First synthesis of the naturally occurring diazocarbonyl compound cremeomycin

Article abstract of DOI:10.1055/s-0028-1083204

The first synthesis of cremeomycin (3-diazo-4-methoxy-2-oxocyclohexa-4,6- dienecarboxylic acid), a naturally occurring diazocarbonyl compound, is described. Starting from methyl 2-hydroxy-4-methoxybenzoate, nitration, hydrolysis, and reduction gave 3-amin

Full text of DOI:10.1055/s-0028-1083204

PAPER
3601
First Synthesis of the Naturally Occurring Diazocarbonyl Compound
Cremeomycin
First Synthesis
o
i
f
a
D
ia
s
zoquinon
a
e
N
atural
P
roductM. Varley, Christopher J. Moody*
School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK
Fax +44(115)9513564; E-mail: c.j.moody@nottingham.ac.uk
Received 7 July 2008
presence of the diazo group being confirmed by IR (2145
cm–1) and 13C NMR (diazo carbon at d = 84.5) spectrosco-
py. Perhaps not surprisingly, attempted hydrolysis of
methyl ester 14 to cremeomycin (9) itself was not success-
Abstract: The first synthesis of cremeomycin (3-diazo-4-methoxy-
2-oxocyclohexa-4,6-dienecarboxylic acid), a naturally occurring
diazocarbonyl compound, is described. Starting from methyl 2-hy-
droxy-4-methoxybenzoate, nitration, hydrolysis, and reduction
gave 3-amino-2-hydroxy-4-methoxybenzoic acid as the key ami- ful, and therefore the hydrolysis step was carried out ear-
nophenol precursor, diazotization of which gave the diazoquinone
natural product.
lier in the sequence. Thus, the nitro ester 12 was
hydrolyzed to the nitro acid 15, reduction of which gave
the corresponding amino acid 16 (Scheme 1). Finally, di-
azotization of aminophenol 16 gave, after workup and
chromatography on phosphate buffered silica gel, cre-
meomycin (9) as a bright yellow solid with spectroscopic
Key words: natural products, total synthesis, diazo compounds,
electrophilic aromatic substitution
Although diazocarbonyl compounds are probably best properties consistent with those reported for the natural
known for their involvement as versatile intermediates in product. In particular, the diazo group showed a strong IR
modern synthetic organic chemistry,1 a number of such absorbance at 2166 cm–1 and the diazo carbon at d = 84.2
compounds also occur naturally. Many of the early exam- in the 13C NMR spectrum, although the spectrometer re-
ples, originally isolated in the 1950s, have antitumor prop- laxation delay had to be increased to 10 seconds to ob-
erties and consist of modified a-amino acids. These
include azaserine (1),2–4 6-diazo-5-oxonorleucine (DON,
2),5,6 alazopeptin (3),7,8 duazomycins A and B (4 and 5),9–11
N-alanylazaserine (LL-D05139b, 6),12 and thrazarine
(FR900840, 7)13–16 (Figure 1). More recently, other diazo-
carbonyl compounds have been isolated from natural
sources including SQ30,957 (8),17 and the more complex
natural product lagunamycin.18,19 We now report the first
synthesis of another diazocarbonyl natural product, cre-
meomycin (9).
O
O
H
CO2H
H
CO2H
NH2
O
N2
NH2
N2
azaserine (1)
6-diazo-5-oxonorleucine (2)
(DON)
O
H
CO2H
O
O
H
CO2H
NHAc
N2
H2N
HN
O
N2
Cremeomycin (9) was originally described in 1967 as a
photodegradable antibiotic, isolated from Streptomyces
cremeus by the Upjohn Company, although no details of
the structure were reported.20 The unusual structure 9 of
the natural product was finally solved by Rinehart and co-
workers using X-ray crystallography some 28 years lat-
er.21 The compound is an example of a diazoquinone, also
known as quinone diazides,22 and as such should be avail-
able by diazotization of the appropriately substituted ami-
nophenol. The starting point for the synthesis was the
nitration of methyl 2-hydroxy-4-methoxybenzoate (10),23
that gave a mixture of the 5-nitro compound 11 (42%) to-
gether with the desired 3-nitro isomer 12 (23–32%). Re-
duction of 12 gave the corresponding amino ester 13, and
this was followed by diazotization to give cremeomycin
methyl ester 14 in modest yield. The diazoketone 14 was
an orange solid that quickly darkened on exposure to light,
but could be characterized spectroscopically, with the
N2
N
H
H
Me
O
alazopeptin (3)
duazomycin A (4)
O
O
H
CO2H
CO2H
H
H
O
Me
N2
HN
O
O
N2
N2
HN
NH2
HO2C
N
H
O
NH2
O
N-alanylazaserine (6)
(LL-D05139β)
duazomycin B (5)
O
OH
O
CO2H
NH2
O
MeO
N2
N2
thrazarine (7)
(FR900840)
SYNTHESIS 2008, No. 22, pp 3601–3604
x
x.
x
x
.
2
0
0
8
SQ30,957 (8)
Advanced online publication: 23.10.2008
DOI: 10.1055/s-0028-1083204; Art ID: P07508SS
© Georg Thieme Verlag Stuttgart · New York
Figure 1 Some naturally occurring diazocarbonyl compounds

3602
L. M. Varley, C. J. Moody
PAPER
mL) was added and the mixture was allowed to stand without stir-
ring for a further 30 min, after which the precipitate was filtered,
rinsed with small amounts of H2O and dried under vacuum to yield
a light brown solid, that was purified by chromatography on silica
gel (15:1 light petroleum–EtOAc, gradient to 100% EtOAc) to yield
methyl 2-hydroxy-4-methoxy-5-nitrobenzoate (11) as a light yel-
low solid (4.17 g, 42%) and the title compound 12 (2.28 g, 23%) as
a light yellow solid.
OH
CO2Me
MeO
OH
NO2
CO2Me
a
11
MeO
OH
11
O2N
CO2Me
10
Mp 156–158 °C (Lit.24 mp 156–157 °C).
1H NMR (400 MHz, CDCl3): d = 11.43 (1 H, s, OH), 8.58 (1 H, s,
6-H), 6.59 (1 H, s, 3-H), 4.00 (3 H, s, OCH3), 3.99 (3 H, s, OCH3).
MeO
12
13C NMR (100 MHz, CDCl3): d = 169.5 (C), 166.9 (C), 159.6 (C),
132.5 (C), 129.9 (CH), 105.1 (C), 101.4 (CH), 57.2 (CH3), 53.1
(CH3).
d
b
MS (ESI): m/z (%) = 228 (M + H+, 100%), 227 (30), 223 (39), 217
(26), 210 (26), 196 (26), 192 (26), 190 (27), 174 (25), 150 (27), 149
(52).
OH
OH
H2N
CO2Me
X
CO2H
HRMS-ESI: m/z calcd for C9H9NO6 + H [M + H]+: 228.0509;
MeO
MeO
13
found: 228.0506.
15 X = NO2
16 X = NH2
e
12
c
Mp 183–186 °C (Lit.25 mp 185–187 °C).
f
IR (CHCl3): 2958, 1682, 1627, 1583, 1541, 1509, 1441, 1377, 1333,
1284, 1240, 1183, 1160, 1144, 1098, 996, 890 cm–1.
O
O
N2
CO2Me
N2
CO2H
1H NMR (400 MHz, CDCl3): d = 11.41 (1 H, s, OH), 7.92 (1 H, d,
J = 9.1 Hz, 6-H), 6.57 (1 H, d, J = 9.1 Hz, 5-H), 3.98 (3 H, s,
OCH3), 3.97 (3 H, s, OCH3).
13C NMR (100 MHz, CDCl3): d = 169.1 (C), 156.0 (C), 154.2 (C),
132.3 (CH), 130.4 (C), 106.6 (C), 102.5 (CH), 56.4 (CH3), 52.4
(CH3).
MeO
MeO
9
14
Scheme 1 Reagents and conditions: (a) HNO3, Ac2O, AcOH (42%
of 11, 23–32% of 12); (b) H2, Pd/C, EtOH (51%); (c) NaNO2, aq HCl,
0 °C, then Na2CO3 (58%); (d) LiOH, aq THF (94%); (e) H2, Pd/C,
EtOH (81%); (f) NaNO2, aq HCl, 0 °C, then Na2CO3 (38%).
MS (ESI): m/z (%) = 250 (M + Na+, 11%), 197 (9), 196 (100), 149
(5).
HRMS-ESI: m/z calcd for C9H9NO6 + Na [M + Na]+: 250.0328;
found: 250.0320.
serve the diazo carbon. As reported for the natural
product, the EI mass spectrum showed a molecular ion
that lost CO2 and then N2.
When the reaction was repeated on a smaller scale (3.0 g of starting
material), the yield of 12 was 32%.
Methyl 3-Amino-2-hydroxy-4-methoxybenzoate (13)
Methyl 2-hydroxy-4-methoxy-3-nitrobenzoate (12; 0.20 g, 0.88
mmol) was hydrogenated under an atmosphere of H2 over 10%
Pd/C (0.10 g) in EtOH (20 mL) for 17 h. The resulting suspension
was filtered through Celite and rinsed with EtOH (3 × 20 mL) and
the solvent was removed to give a brown solid, which was purified
by column chromatography on silica gel (10:1 light petroleum–
EtOAc) to give the title compound (88 mg, 51%) as a light brown
solid; mp 72–75 °C (Lit.25 mp 72–74 °C).
Commercially available reagents were used throughout without pu-
rification unless otherwise stated. Light petroleum refers to the frac-
tion with bp 40–60 °C. Reactions were routinely carried out under
N2 or argon. Analytical TLC analyses were carried out on alumi-
num-backed plates coated with silica gel, and visualized under UV
light at 254 and/or 360 nm. Chromatography was carried out on sil-
ica gel unless otherwise stated. Fully characterized compounds are
chromatographically homogeneous. IR spectra were recorded in the
range of 4000–600 cm–1. NMR spectra were recorded at 400 and
500 MHz (1H frequencies, corresponding 13C frequencies at 100 and
125 MHz). Chemical shifts are quoted in ppm and are referenced to
residual H in the deuterated solvent as the internal standard. J values
are recorded in Hz. High- and low-resolution mass spectra were re-
corded on a time-of-flight mass spectrometer.
IR (CHCl3): 3457, 3371, 3193, 3011, 2956, 2912, 2844, 1670, 1624,
1603, 1577, 1508, 1461, 1440, 1396, 1312, 1291, 1253, 1193, 1147,
1071, 1020 cm–1.
1H NMR (400 MHz, CDCl3): d = 10.83 (1 H, s, OH), 7.30 (1 H, d,
J = 9.0 Hz, 6-H), 6.45 (1 H, d, J = 9.0 Hz, 5-H), 3.97 (6 H, s, 2 ×
OCH3), 3.84 (2 H, br s, NH2).
13C NMR (100 MHz, CDCl3): d = 170.9 (C), 151.5 (C), 149.5 (C),
124.1 (C), 119.2 (CH), 106.1 (C), 102.3 (CH), 55.8 (CH3), 52.0
(CH3).
MS (ESI): m/z (%) = 198 (M + H+, 8%), 194 (15), 167 (9), 166
(100).
HRMS-ESI: m/z calcd for C9H11NO4 + H [M + H]+: 198.0766;
found: 198.0749.
Methyl 2-Hydroxy-4-methoxy-3-nitrobenzoate (12)
Methyl 2-hydroxy-4-methoxybenzoate (10; 8.00 g, 43.92 mmol)
was dissolved in a mixture of glacial AcOH (57.6 mL) and Ac2O
(29.6 mL). After cooling the mixture to 0 °C, a mixture of fuming
HNO3 (70%; 4.3 mL, 48.32 mmol) in glacial AcOH (26.4 mL) was
added with stirring over 5 min. When addition was complete, the
light yellow solution was allowed to reach r.t. and was stirred for 25
min, after which a light brown suspension had formed. H2O (110
Synthesis 2008, No. 22, 3601–3604 © Thieme Stuttgart · New York

PAPER
First Synthesis of a Diazoquinone Natural Product
3603
Methyl 3-Diazo-4-methoxy-2-oxocyclohexa-4,6-dienecarboxy-
late (Cremeomycin Methyl Ester, 14)
13C NMR (100 MHz, DMSO-d6): d = 172.9 (C), 151.3 (C), 149.8
(C), 124.2 (C), 119.3 (CH), 106.7 (C), 103.0 (CH), 56.2 (CH3).
To a stirred solution of methyl 3-amino-2-hydroxy-4-methoxyben-
zoate (13; 77 mg, 0.39 mmol) in aq 2 M HCl (0.86 mL), cooled to
0 °C was slowly added a solution of NaNO2 (27 mg, 0.39 mmol) in
H2O (1.4 mL). The mixture was stirred at 0 °C with the exclusion of
light for 1.5 h, and then solid Na2CO3 was added to neutralize the
reaction. The resulting slurry was then reacidified to pH 2 with aq 2
M HCl and extracted with CH2Cl2 (3 × 10 mL). Drying (Na2SO4)
and removal of the solvent under reduced pressure yielded a bright
orange solid. Purification by chromatography on silica gel (100%
CH2Cl2, then 100:1 CH2Cl2–MeOH) yielded the title compound (47
mg, 58%) as a bright orange solid that quickly turned brown upon
exposure to light; mp 150–153 °C.
MS (ESI): m/z (%) = 182 (M – H, 100%), 181 (42), 175 (7), 167
(41), 123 (44), 122 (20).
HRMS-ESI: m/z calcd for C8H9NO4 – H [M – H]+: 182.0453; found:
182.0457.
Anal. Calcd for C8H9NO4: C, 52.5; H, 5.0; N, 7.7. Found: C, 52.2;
H, 4.8; N, 7.3.
3-Diazo-4-methoxy-2-oxocyclohexa-4,6-dienecarboxylic Acid
(Cremeomycin, 9)
To a stirred solution of 3-amino-2-hydroxy-4-methoxybenzoic acid
(16; 20 mg, 0.11 mmol) in aq 2 M HCl (1.0 mL), cooled to 0 °C was
slowly added a solution of NaNO2 (7.5 mg, 0.11 mmol) in H2O (0.2
mL). The mixture was stirred at 0 °C with the exclusion of light for
40 min, and then solid Na2CO3 was added to neutralize the reaction.
The resulting slurry was then reacidified to pH 2 with aq 2 M HCl
and extracted with CH2Cl2 (3 × 5 mL). Drying (Na2SO4) and remov-
al of the solvent under reduced pressure yielded a brown solid. Pu-
rification by chromatography on phosphate buffered silica gel
[prepared using KH2PO4 (2.45 g) and Na2HPO4 (0.024 g) in H2O for
silica gel (45 g), eluent: 100% CH2Cl2, then 99:1 CH2Cl2–MeOH)]
yielded the title compound (8 mg, 38%) as a bright yellow solid; mp
139–141 °C (Lit.20 mp 142–143 °C).
IR (CHCl3): 3010, 2953, 2145, 1720, 1694, 1623, 1566, 1526, 1445,
1424, 1336, 1295, 1278, 1193, 1171, 1104 cm–1.
1H NMR (400 MHz, CDCl3): d = 8.16 (1 H, d, J = 8.6 Hz, 6-H),
5.74 (1 H, d, J = 8.6 Hz, 5-H), 3.99 (3 H, s, OCH3), 3.88 (3 H, s,
OCH3).
13C NMR (100 MHz, CDCl3): d = 173.5 (C), 166.0 (C), 161.2 (CH),
116.9 (C), 92.7 (CH), 84.5 (C), 57.0 (CH3), 52.0 (CH3).
MS (EI): m/z (%) = 208 (M+, 100%), 182 (7), 177 (14), 165 (92),
149 (34), 137 (97), 109 (23).
HRMS-EI: m/z calcd for C9H8N2O4 + H [M]+: 208.0484; found:
208.0476.
IR (CHCl3): 3012, 2166, 1736, 1604, 1538, 1459, 1443, 1339, 1270,
1239, 1168, 1101, 966 cm–1.
1H NMR (500 MHz, CDCl3): d = 13.75 (1 H, s, OH), 8.37 (1 H, d,
J = 8.6 Hz, 6-H), 5.98 (1 H, d, J = 8.6 Hz, 5-H), 4.06 (3 H, s,
OCH3).
13C NMR (125 MHz, CDCl3): d = 177.2 (C), 165.9 (C), 162.0 (C),
146.5 (CH), 114.6 (C), 95.0 (CH), 84.2 (C), 57.6 (CH3).
MS (ESI): m/z (%) = 195 (M + H+, 8%), 177 (53), 153 (6), 150 (9),
149 (100), 139 (13), 134 (31).
HRMS-ESI: m/z calcd for C8H6N2O4 + H [M + H]+: 195.0406;
found: 195.0397.
UV (MeCN): lmax (log e) = 270 (3.10), 423 nm (2.76).
2-Hydroxy-4-methoxy-3-nitrobenzoic Acid (15)
To a solution of 12 (0.50 g, 2.20 mmol) dissolved in a mixture of
THF (100 mL) and H2O (25 mL) was added LiOH (2.90 g, 121.05
mmol). The resulting mixture was heated under reflux with stirring
for 18 h. After cooling to r.t., the mixture was acidified to pH 2 with
aq 2 M HCl (2 M), and then extracted with CH2Cl2 (3 × 50 mL). The
organic extracts were combined, dried (Na2SO4), and concentrated
in vacuo to yield the title compound (0.44 g, 94%) as a light yellow
solid; mp 210–211 °C.
UV (MeOH): lmax (log e) = 206 (3.73), 260 (3.14), 289 (3.04), 413
nm (3.04).
IR (CHCl3): 3690, 3606, 3507, 3012, 2928, 2855, 1688, 1602, 1542,
1508, 1458, 1374, 1302, 1240, 1181, 1159, 1126, 1098, 904 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 7.95 (1 H, d, J = 9.1 Hz, 6-H),
6.86 (1 H, d, J = 9.1 Hz, 5-H), 3.94 (3 H, s, OCH3).
13C NMR (100 MHz, DMSO-d6): d = 170.8 (C), 155.3 (C), 153.7
(C), 133.2 (CH), 130.2 (C), 107.6 (C), 103.6 (CH), 57.1 (CH3).
References
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MS (ESI): m/z (%) = 258 (M + Na2, 100%), 236 (M + Na, 30), 234
(37), 227 (35), 223 (64), 196 (60), 150 (47), 149 (36), 131 (31).
HRMS-ESI: m/z calcd for C8H7NO6 + Na [M + Na]+: 236.0171;
found: 236.0148.
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Synthesis 2008, No. 22, 3601–3604 © Thieme Stuttgart · New York

3604
L. M. Varley, C. J. Moody
PAPER
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Synthesis 2008, No. 22, 3601–3604 © Thieme Stuttgart · New York