Unusual intramolecular N→O acyl group migration occurring during conjugation of (-)-DHMEQ with cysteine

Article abstract of DOI:10.1016/j.bmcl.2009.07.123

Previously we found that (-)-DHMEQ, a specific NF-κB inhibitor, covalently bound to a specific cysteine of NF-κB component proteins. In the course of formation of the (-)-DHMEQ and protected cysteine conjugate, we observed an unusual intramolecular N→O ac

Full text of DOI:10.1016/j.bmcl.2009.07.123

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5380–5382
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Bioorganic & Medicinal Chemistry Letters
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Unusual intramolecular N?O acyl group migration occurring during
conjugation of (ꢀ)-DHMEQ with cysteine
Ikuko Kozawa a, Kuniki Kato a, Toshiaki Teruya b, Kiyotake Suenaga b, Kazuo Umezawa a,
*
a Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
b Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Previously we found that (ꢀ)-DHMEQ, a specific NF-
jB inhibitor, covalently bound to a specific cysteine
B component proteins. In the course of formation of the (ꢀ)-DHMEQ and protected cysteine con-
Received 13 June 2009
Revised 23 July 2009
Accepted 27 July 2009
Available online 30 July 2009
of NF-
j
jugate, we observed an unusual intramolecular N?O acyl group migration.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
(ꢀ)-DHMEQ
Cysteine
NF-jB
Bioconjugate
Rearrangement
The inducible transcription factor nuclear factor-kappa B (NF-
B) plays an important role in the regulation of immune, inflam-
matory, and carcinogenic responses.1 Whereas the normal activa-
tion of NF- is necessary for cell survival and immunity,
deregulated NF- B expression is a characteristic phenomenon in
inflammatory diseases and cancer development.2 Hence, NF-
N?O acyl group migration reaction observed in the course of the
formation of the DHMEQ–cysteine conjugate.
j
When DHMEQ (1) was treated with N-(tert-butoxycarbonyl)-L-
jB
cysteine methyl ester (Boc-Cys-Me 2) in DMF/0.1 M sodium phos-
phate buffer (1:1, pH 6.0) at room temperature, compound 311 was
obtained in 87% yield with complete consumption of 1 within
25 min (Scheme 1).8
j
jB
has become a major target in drug discovery, and several natural
and synthetic compounds have been investigated for their poten-
tial to inhibit NF-
published class of natural products cited as NF-
j
B.3 Sesquiterpene lactones are the most widely
OH
H
j
B inhibitors, and
MeO2C
HN
HO
6
1
HO
S
N
SH
Boc
OH
O
their bioactivity can be explained by a Michael-type conjugate
H
5
N
addition of the nucleophilic cysteine sulfhydryl groups of Cys38
2
3
O
OH
2
4
Boc
HN
O
and Cys120 in the p65 monomer of NF-
jB to one or more ab-unsat-
O
a
O
urated carbonyl moieties of the inhibitor.4 Recently we designed
and synthesized (ꢀ)-dehydroxymethylepoxyquinomicin ((ꢀ)-
MeO
OH
O
DHMEQ, 1) as a potent and specific inhibitor of NF-j
B.5 Animal
(-)-DHMEQ (1)
3
and cellular studies have shown 1 to be a promising drug as an
anticancer and/or anti-inflammatory agent.6 Very recently, we
clarified that the bioactivity of 1 could be explained by the covalent
binding of the nucleophilic cysteine sulfhydryl group of Cys38 of
p65 to an oxirane ring of 1, as judged from the data obtained by
MALDI-TOF MS analysis.7 Inter alia we were intrigued by the struc-
ture of the conjugate compound and the mechanism of oxirane
ring opening by the sulfhydryl group. In this Letter, we report a
regioselective oxirane ring opening and an unusual intramolecular
O
MeO2C
HN
OH
SH
b
6
1
O
S
NH2
Boc
5
2
2
3
4
Boc
HN
O
MeO
O
4
Scheme 1. Reagents and conditions: (a) compound 2, DMF/0.1 M sodium phos-
phate buffer (pH 6.0) (1:1), rt, 25 min; (b) 2, DMF/0.1 M sodium phosphate buffer
(pH 7.0) (1:1), rt, 4 h.
* Corresponding author.
E-mail address: umezawa@applc.keio.ac.jp (K. Umezawa).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.123

I. Kozawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5380–5382
5381
protons, that is, OH at C5 at d 5.74 ppm and NH of NHC@O at d
11.82 ppm in 3 disappeared and that resonances of protons H-2
and H-5 appeared at d 4.99 ppm and d 5.51 ppm, respectively.
However, the structure of 4 presumed by 2D NMR analysis was still
open to question because of the absence of the desirable long-
range C–H correlation between the carbon of the C@O group at d
168.7 ppm and H-5 at d 5.51 ppm. So we prepared the N-,O-isopro-
pylidene derivative 613 from 4 in order to determine the structures
unambiguously (Scheme 2). NMR studies of this product revealed
that the proton on NH at d 9.39 ppm showed a long-range C–H cor-
relation to the isopropyl quaternary carbon at d 97.2 ppm and that
proton H-5 at d 5.75 ppm showed a long-range C–H correlation to
the carbon of the C@O group at d 167.2 ppm (Fig. 1). Finally, we se-
cured positive evidence for the correct structure of 4, which was
derived from 3 via an unusual intramolecular N?O acyl group
migration reaction in accordance with the hypothetical mecha-
nism depicted in Figure 3.10
This unusual oxirane ring opening of DHMEQ with other sulfhy-
dryl compounds followed by this N?O acyl migration reaction was
recognized. Reaction of 1 with mercaptoethanol or benzyl mercap-
tan in DMF/0.1 M sodium phosphate buffer (pH 7.0) (1:1) for 4 h at
room temperature afforded compounds 7a and 8a in a 1:7 ratio in
88% yield or compounds 7b and 8b in a 1:1 ratio in 87% yield,
respectively (Scheme 3).
OH
6
OH O
Boc
HN
H
N
O
1
5
HO
RS
6
1
O
H
NH
R =
2
5
4
3
O
OH
3
H
4
2
MeO
O
RS
O
O
3
6
Figure 1. Long-range C–H correlations observed for 3 and 6.
HO
O
H
O
N
Me2C(OMe)2 / p-TsOH
acetone, rt, 1 hr
3
4
O
5
RS
Boc
O
O
HN
R =
OH
O
MeO
O
O
NH
Me2C(OMe)2 / p-TsOH
acetone, rt, 1 hr
RS
O
6
Scheme 2. Synthesis of acetonides 5 and 6.
We previously identified the p65 peptide-(ꢀ)-DHMEQ adduct
with the dehydrated DHMEQ structure.7 In this case the peptide
adduct had been treated with an acid TFA, then without acidic
treatment the intact DHMEQ structure would be likely to be main-
tained. Treatment of the model adduct molecule was also shown to
be dehydrated by the acidic condition.7 Compared with 3, it may be
more difficult to dehydrate 4. From this view point, the intact
DHMEQ structure without migration may be more likely to exist
in the protein. However, time dependent migration of 3 to 4 in
the protein is still possible to occur. Crystallographic analysis of
the protein adduct may identify the main structure.
Confirmation of the structure of 3 was provided by MS and 2D
NMR spectral analysis using 1H–1H COSY, HMQC, and HMBC. Pro-
tons H-5, H-4 and carbon C4 resonances appeared at d 4.12–
4.17 ppm, d 3.38 ppm, and d 51.6 ppm, respectively; and a long-
range C–H correlation between H-2 and C4 was observed in the
HMBC spectrum (Fig. 1).
Furthermore, formation of O-,O-isopropylidene derivative 5
from 3 (Scheme 2) confirmed the regio- and stereochemistry be-
tween the two hydroxy groups at C5 and C6 to be in a cis-vicinal
relationship.
A mechanistic rationalization for the regioselective opening at
C4 of the oxirane ring by the sulfhydryl group of 2 is given in Figure
2.9
To our surprise, we noticed that the amount of compound 3 de-
creased with time and therewith a new spot on TLC appeared un-
der the same reaction conditions. After completion of the reaction
in 4 h, the unexpected new compound was obtained in 60% yield.
NMR studies of compound 412 revealed that 2 characteristic
OH
O
OH
OH
H
N
O
NH2
HO
RS
RSH
+
1
O
OH
RS
8a or b
7a or b
O
O
Scheme 3. Reagents and conditions: compound 1, RSH: a = HOCH2CH2SH, b = C6H5
CH2SH, DMF/0.1 M sodium phosphate buffer (pH 7.0) (1:1), rt, 4 h.
OH
O
OH
OH
OH
H
N
H
N
H
N
H
N
HO
RS
HO
O
O
O
O
OH
O
OH
O
OH
OH
RS
OH
OH
RS
O
RSH
3
RSH = 2
Figure 2. Plausible mechanism of regioselective oxirane ring opening with sulfhydryl group.
OH
HO
O
HO
O
O
O
HO
O
OH
HO
RS
NH2
HO
RS
NH
HO
RS
NH
4
3
O
O
Figure 3. Plausible mechanism of intramolecular N?O acyl group migration.

5382
I. Kozawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5380–5382
was dried and concentrated in vacuo. The residue was purified by column
In summary, we identified an unusual intramolecular N?O acyl
chromatography on silica gel (EtOAc/hexane, 1:1?5:1) to provide 82.5 mg (87%)
of 3 as a colorless oil.: TLC Rf = 0.58 (toluene/EtOH, 3:1); 1H NMR (300 MHz,
DMSO-d6,) d 1.39 (s, 9H, Boc), 2.97 (m, 2H, cys b), 3.38 (d, 1H, J = 2.1 Hz, H-4), 3.65
group migration reaction resulting in compound 4 via compound 3
in the course of the formation of (ꢀ)-DHMEQ–cysteine conjugates;
however, the detailed reaction mechanism must be the subject of
forthcoming investigations. We are intrigued with the speculation
that the same migration reaction would happen in the case of the
(s, 3H, COOMe), 4.12–4.17 (m, 2H, cys a, H-5), 4.56 (ddd, 1H, J = 7.8, 2.1, 2.1 Hz, H-
6), 5.74 (d, 1H, J = 3.9 Hz, C-5-OH), 6.16 (d, J = 7.8 Hz, C-6-OH), 6.92 (br s, 1H, H-2),
6.96 (d, 1H, J = 8.1 Hz, H-50), 6.99 (dd, J = 8.1 Hz, H-40), 7.41 (d, 1H, J = 8.1 Hz,
NHBoc), 7.44 (ddd, J = 8.1, 8.1, 1.7 Hz, H-60), 7.94 (dd, 1H, J = 8.1, 1.7 Hz, H-70),
11.16 (br s, 1H, C30-OH), 11.82 (br s, 1H, C-1-NH);13C NMR (75 MHz, DMSO-d6) d
28.1 ꢁ 3, 32.2, 51.6, 52.1, 53.5, 65.7, 72.4, 78.6, 107.2, 117.0 , 118.4, 119.8, 131.2,
134.2, 152.5, 156.2, 156.9, 164.8, 171.3, 193.8; MS (FAB) m/z 497 [M+H]+.
covalent binding of (ꢀ)-DHMEQ with NF-
jB.
References and notes
12. For the synthesis of 4: To a stirred solution of 2 (47 lL, 0.23 mmol) in DMF
(4.76 mL) and 0.1 M sodium phosphate buffer (5.76 mL, pH 7.0) was added 1
(30.0 mg, 0.115 mmol) in DMF (1 mL). The mixture was stirred at rt for 4 h.
Then the mixture was diluted with EtOAc (30 mL) and washed with H2O
(20 mL ꢁ 2). The organic layer was dried and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (EtOAc/hexane,
2:1?5:1) to yield 34.4 mg (60%) of 4 as a colorless oil.: TLC Rf = 0.60 (toluene/
EtOH, 3:1); 1H NMR (270 MHz, DMSO-d6,) d 1.36 (s, 9H, Boc), 3.06 (d, 2H,
J = 6.6 Hz, cys b), 3.51 (d, 1H, J = 3.5 Hz, H-4), 3.64 (s, 3H, COOMe), 4.24 (ddd,
1. Karin, M. Nature 2006, 441, 431.
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1H, J = 8.1, 6.6, 6.6 Hz, cys a), 4.76 (m, 1H, H-6), 4.99 (s, 1H, H-2), 5.51 (dd, 1H,
J = 3.5, 3.5 Hz, H-5), 6.19 (d, J = 7.3 Hz, C-6-OH), 6.95 (t, J = 7.3 Hz, H-60), 6.99 (d,
J = 8.0 Hz, H-40), 7.49 (d, J = 8.1 Hz, NHBoc), 7.53 (ddd, 1H, 8.0, 8.0, 1.5 Hz, H-50),
7.64, (dd, 1H, J = 8.0 1.5 Hz, H-70), 10.35 (br s, 1H, C30-OH);13C NMR (68 MHz,
DMSO-d6) d 28.1 ꢁ 3, 33.0, 49.1, 52.1, 53.9, 63.8, 75.8, 78.5, 93.5, 113.0, 117.4,
119.4, 130.1, 135.9, 155.3,160.1, 164.7, 167.8, 171.4, 187.0; MS (FAB) m/z 497
[M+H]+.
6. Suzuki, Y.; Sugiyama, C.; Ohno, O.; Umezawa, K. Tetrahedron 2004, 60, 7061;
Umezawa, K. Cancer Sci. 2006, 97, 990; Miyake, A.; Dewan, Z.; Ishida, T.;
Watanabe, M.; Honda, M.; Sata, T.; Yamamoto, N.; Umezawa, K.; Watanabe, T.;
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S.; Suzuki, T.; Itoh, T.; Taniguchi, M.; Shimamura, T.; Furukawa, H.; Ozaki, M.;
Umezawa, K.; Todo, S. Transplantation 2006, 82, 1720.
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13. For the synthesis of 6: To a stirred solution of 4 (52.3 mg, 0.105 mmol) in
acetone/Me2C(OMe) 2 (2 mL, 1:1) was added a catalytic amount of p-TsOH. The
mixture was stirred at rt for 1 h. Then the mixture was diluted with EtOAc
(20 mL) and washed with H2O (10 mL ꢁ 2). The organic layer was dried and
concentrated in vacuo. The residue was purified by column chromatography on
slica gel (EtOAc/hexane, 1:1?2:1) to obtain 18.6 mg (33%) of 6 as a colorless
oil.: TLC Rf = 0.2 (EtOAc /hexane, 2:1); 1H NMR (300 MHz, DMSO-d6) d 1.26 (s,
3H), 1.36 (s, 9H), 1.42 (s, 3H), 3.06 (2d, 2H, J = 5.2 Hz), 3.11 (d, 1H, J = 2.4 Hz),
3.64 (s, 3H), 4.24 (ddd, 1H, J = 8.1, 5.2, 5.2 Hz), 4.85 (s, 1H), 5.32 (d, 1H,
J = 2.4 Hz), 5.75 (dd, J = 2.4, 2.4 Hz), 6.93 (t, J = 7.2 Hz), 6.99 (d, J = 8.3 Hz), 7.50
(d, J = 8.1 Hz), 7.51 (ddd, 1H, J = 8.3, 8.3, 1.7 Hz), 7.58, (dd, 1H, J = 8.3, 1.7 Hz),
9.39 (s, 1H), 10.28 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d 26.4, 27.8, 28.1 ꢁ 3,
33.7, 48.4, 52.1, 53.9, 72.0, 74.6, 78.5, 87.2, 97.2, 112.8, 117.6, 119.5, 129.6,
135.9, 155.3, 160.1, 160.3, 167.2, 171.3, 187.5.
8. At pH 7.0, this reaction was completed in
a very short time to provide
compound 3.
9. Wipf, P.; Jeger, P.; Kim, Y. Bioorg. Med. Chem. Lett. 1998, 8, 351.
10. Wakamiya, T.; Tarumi, Y.; Shiba, T. Bull. Chem. Soc. Jpn. 1974, 47, 2686. This
paper reported the N,O-acyl migration reaction of b-hydroxy-
with concentrated sulfuric acid via synchronous SNi type reaction
a-amino acids
a
mechanism. So, this reaction is not a real one but an apparent intramolecular
N?O acyl group migration.
11. For the synthesis of 3 at pH 6.0: To a solution of N-(tert-butoxycarbonyl)-
L-
cysteine methyl ester 2 (78 L, 0.38 mmol) in DMF (8.6 mL) and 0.1 M sodium
l
phosphate buffer (9.6 mL, pH 6.0) was added (ꢀ)-DHMEQ (50.0 mg, 0.191 mmol)
in DMF (1 mL). The mixture was stirred at rt for 25 min. Then the mixture was
diluted with EtOAc (50 mL) and washed with H2O (30 mL ꢁ 2). The organic layer