Synthesis of hipposudoric and norhipposudoric acids: The pigments responsible for the color reaction of the red sweat of Hippopotamus amphibius

Article abstract of DOI:10.1016/j.tetlet.2006.02.042

Highly unstable pigments, hipposudoric acid and norhipposudoric acid, isolated from the red sweat of hippopotamus were synthesized using the Pschorr reaction for the construction of the fluorene nucleus as the key step and the careful oxidation in the las

Full text of DOI:10.1016/j.tetlet.2006.02.042

Tetrahedron Letters 47 (2006) 2535–2538
Synthesis of hipposudoric and norhipposudoric acids:
the pigments responsible for the color reaction of the red sweat
of Hippopotamus amphibius
Yoko Saikawa, Kai Moriya, Kimiko Hashimoto*,ꢀ and Masaya Nakata*
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi,
Kohoku-ku, Yokohama 223-8522, Japan
Received 10 January 2006; revised 7 February 2006; accepted 9 February 2006
Available online 28 February 2006
Abstract—Highly unstable pigments, hipposudoric acid and norhipposudoric acid, isolated from the red sweat of hippopotamus
were synthesized using the Pschorr reaction for the construction of the fluorene nucleus as the key step and the careful oxidation
in the last step.
Ó 2006 Elsevier Ltd. All rights reserved.
Hippopotamuses secrete a viscous alkaline liquid over
their face and back. The colorless sweat (secretion) turns
red within a few minutes, then gradually becomes brown
polymers. The colored sweat has been said to protect the
hippopotamus’ skin from sunlight and from infection by
microbes.1 We focused on this color reaction of the
sweat, and recently isolated the red and orange pigments
responsible for the color reaction, determined their
structures, and partly clarified their functions.2 We desig-
nated the red and orange pigments, hipposudoric acid
(1) and norhipposudoric acid (2), respectively (Fig. 1).
In order to investigate in more detail the chemical and
biological properties of these scarcely available and
ephemeral compounds, we decided to synthesize these
pigments. The synthetic strategy is outlined in Scheme
1. The easily polymerizable target compounds 1 and 2
would be obtained by oxidation of the corresponding
hydroquinones 3 and 4 with spontaneous enolization.2
Each carboxylic acid would be available by the one-car-
bon elongation (for 4) or double one-carbon elongation
CO H
2
O
O
O
O
HO C
HO C
2
2
O
HO
O
HO
Hipposudoric acid (1)
Norhipposudoric acid (2)
Figure 1.
(for 3) of the key intermediate 5. Furthermore, the fluo-
renone skeleton of 5 would be constructed from 6 by
the Pschorr reaction.3 Thus the precursor of 6 was ana-
lyzed into 7 and 8.
The commercially available 2,5-dimethoxytoluene (7)
was brominated with Br2 in CH2Cl2 to afford the desired
substitution product, 4-bromo-2,5-dimethoxytoluene
(9)4 (Scheme 2). The other component, 3,6-dimethoxy-
2-nitrobenzaldehyde (10), was synthesized from the
commercially available 2,5-dimethoxybenzaldehyde (8)
by nitration with nitric acid,5 giving a 3:1 mixture of
the o- and p-nitro products, 10 and 11, respectively;6
from which the desired 10 was obtained by recrystalliza-
tion. The coupling reaction of the aryllithium derived
from bromide 9 and t-BuLi with aldehyde 10 in THF7
followed by oxidation using PCC in CH2Cl2 afforded
ketone 12 in excellent yield (96% for two steps). The nitro
group of 12 was reduced with iron in aqueous AcOH to
give amine 6 in 86% yield,8 which was oxidized to the
Keywords: Hippopotamus amphibius; Hipposudoric acid; Norhippo-
sudoric acid; Fluorene; Pschorr reaction.
*
Corresponding authors. Tel.: +81 75 595 4673; fax: +81 75 595 4763
(K.H.); tel.: +81 45 566 1577; fax: +81 45 566 1551 (M.N.); e-mail
addresses: kimikoh@mb.kyoto-phu.ac.jp; msynktxa@applc.keio.
ac.jp
ꢀ Present address: Kyoto Pharmaceutical University, 1 Shichono-cho,
Misasagi, Yamashina-ku, Kyoto 607-8412, Japan.
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.02.042

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Y. Saikawa et al. / Tetrahedron Letters 47 (2006) 2535–2538
R
R
O
O
HO
OH
oxidation
HO C
HO C
2
2
O
HO
OH HO
1: R = CO H
3: R = CO H
2
2
2: R = H
4: R = H
double one-carbon
elongation for 3
one-carbon
elongation
for 4
O
OMe O
OMe
OMe
MeO
Pschorr
reaction
H N
2
OMe
OMe
OMe OMe
6
5
OMe
OMe
OHC
+
OMe
OMe
8
7
Scheme 1. Retrosynthesis of hipposudoric acid (1) and norhipposudoric acid (2).
OMe
OMe
OMe
OMe
OMe
OHC
OHC
OHC
Br
a
b
+
NO
O N
2
2
OMe
OMe
OMe
OMe
OMe
11
7
9
8
10
O
OMe O
OMe
OMe
OMe O
OMe
OMe
MeO
OMe
c, d
f
e
9
H N
O N
X
2
2
OMe
OMe
OMe OMe
12
6
5: X = H
13: X = Br
g
Scheme 2. Synthesis of the key intermediate 13. Reagents and conditions: (a) Br2 (1.05 mol amt), CH2Cl2, rt, 10 h, 75%; (b) concd HNO3, 0 °C,
5 min, 57% of 10 (after recrystallization from hexane–CHCl3); (c) 9 (1.0 mol amt), t-BuLi (1.5 mol amt)/pentane, THF, À78 °C, 15 min, then 10
(0.8 mol amt), À78 °C to rt, 2 h; (d) PCC (1.6 mol amt), NaOAc, CH2Cl2, rt, 12 h, 96% (two steps); (e) Fe (3.6 mol amt), AcOH–H2O (9:1), 80 °C,
1 h, 86%; (f) isoamyl nitrite (2.0 mol amt), AcOH, rt, 0.5 h, then hydroquinone (1.2 mol amt)/acetone, rt, 1 h, 79%; (g) NBS (1.0 mol amt), BPO
(0.06 mol amt), benzene, reflux, 13 h, 92%. PCC = pyridinium chlorochromate, NBS = N-bromosuccinimide, BPO = benzoyl peroxide.
diazonium salt and then smoothly converted to the de-
sired fluorenone 5 (79%) by the modified Pschorr ring-
closing reaction promoted by hydroquinone.9 Benzylic
bromination of 5 with NBS in the presence of a catalytic
amount of BPO in benzene gave 13 in 92% yield as the
key intermediate for the synthesis of 1 and 2.
For the synthesis of hipposudoric acid (1), the double
one-carbon elongation on 13 was realized. The simulta-
neous substitution and addition of the cyano groups
using TMSCN afforded the cyanohydrin TMS ether 14
in 69% yield (Scheme 3).10,11 In this reaction, the cyanide
ion released from TMSCN and TBAF is used for the

Y. Saikawa et al. / Tetrahedron Letters 47 (2006) 2535–2538
2537
vis spectra.15 We examined the milder and more efficient
oxidation methods and found that the Cu2+-catalyzed
air oxidation16 was the more appropriate method
because of lower degree of polymerization. Interestingly,
the results of this oxidation depended on the pH of the
reaction medium. The oxidation of 3 under the basic
conditions (0.5 M aqueous NaHCO3, pH 8.3) afforded
1 in 35% yield from 15 along with the negligible yield
of 2.15 On the other hand, the same oxidation in an
acidic buffer solution (0.2 M phosphate buffer, pH 6.1)
led to the formation of 1 (8.4%) along with 2 (6.8%).15
The orange pigment 2 would occur due to the competi-
tive decarboxylative enolization after oxidation.
NC
R
O
OMe
MeO
MeO
OMe
a
Br
NC
b
OMe OMe
OMe OMe
13
14: R = OTMS
15: R = H
c
CO H
CO H
2
2
O
OH
O
HO
d
The other target molecule, norhipposudoric acid (2) (the
orange pigment), was also synthesized from the inter-
mediate 13 (Scheme 4). The carbonyl group of 13 was
reduced with Et3SiH in TFA17 to afford fluorene 16 in
79% yield. Substitution of the bromide with the cyanide
was performed by the standard method (NaCN, DMF)
to give benzylnitrile 17 in 80% yield. Hydrolysis of the
nitrile and demethylation of 17 were conducted using a
similar method as that in the case of 15, giving hydro-
quinone 4. This hydroquinone was also unstable; there-
fore, the crude product was immediately oxidized with
CuSO4 in 0.2 M phosphate buffer (pH 6.1) to produce
the orange solution. This solution was immediately sub-
jected to filtration through a cation exchange resin (CM
Sephadex, 0.2 M phosphate buffer, pH 6.1) followed by
anion exchange chromatography (QAE Sephadex,
2.3 M NaCl in 0.2 M phosphate buffer, pH 6.1) to afford
norhipposudoric acid (2) in 36% yield from 17 as a dilute
buffer solution.15 The analytical data of these synthe-
sized pigments were identical to those of the natural
products.
HO C
HO C
2
2
HO
HO
OH
O
Hipposudoric acid (1)
3
Scheme 3. Synthesis of hipposudoric acid (1). Reagents and condi-
tions: (a) 1 M TBAF in THF (1.6 mol amt), TMSCN (solvent,
18 mol amt), rt, 15 min, 69%; (b) Et3SiH (5.0 mol amt), BF3ÆEt2O
(5.0 mol amt), CH2Cl2, 0 °C, 1 h, 99%; (c) 48% aq HBr–AcOH (3:1),
reflux, 4 h; (d) CuSO4 (1.0 mol amt), 0.5 M aq NaHCO3, rt, 2 min,
35% (two steps). TBAF = tetrabutylammonium fluoride, TMS =
trimethylsilyl.
substitution reaction10 and also catalyzes the addition of
TMSCN to the ketone.11 The resulting cyanohydrin 14
was deoxygenated with Et3SiH and BF3ÆEt2O to quanti-
tatively give nitrile 15.12 Having the desired compound
with the appropriate skeleton, the synthesis reached its
final stage. Both hydrolysis of the two nitrile groups
and cleavage of the four methyl ethers were achieved
using aqueous HBr–AcOH to afford the highly unstable
1
hydroquinone 3. Even if H NMR analysis of the crude
product showed the quantitative formation of 3, purifi-
cation by SiO2 or ODS column chromatography
resulted in the decomposition of the product because of
its instability in air. Therefore, the crude product was
directly subjected to the last step. Oxidation of the two
hydroquinone groups under the usual conditions such
as FeCl3–HCl13 and Ag2O14 resulted in failure, giving
brown polymers. In some cases (FeCl3–MeOH, FeCl3–
MeCN), the desired oxidation should have proceeded
judging from the appearance of the red coloration of
the solution; however, the subsequent polymerization
was too fast to isolate the red compound. Simple dilute
conditions had no effect on retarding the polymeriza-
tion. After considerable efforts, we succeeded in this oxi-
dation using FeCl3 in a viscous solvent, glycerol–H2O
(10:1), in order to prevent the substrates from approach-
ing each other. To obtain a sufficient amount of 1, care-
ful treatment during the purification step was required.
The resulting reaction mixture was immediately filtered
through a cation exchange resin (CM Sephadex, 0.2 M
phosphate buffer, pH 6.1) to remove the iron ion. The
obtained red solution was subjected to anion exchange
chromatography similar to the purification of the natu-
ral product (QAE Sephadex, 1.7 M NaCl in 0.2 M phos-
phate buffer, pH 6.1)2 to afford highly unstable
hipposudoric acid (1) as a dilute buffer solution in 10%
In conclusion, the syntheses of hipposudoric acid (1)
and norhipposudoric acid (2) were performed by fea-
turing the Pschorr ring-closing reaction, cyanation to
O
OMe
MeO
MeO
OMe
a
Br
R
b
OMe OMe
13
OMe OMe
16: R = Br
17: R = CN
c
O
OH
O
HO
d
HO C
HO C
2
2
HO
HO
OH
O
Norhipposudoric acid (2)
4
Scheme 4. Synthesis of norhipposudoric acid (2). Reagents and
conditions: (a) Et3SiH (5.0 mol amt), TFA, 0 °C, 15 min, 79%; (b)
NaCN (1.8 mol amt), DMF, 50 °C, 0.5 h, 80%; (c) 48% aq HBr–AcOH
(3:1), reflux, 4 h; (d) CuSO4 (1.0 mol amt), 0.2 M phosphate buffer, rt,
2 min, 36% (two steps).
1
yield (from 15) judging from the H NMR and UV–

2538
Y. Saikawa et al. / Tetrahedron Letters 47 (2006) 2535–2538
´ ´
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and their analogs are now under way.
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Acknowledgments
This research was partially supported by a Grant-in-Aid
for Scientific Research (B) and a Grant-in-Aid for Scien-
tific Research on Priority Areas (A) ‘Targeted Pursuit of
Challenging Bioactive Molecules’ from the Ministry of
Education, Culture, Sports, Science and Technology,
Japan.
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