR spectra. UV–visible spectra were recorded
on a Perkin–Elmer Lambda-12 spectrophotometer. Melting
points were taken on a Mel Temp capillary apparatus and
are corrected. Combustion analyses were carried out by Des-
ert Analytics, Tucson, AZ. Analytical thin layer chromato-
graphy was carried out on J. T. Baker silica gel IB-F plates
(125 m layers). Radial chromatography was carried out on
Merck silica gel PF254 with gypsum preparative layer grade,
using a Chromatotron (Harrison Research, Palo Alto, CA).
Spectral data were obtained in spectral grade solvents
(Aldrich or Fisher). Deuterated chloroform and dimethyl
sulfoxide were from Cambridge Isotope Laboratories.
(4Z)-3,8-Diethyl-2,7,9-trimethyl-10H-dipyrrin-1-one (kryp-
topyrromethenone)8c,9 and etiobiliverdin-IVg (1),8 (4Z)-
3-ethyl-8-(5-carbomethoxypentyl)-2,7,9-trimethyl-10H-di-
pyrrin-1-one,8b mesobiliverdin-XIIIa-8,12-dihexanoic acid
dimethyl ester (2),8b 3,4-diethyl-5-methylpyrrole-2-carbal-
dehyde,17 and 2,5-dimethylpyrrole-3-carbaldehyde18 were
prepared as described in the literature. Barbituric, 1,3-dime-
thylbarbituric, thiobarbituric, and 1,3-diethylthiobarbituric
acids were from Aldrich and used as received, Meldrum’s
acid was synthesized according to a literature procedure,28
and biliverdin-IXa dimethyl ester was obtained by esterifi-
cation of bilirubin followed by oxidation.2
Anticipating an absorption band near 450 nm, we found it
near 460 nm for 14 and near 430 nm for 15. Strikingly, how-
ever, the 3 value of this band in 14 exceeded 100,000, while
that from 15 was close to 40,000 (Fig. 5). The 460 nm band
of 14 is very sharp and very intense, e.g., 14a in methanol
3¼130,200 (456 nm). In contrast, 15a in methanol has
3¼44,000 (428 nm), an 3 value that is still higher than
from a typical dipyrrinone. The observed strong intensity
of 14 is very much related to its narrow shape. In comparing
the integrated intensities of 14 and 15, and also those of the
dipyrrinone adduct 4 and xanthobilirubinic acid methyl es-
ter, one finds nearly identical values, with that of 14 being
onlyw10% higher. Thus, the observed difference in 3 values
due to TBA attachment to an a- versus a b-pyrrole position
in 14 versus 15 is clarified; the dipole strengths are actually
quite similar.
In contrast to the long wavelength band in 14 or 15, which is
shifted byw100 nm from that of the dipyrrinone adducts of
TBA, the shorter wavelength band lies (in 14) at nearly the
same wavelength (near 325 nm) but is shrunk considerably
in intensity, down to 3 w6000. Apparently the intensity of
the last is very much related to the presence of the dipyrri-
none unit, as is the long wavelength band hypsochromic shift
ofw100 nm.
4.2. General procedure for biliverdin cleavage
To a solution of 249 mg (0.5 mmol) of etiobiliverdin-IVg
(1)8 in 60 mL of methanol (obtained within 45–60 min)
was added a solution of 1.2 mmol of carbon acid (thiobarbi-
turic acid, TBA; diethyl TBA; barbituric acid, BA; dimethyl
BA) in 60 mL of methanol, and the mixture was stirred at
25 ꢁC for 1.5–2.5 h. Then the solvent was evaporated under
vacuum (rotovap), and the residue was purified by radial
chromatography. In reactions using TBA and diethyl TBA,
radial chromatography was performed after filtration of the
magenta-colored, partially insoluble adducts. The adduct
from reaction of the verdin with BA was separated by filtra-
tion, and after evaporation the filtrate was chromatographed
to yield 9-H dipyrrinones. Using the same stoichiometry, re-
action conditions and work-up, verdin 2 and biliverdin
dimethyl ester were reacted and worked up similarly.
3. Concluding comments
The current work elaborates on the mechanism and useful-
ness of the smooth cleavage of biliverdins by selected carbon
acids to 9-H dipyrrinones and the carbon acid (Knovenagel)
€
adducts of 9-CHO dipyrrinones. The reaction is improved by
changes in solvent from the originally used ethyl acetate to
methanol or DMSO and by choosing from among thiobarbi-
turic acid (TBA), diethyl TBA, barbituric acid (BA), di-
methyl BA, and Meldrum’s acid—depending on the
requirements of product isolation. For example, it is easy
to isolate the adduct in high yield (>90%) when BA in
CH3OH is used to cleave the verdin, as the product precipi-
tates almost entirely and in high purity (>95%) from the re-
action mixture. From this carbon acid, the 9-H dipyrrinone
may be isolated relatively easily and in high yield by chro-
matography of the mother liquor. The adduct, which shows
tight intramolecular hydrogen bonding between the dipyrri-
none pyrrole NH and a proximal C]O of the carbon acid,
4.2.1. 3,8-Diethyl-2,7-dimethyl-(10H)-dipyrrin-1-one (3).
Neokryptopyrromethenone (3) was isolated (eluant CH2Cl2/
AcOH/CH3OH¼100:2:1 to 100:2:3) in 76% yield following
cleavage of etiobiliverdin-IVg (1). It had mp 202–204 ꢁC af-
ter crystallization from CH3OH/CH2Cl2 (lit. mp 197 ꢁC11);
1H NMR ((CD3)2SO) d: 1.08 (3H, t, J¼7.6 Hz), 1.09 (3H,
t, J¼7.6 Hz), 1.77 (3H, s), 2.02 (3H, s), 2.33 (2H, q,
J¼7.6 Hz), 2.49 (2H, q, J¼7.6 Hz), 5.95 (1H, s), 6.71 (1H,
d, J¼2.5 Hz), 9.69 (1H, s), 10.46 (1H, s) ppm; 13C NMR
((CD3)2SO) d: 8.0, 9.0, 14.6, 14.8, 17.1, 18.0, 97.7, 118.8,
121.0, 123.4, 123.8, 125.3, 128.5, 147.3, 172.0 ppm; NMR
data in CDCl3 are in Table 1. Anal. Calcd for C15H20N2O
(244.3): C, 73.73; H, 8.25; N, 11.47. Found: C, 73.45; H,
8.13; N, 11.64.
€
will undergo a retro-Knovenagel reaction, from which a
9-CHO dipyrrinone may be isolated in 26–66% yield.
4. Experimental section
4.1. General procedures
Nuclear magnetic resonance (NMR) spectra were obtained
on a Varian Unity Plus 500 MHz spectrometer in CDCl3
solvent (unless otherwise specified) at 25 ꢁC. Chemical
shifts were reported in d ppm referenced to the residual
CHCl3 1H signal at 7.26 ppm and 13C signal at 77.0 ppm.
4.2.2. 3,8-Diethyl-2,7-dimethyl-9-(40,60-dioxo-20-thioxo-
(10H,30H,50H)-pyrimidin-50-ylidene)methyl-(10H)-dipyr-
rin-1-one (4). This pigment adduct was obtained (eluant

8974
S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
CH2Cl2/AcOH/CH3OH ¼100:2:1 to 100:2:3) in 87% yield
from 1. It had mp 334–336 ꢁC after crystallization from
5.95 (1H, s), 8.15 (1H, br s), 8.24 (1H, s), 14.35 (1H, br s)
1
ppm; H NMR ((CD3)2SO) d: 1.12 (3H, t, J¼7.6 Hz), 1.13
1
EtOAc/hexane; H NMR d: 1.17 (3H, t, J¼7.6 Hz), 1.21
(3H, t, J¼7.6 Hz), 1.20 (3H, t, J¼7.0 Hz), 1.24 (3H, t, J¼
7.0 Hz), 1.85 (3H, s), 2.14 (3H, s), 2.58 (2H, q, J¼7.6 Hz),
2.69 (2H, q, J¼7.6 Hz), 4.45 (2H, q, J¼7.0 Hz), 4.50 (2H, q,
J¼7.0 Hz), 6.21 (1H, s), 8.02 (1H, s), 9.92 (1H, s), 13.36 (1H,
s) ppm; 13C NMR d: 8.4, 9.2, 12.3, 12.4, 14.4, 16.1, 18.0, 18.1,
43.8, 44.0, 93.8, 104.5, 126.0, 129.3, 130.9, 135.7, 140.3,
141.0, 145.6, 147.4, 161.9, 162.1, 172.6, 177.9 ppm; 13C
NMR ((CD3)2SO) data are in Table 2. Anal. Calcd for
C24H30N4O3S (454.6): C, 63.41; H, 6.65; N, 12.33. Found:
C, 63.39; H, 6.83; N, 12.33.
(3H, t, J¼7.6 Hz), 1.98 (3H, s), 2.16 (3H, s), 2.55 (2H, q,
J¼7.6 Hz), 2.71 (2H, q, J¼7.6 Hz), 5.94 (1H, s), 8.00 (1H,
s), 8.37 (1H, br s), 9.60 (1H, br s), 9.65 (1H, br s), 14.08
(1H, br s) ppm; 1H NMR ((CD3)2SO) d: 1.11 (3H, t,
J¼7.6 Hz), 1.12 (3H, t, J¼7.6 Hz), 1.85 (3H, s), 2.13 (3H,
s), 2.57 (2H, q, J¼7.6 Hz), 2.67 (2H, q, J¼7.6 Hz), 6.16
(1H, s), 7.92 (1H, s), 9.93 (1H, s), 12.23 (1H, s), 12.36
(1H, s), 13.45 (1H, s) ppm; 13C NMR ((CD3)2SO) data are
in Table 2. Anal. Calcd for C20H22N4O3S (398.5): C,
60.28; H, 5.57; N, 14.06. Found: C, 59.96; H, 5.69; N, 13.91.
4.2.6. 3,8-Diethyl-2,7-dimethyl-9-(20,40,60-trioxo-(10H,
30H,50H)-pyrimidin-50-ylidene)methyl-(10H)-dipyrrin-1-
one (8). Obtained in 94% yield from 1 and barbituric acid,
this pigment adduct had mp 347–349 ꢁC (dec) after crys-
tallization from (CH3)2SO/CH3OH+CHCl3; 1H NMR
((CD3)2SO) d: 1.109 (3H, t, J¼7.6 Hz), 1.114 (3H, t, J¼
7.6 Hz), 1.84 (3H, s), 2.12 (3H, s), 2.56 (2H, q, J¼7.6 Hz),
2.66 (2H, q, J¼7.6 Hz), 6.13 (1H, s), 7.96 (1H, s), 9.81
(1H, s), 11.09 (1H, s), 11.25 (1H, s), 13.45 (1H, s) ppm;
13C NMR ((CD3)2SO) data are in Table 2. Anal. Calcd for
C20H22N4O4 (382.4): C, 62.81; H, 5.80; N, 14.65. Calcd
for C20H22N4O4$1/4H2O (386.9): C, 62.08; H, 5.86; N,
14.48. Found: C, 62.34; H, 6.12; N, 14.62.
4.2.3. 2,7-Dimethyl-3-ethyl-8-(5-methoxycarbonylpen-
tyl)-(10H)-dipyrrin-1-one (5). Neodipyrrinone 5 was iso-
lated (eluant CHCl3/CH2Cl2/AcOH/CH3OH¼50:50:2:1 to
50:50:2:2.5) in 76% yield from cleavage of mesobiliver-
din-XIIIa-bis-hexanoate (2). It had mp 129–130 ꢁC (from
CH3OH/CH2Cl2); 1H NMR ((CD3)2SO) d: 1.08 (3H, t,
J¼7.6 Hz), 1.29 (2H, m), 1.46 (2H, m), 1.54 (2H, m), 1.77
(3H, s), 2.02 (3H, s), 2.28 (2H, t, J¼7.5 Hz), 2.31 (2H, t, J¼
7.6 Hz), 2.50 (2H, q, J¼7.6 Hz), 3.57 (3H, s), 5.95 (1H, s),
6.72 (1H, d, J¼2.8 Hz), 9.70 (1H, s), 10.48 (1H, s) ppm;
13C NMR ((CD3)2SO) d: 8.0, 9.1, 14.8, 17.1, 24.3, 24.6,
28.3, 29.5, 33.2, 51.1, 97.7, 119.5, 121.2, 123.3, 123.4,
1
123.7, 128.5, 147.3, 172.0, 173.3 ppm; H and 13C NMR
data in CDCl3 are in Table 1. Anal. Calcd for C20H28N2O3
(344.4): C, 69.74; H, 8.19; N, 8.13. Found: C, 70.00; H,
8.19; N, 8.40.
4.2.7. 3,8-Diethyl-2,7-dimethyl-9-(10,30-dimethyl-20,40,60-
trioxo-(10H,30H,50H)-pyrimidin-50-ylidene)methyl-
(10H)-dipyrrin-1-one (9). Isolated (eluant CH2Cl2/
CH3OH¼100:0.5 to 100:1.5) in 88% yield from 1, this pig-
ment adduct had mp 233–234 ꢁC after crystallization from
4.2.4. 2,7-Dimethyl-3-ethyl-8-(5-methoxycarbonylpen-
tyl)-9-(40,60-dioxo-20-thioxo-(10H,30H,50H)-pyrimidin-50-
ylidene)methyl-(10H)-dipyrrin-1-one (6). Isolated (eluant
CHCl3/CH2Cl2/AcOH/CH3OH¼50:50:2:1 to 50:50:2:2.5)
in 95% yield from 2, the desired adduct pigment (6) had
mp 223–225 ꢁC after crystallization from EtOAc/hexane;
1H NMR d 1.21 (3H, t, J¼7.6 Hz), 1.36 (2H, m), 1.52 (2H,
m), 1.64 (2H, m), 1.94 (3H, s), 2.15 (3H, s), 2.30 (2H, t, J¼
7.4 Hz), 2.54 (2H, q, J¼7.6 Hz), 2.66 (2H, t, J¼7.4 Hz), 3.66
(3H, s), 5.94 (1H, s), 7.90 (1H, s), 8.95 (1H, br s), 10.24 (1H,
1
EtOAc/hexane; H NMR d: 1.18 (3H, t, J¼7.6 Hz), 1.20
(3H, t, J¼7.6 Hz), 1.95 (3H, s), 2.15 (3H, s), 2.55 (2H, q,
J¼7.6 Hz), 2.73 (2H, q, J¼7.6 Hz), 3.39 (3H, s), 3.43 (3H,
s), 5.94 (1H, s), 8.06 (1H, br s), 8.24 (1H, s), 14.13 (1H, br
s) ppm; 13C NMR d: 8.5, 9.2, 14.4, 16.2, 17.9, 18.0, 28.7,
28.8, 93.9, 103.2, 125.35, 129.1, 129.7, 135.6, 138.9, 140.1,
145.0, 147.4, 151.5, 163.7, 164.4, 172.5 ppm; 1H NMR
((CD3)2SO) d: 1.12 (6H, t, J¼7.6 Hz), 1.85 (3H, s), 2.12
(3H, s), 2.57 (2H, q, J¼7.6 Hz), 2.67 (2H, q, J¼7.6 Hz),
3.22 (3H, s), 3.26 (3H, s), 6.16 (1H, s), 8.04 (1H, s), 9.84
(1H, s), 13.30 (1H, s) ppm; 13C NMR ((CD3)2SO) data are
in Table 2. Anal. Calcd for C22H26N4O4 (410.5): C, 64.37;
H, 6.39; N, 13.65. Found: C, 64.37; H, 6.20; N, 13.57.
1
br s), 10.59 (1H, br s), 13.89 (1H, br s) ppm; H NMR
((CD3)2SO) d: 1.11 (3H, t, J¼7.6 Hz), 1.31 (2H, m), 1.48
(2H, m), 1.55 (2H, m), 1.85 (3H, s), 2.12 (3H, s), 2.28 (2H,
t, J¼7.3 Hz), 2.57 (2H, q, J¼7.6 Hz), 2.65 (2H, t, J¼7.4 Hz),
3.56 (3H, s), 6.16 (1H, s), 7.91 (1H, s), 9.93 (1H, s), 12.23
(1H, s), 12.36 (1H, s), 13.46 (1H, s) ppm; 13C NMR d: 8.7,
9.5, 14.3, 17.9, 24.6, 24.7, 28.9, 31.2, 33.9, 51.5, 94.1, 102.6,
126.9, 130.5, 131.4, 133.6, 142.4, 142.5, 144.5, 147.3, 163.0,
163.2, 173.7, 174.0, 175.4 ppm; 13C NMR ((CD3)2SO)
data are in Table 2. Anal. Calcd for C25H30N4O5S (498.6):
C, 60.22; H, 6.07; N, 11.24. Found: C, 59.94; H, 5.83; N,
11.16.
4.3. 3,8-Diethyl-2,7-dimethyl-9-(20,20-dimethyl-40,60-di-
oxo-10,30-dioxan-50-ylidene)methyl-(10H)-dipyrrin-1-
one (10)
To a solution of 249 mg (0.5 mmol) etiobiliverdin-IVg (1) in
55 mL of methanol were added a solution of 432 mg
(1.5 mmol) 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s
acid28) in 20 mL of methanol and 0.23 mL (1.5 mmol) of
DBU, and the mixturewas heated at reflux for 5 h. After cool-
ing, the mixture was diluted with 200 mL of CH2Cl2 and
washed with 0.5% aq HCl (70 mL) and water (3ꢃ100 mL).
After drying (Na2SO4), filtration, and evaporation of the
solvent, the residue was purified by radial chromatography
on silica gel using as gradient CH2Cl2/CHCl3/CH3OH¼
80:20:0.5 to 80:20:3. Crystallization from ethyl acetate/
hexane gave 91 mg (46%) of 10 as a purple solid. It had mp
228–229 ꢁC (dec); 1H NMR d: 1.18 (3H, t, J¼7.6 Hz), 1.20
4.2.5. 3,8-Diethyl-2,7-dimethyl-9-(10,30-diethyl-40,60-dioxo-
20-thioxo-(10H,30H,50H)-pyrimidin-50-ylidene)methyl-
(10H)-dipyrrin-1-one (7). Obtained (eluant CH2Cl2/
CH3OH¼100:0.5 to 100:2) in 92% yield from 1, this adduct
pigment had mp 206–207 ꢁC after crystallization from
EtOAc/hexane; 1H NMR d: 1.20 (3H, t, J¼7.6 Hz), 1.21 (3H,
t, J¼7.6 Hz), 1.33 (3H, t, J¼7.0 Hz), 1.35 (3H, t, J¼7.0 Hz),
1.93 (3H, s), 2.16 (3H, s), 2.55 (2H, q, J¼7.6 Hz), 2.75 (2H, q,
J¼7.6 Hz), 4.60 (2H, q, J¼7.0 Hz), 4.67 (2H, q, J¼7.0 Hz),

S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
8975
(3H, t, J¼7.6 Hz), 1.77 (6H, s), 1.96 (3H, s), 2.14 (3H, s), 2.54
(2H, q, J¼7.6 Hz), 2.72 (2H, q, J¼7.6 Hz), 5.91 (1H, s), 7.56
(1H, br s), 8.15 (1H, s), 13.33 (1H, br s) ppm; 13C NMR d: 8.6,
9.2, 14.4, 16.0, 17.90, 17.92, 27.1, 93.5, 97.5, 104.1, 125.0,
128.7, 129.3, 136.0, 139.2, 140.1, 145.3, 147.3, 164.9,
165.6, 172.1 ppm; 13C NMR data from (CD3)2SO are in
Table 2. Anal. Calcd for C22H26N2O5: C, 66.31; H, 6.58;
N, 7.03. Found: C, 65.79; H, 6.69; N, 6.93.
4.4.3. 2,5-Dimethyl-3-(40,60-dioxo-20-thioxo-(10H,30H,50H)-
pyrimidin-50-ylidene)methyl-(1H)-pyrrole (15a). Isolated
from reaction of 2,5-dimethylpyrrole-3-carbaldehyde with
TBA in 92% yield, the yellow pigment had mp 286–288 ꢁC
1
(dec) after crystallization from CH3OH; H NMR d: 2.27
(3H, d, J¼0.9 Hz), 2.57 (3H, s), 7.40 (1H, br s), 8.44 (1H,
4
s), 8.53 (1H, v br s), 8.77 (1H, br s), 8.83 (1H, br s) ppm; 1H
4
NMR ((CD3)2SO) d: 2.16 (3H, d, J¼0.9 Hz), 2.43 (3H, s),
7.25 (1H, br q), 8.19 (1H, s), 11.87 (1H, s), 11.94 (1H, br s),
11.99 (1H, s) ppm; 13C NMR ((CD3)2SO) d: 11.4, 12.4, 105.8,
110.7, 119.3, 129.9, 147.1, 147.9, 160.4, 163.3, 177.6 ppm.
Anal. Calcd for C11H11N3O2S (249.3): C, 53.00; H, 4.45;
N, 16.86. Calcd for C11H11N3O2S$1/4H2O (253.8): C,
52.06; H, 4.57; N, 16.56. Found: C, 52.19; H, 4.74; N, 16.46.
4.4. General procedure for condensation of pyrrolecarb-
aldehydes with thiobarbituric acid
To a solution of 2.0 mmol of pyrrolecarbaldehyde in 7 mL of
ethanol was added a solution of 2.1 mmol of TBA or diethyl
TBA in 23 mL of ethanol, and the mixture was stirred at
55 ꢁC for 3 h. About 10–13 mL of the solvent was removed
during the last 1 h of the reaction by blowing a slow stream
of nitrogen close to the surface. The mixture was cooled;
then 3 mL of water was added slowly, and the mixture was
chilled at ꢀ15 ꢁC for 1 h. The precipitated product was col-
lected by filtration and purified by two recrystallizations
from methanol (with dichloromethane co-solvent) to obtain
a clear solution, which was partially evaporated by boiling to
remove CH2Cl2.
4.4.4. 2,5-Dimethyl-3-(10,30-diethyl-40,60-dioxo-20-thioxo-
(10H,30H,50H)-pyrimidin-50-ylidene)-methyl-(1H)-pyr-
role (15b). The desired compound was isolated (eluant
CH2Cl2/CH3OH¼100:0.3 to 100:0.5) following reaction of
2,5-dimethylpyrrole-3-carbaldehyde with diethyl TBA in
83% yield. It had mp 198–200 ꢁC (dec) after crystallization
1
from EtOAc/hexane; H NMR (CDCl3) d: 1.31 (3H, t, J¼
4
7.0 Hz), 1.32 (3H, t, J¼7.0 Hz), 2.26 (3H, d, J¼1.2 Hz),
2.53 (3H, s), 4.57 (2H, q, J¼7.0 Hz), 4.59 (2H, q, J¼
7.0 Hz), 7.40 (1H, br q), 8.50 (1H, s), 8.61 (1H, br s) ppm;
1H NMR ((CD3)2SO) d: 1.18 (3H, t, J¼7.0 Hz), 1.19 (3H,
4.4.1. 3,4-Diethyl-5-methyl-(40,60-dioxo-20-thioxo-
(10H,30H,50H)-pyrimidin-50-ylidene)methyl-(1H)-pyrrole
(14a). This product was obtained from 3,4-diethyl-5-meth-
ylpyrrole-2-carbaldehyde and TBA in 91% yield as an or-
ange solid; mp 323–325 ꢁC (dec) (from CH3OH/CH2Cl2);
1H NMR d: 1.12 (3H, t, J¼7.6 Hz), 1.21 (3H, t, J¼7.6 Hz),
2.45 (3H, s), 2.48 (2H, q, J¼7.6 Hz), 2.73 (2H, q, J¼7.6 Hz),
8.06 (1H, s), 8.89 (1H, br s), 9.22 (1H, br s), 13.45 (1H, br s)
4
t, J¼7.0 Hz), 2.18 (3H, d, J¼0.9 Hz), 2.46 (3H, s), 4.41
(2H, q, J¼7.0 Hz), 4.42 (2H, q, J¼7.0 Hz), 7.28 (1H, br),
8.32 (1H, s), 12.06 (1H, br s) ppm; 13C NMR d: 12.2, 12.4,
12.5, 12.8, 43.2, 43.8, 108.1, 111.3, 120.1, 129.5, 146.2,
150.4, 159.6, 162.6, 179.1 ppm; 13C NMR ((CD3)2SO) d:
11.5, 12.2, 12.28, 12.33, 42.3, 49.9, 105.3, 110.8, 119.9,
130.3, 149.07, 149.08, 158.7, 161.7, 178.3 ppm. Anal. Calcd
for C15H19N3O2S (305.4): C, 58.99; H, 6.27; N, 13.76.
Found: C, 59.05; H, 6.10; N, 13.78.
1
ppm; H NMR ((CD3)2SO) d: 1.05 (3H, t, J¼7.6 Hz), 1.12
(3H, t, J¼7.6 Hz), 2.41 (3H, s), 2.44 (2H, q, J¼7.6 Hz),
2.64 (2H, q, J¼7.6 Hz), 7.88 (1H, s), 12.16 (1H, s), 12.20
(1H, s), 13.43 (1H, s) ppm; 13C NMR ((CD3)2SO) d: 12.6,
15.1, 16.8, 17.3, 17.4, 101.3, 126.4, 129.7, 133.3, 145.2,
146.9, 162.90, 162.94, 176.9 ppm. Anal. Calcd for
C14H17N3O2S (291.4): C, 57.71; H, 5.88; N, 14.42. Found:
C, 57.51; H, 5.89; N, 14.04.
4.5. Condensation of o-tolualdehyde with thiobarbituric
acid
A mixture of 3.0 mmol TBA or diethyl TBA, 3.5 mmol of
freshly purified o-tolualdehyde, 4 mL of acetic acid, and
0.4 mL of acetic anhydride was heated under N2 at gentle re-
flux for 2 h. When TBAwas used, after cooling, the reaction
mixture was diluted with anhyd Et2O (3 mL), and the prod-
uct was separated by filtration. When diethyl TBAwas used,
the diethyl TBA product was extracted with CHCl3 after
diluting with H2O, and purified by radial chromatography
(eluant CH2Cl2/CH3OH¼100:0.25) and recrystallized from
ethyl acetate/hexane.
4.4.2. 3,4-Diethyl-5-methyl-(10,30-diethyl-40,60-dioxo-20-
thioxo-(10H,30H,50H)-pyrimidin-50-ylidene)methyl-(1H)-
pyrrole (14b). Obtained from 3,4-diethyl-5-methylpyrrole-
2-carbaldehyde and diethyl TBA (eluant CH2Cl2/CH3OH¼
100:0.25) in 91% yield, this yellow pigment had mp 163–
164.5 ꢁC after crystallization from EtOAc/hexane; 1H NMR
d: 1.11 (3H, t, J¼7.6 Hz), 1.20 (3H, t, J¼7.6 Hz), 1.31 (3H, t,
J¼7.0 Hz), 1.32 (3H, t, J¼7.0 Hz), 2.43 (3H, s), 2.47 (2H, q,
J¼7.6 Hz), 2.72 (2H, q, J¼7.6 Hz), 4.58 (2H, q, J¼7.0 Hz),
4.62 (2H, q, J¼7.0 Hz), 8.20 (1H, s), 13.54 (1H, br s) ppm;
1H NMR ((CD3)2SO) d: 1.08 (3H, t, J¼7.6 Hz), 1.16 (3H, t,
J¼7.6 Hz), 1.22 (6H, br), 2.45 (3H, s), 2.47 (2H, q,
J¼7.6 Hz), 2.68 (2H, q, J¼7.6 Hz), 4.45 (2H, br), 4.51
(2H, br), 8.03 (1H, s), 13.22 (1H, br s) ppm; 13C NMR d:
12.3, 12.5, 13.1, 15.1, 17.3, 17.4, 18.0, 43.4, 43.9, 101.9,
127.8, 130.0, 136.1, 146.2, 146.6, 161.8, 162.3, 178.2 ppm;
13C NMR ((CD3)2SO) d: 11.8, 11.9, 12.4, 14.4, 16.4, 16.6,
17.1, 42.4, 42.7, 100.8, 126.7, 129.6, 134.4, 145.7, 147.4,
160.8, 160.9, 177.4 ppm. Anal. Calcd for C18H25N3O2S
(347.5): C, 62.22; H, 7.25; N, 12.09. Found: C, 62.42; H,
7.22; N, 12.19.
4.5.1. 5-(2-Methylphenyl)methylidene-4,6-dioxo-2-thi-
oxo-(1H,3H,5H)-pyrimidine (16a). This yellow conjugate
was isolated in 89% yield. It had mp 255–257 ꢁC (lit.29
1
mp 200 ꢁC); H NMR ((CD3)2SO) d: 2.29 (3H, s), 7.19
(1H, m), 7.26 (1H, m), 7.35 (1H, m), 7.64 (1H, d, J¼
6.9 Hz), 8.41 (1H, s), 12.25 (1H, s), 12.44 (1H, s) ppm;
13C NMR ((CD3)2SO) d: 19.7, 120.4, 124.8, 129.6, 130.2,
130.8, 132.9, 138.0, 154.4, 159.0, 161.3, 178.8 ppm.
4.5.2. 5-(2-Methylphenyl)methylidene-1,3-diethyl-4,6-di-
oxo-2-thioxo-(1H,3H,5H)-pyrimidine (16b). This yellow
conjugate was isolated in 57% yield. It had mp 77–79 ꢁC
and 1H NMR d: 1.26 (3H, t, J¼7.0 Hz), 1.34 (3H, t,

8976
S. E. Boiadjiev, D. A. Lightner / Tetrahedron 63 (2007) 8962–8976
Grubmayr, K. J. Chem. Soc., Perkin Trans. 2 1979, 999–
ꢁ
J¼7.0 Hz), 2.39 (3H, s), 4.48 (2H, q, J¼7.0 Hz), 4.57 (2H, q,
J¼7.0 Hz), 7.24 (1H, m), 7.26 (1H, m), 7.38 (1H, m), 7.69
(1H, d, J¼7.6 Hz), 8.76 (1H, s) ppm; 13C NMR d: 12.38,
12.39, 20.3, 43.5, 44.0, 119.3, 125.2, 130.0, 130.1, 131.7,
132.9, 138.7, 157.9, 159.5, 160.3, 179.0 ppm.
1004; (c) Hori, A.; Mangani, S.; Pepe, G.; Meyer, E. F., Jr.;
Cullen, D. L.; Falk, H.; Grubmayr, K. J. Chem. Soc., Perkin
Trans. 2 1981, 1525–1528; (d) Huggins, M. T.; Lightner,
D. A. Monatsh. Chem. 2001, 132, 203–221; (e) Tipton,
A. K.; Lightner, D. A. Monatsh. Chem. 1999, 130, 425–440.
16. Kaplan, D.; Navon, G. J. Chem. Soc., Perkin Trans. 1 1981,
1374–1383.
Acknowledgements
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Kay, I. T. J. Chem. Soc. C 1966, 30–40.
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We thank the U.S. National Institutes of Health (HD 17779)
for generous support of this research. Dr. S.E.B. is on leave
from the Institute of Organic Chemistry, Sofia, Bulgaria.
19. Bijev, A. T.; Prodanova, P. P. Bulg. Chem. Commun. 2004, 36,
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