Improved synthesis of DCDHF fluorophores with maleimide functional groups

Article abstract of DOI:10.1016/j.tetlet.2006.07.142

A group of dicyanodihydrofuran (DCDHF) fluorophores with thiol-reactive maleimide functionality has been synthesized. One of the methods involves aromatic nucleophilic substitution reaction between an arylfluoride containing DCDHF and an amine containing

Full text of DOI:10.1016/j.tetlet.2006.07.142

Tetrahedron Letters 47 (2006) 7213–7217
Improved synthesis of DCDHF fluorophores with
maleimide functional groups
Zhikuan Lu, Ryan Weber and Robert J. Twieg*
Department of Chemistry, Kent State University, Kent, OH 44242-0001, USA
Received 6 July 2006; revised 26 July 2006; accepted 27 July 2006
Available online 17 August 2006
Abstract—A group of dicyanodihydrofuran (DCDHF) fluorophores with thiol-reactive maleimide functionality has been synthe-
sized. One of the methods involves aromatic nucleophilic substitution reaction between an arylfluoride containing DCDHF and
an amine containing protected maleimide. An alternative and generally useful method involves combination of the Mitsunobu reac-
tion of a DCDHF-OH with a furan or 2-methylfuran protected maleimide and then subsequent retro Diels–Alder reaction.
Ó 2006 Elsevier Ltd. All rights reserved.
Single molecule fluorescence imaging is a powerful tech-
nique that reveals the subtle characteristics of structure
and dynamics in complex condensed phases that are
otherwise obscured in ensemble measurements.1 For
example, a fluorescent Nile Red dye was recently
employed to probe the sequence of conformationally
induced polarity changes in the molecular chaperonin
GroEL.2 We have identified a new class of DCDHF
fluorophores that are particularly well suited for sin-
gle-molecule studies.3 These dipolar molecules contain
an amine donor (usually a dialkylamine, R1R2N), a con-
jugated p-system (comprised of units including 1,4-benz-
ene, 2,6-naphthalene, 2,5-thiophene, alkene, etc.) and
the unique dicyanodihydrofuran DCDHF acceptor
(with R3, R4 groups, which are again usually alkyl
groups). The generic DCDHF chromophore structure
is shown in Figure 1 wherein these three substructures
can all be systematically varied to tune a wide range of
properties. Additional groups can be appended to any
of these substructures to provide additional function.
We seek to specifically control and optimize the intro-
duction of the maleimide functional group that is widely
used as a Michael addition substrate for thiol groups in
proteins.4–6 The maleimide may be directly attached to
the fluorophore via the aromatic ring or through an ali-
phatic spacer.7 Here we describe an efficient method for
the introduction of the maleimide through an aliphatic
spacer.
Several methods have been reported to create the malei-
mide functional group: (1) Cyclization of the corre-
sponding maleamic acid generated by condensation of
amine and maleic anhydride under acidic conditions.8
(2) Alkylation of a furan-protected maleimide with an
alkyl halide in the presence of base and subsequent
retro-Diels–Alder (retro-D–A) deprotection.9 (3) Mitsu-
nobu reaction of pyrrole-2,5-dione (parent maleimide)
or a protected maleimide with an alcohol.10,11
The maleimide unit was first introduced on the amine
donor side (R1 or R2) in a DCDHF dye with a benzene-
conjugating unit. Since we had synthesized the aryl
fluoride intermediate 6 before, and considering the insta-
bility of the maleimide to nucleophiles, we adapted a
SNAr reaction between a secondary amine with a
furan-protected maleimide 5 and the aryl fluoride 6 to
obtain the furan-protected maleimide 7.12 The furan-
protected maleimide 5 was synthesized starting from 1,
the monotosylate of 1,6-hexanediol. The furan-pro-
tected maleimide was then alkylated with monotosylate
1 and the remaining alcohol in 3 was subsequently tosyl-
ated and treated with N-propylamine to afford dialkyl-
amine 5. After a retro-D–A reaction of 7, the DCDHF
fluorophore 8 with maleimide attached on the terminus
NC
CN
NC
O
R2R1NAr
n
R3 R4
Figure 1. The generic DCDHF chromophore with sites R1–4 and Ar
available for synthetic modification.
*
Corresponding author. Tel.: +1 330 672 2791; fax: +1 330 672
3816; e-mail: rtwieg@lci.kent.edu
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.07.142

7214
Z. Lu et al. / Tetrahedron Letters 47 (2006) 7213–7217
O
O
NH
TsCl, Py
O
OH
TsO
HO
OH
2
0˚C, 55%
O
O
N
OH
1
K2CO3, DMF, 50˚C
O
3
O
O
CN
CN
NC
TsCl, Py
0˚C
NH2
O
N
OTs
O
N
NH
F
THF
O
4
(57% from 1)
O
6
O
5
Py, AcOH (cat.) rt, 48h
CN
CN
O
NC
CN
NC
O
xylene
O
N
N
CN
O
N
N
O
150-155˚C
(72%)
8
O
7
(38% yield from 4)
O
Scheme 1. Synthesis of DCDHF with maleimide on the donor side.
of the amine donor has been obtained as is summarized
overall in Scheme 1.13
The limited success here prompted us to seek improved
methods for synthesis of this kind of molecule. The
Mitsunobu reaction of a furan-protected maleimide in
combination with the retro-D–A reaction avoids the
susceptibility of the maleimide to nucleophilic attack.11
We have examined different pyrrole-2,5-dione adducts
of furan, 2-methylfuran, and 2,5-dimethylfuran made
by the procedures reported by Kwart and Burchuk.14,15
The pyrrole-2,5-dione adduct of furan has poor solubility
in chloroform and other ordinary organic solvents,
which limits its use while the 2-methylfuran protected
maleimide has much improved solubility. The pure endo,
exo isomers or a mixture of both isomers were used in the
Mitsunobu reaction and the retro-D–A reaction. The
thermal stability of the D–A adducts is furan > 2-methyl-
furan > 2,5-dimethylfuran and the latter adducts are
not convenient to use, as partial retro-D–A reaction
was often observed during their handling and purifica-
tion. We examined the model Mitsunobu reaction be-
tween 4-biphenylmethanol and furan or 2-methylfuran
protected maleimide and found they are equivalent in
terms of the yield of the products (Scheme 3). However,
one of the major drawbacks of the Mitsunobu reaction is
the necessity to remove hydrazide and phosphine oxide
byproducts.16 The availability of the different furan
D–A adducts provides versatility for chromatographic
This same method cannot be applied if the maleimide is
attached on the acceptor side (R3, R4) because DCDHF
fluorophores are not compatible with either primary
amines, which are the starting material for method (1),
or the basic conditions for the alkylation, which are
required for method (2). Therefore, as an alternative,
the Mitsunobu coupling with pyrrole-2,5-dione appears
very attractive. Indeed, using the method of Walker,10
DCDHFs with a maleimide group were isolated but only
in moderate (35% for Ar = 1,4-phenyl) to low (7% for
Ar = 2,6-naphthyl) yields probably due to the sensitivity
of the unprotected maleimide double bond to the nucleo-
philes existing in the reaction system (Scheme 2).
CN
CN
NC
CN
CN
NC
PPh3, DEAD,
neopentyl alcohol
(C6H13)2N Ar
(C6H13)2N Ar
O
O
O
maleimide, -78 °C to r.t.
OH
N
Ar = 1,4-phenyl, 2,6-naphthyl
O
Scheme 2. Synthesis of a maleimide substituted DCDHF fluorophore
by direct Mitsunobu reaction of pyrrole-2,5-dione.
O
O
OH
+
Ph3P, DEAD, THF
O
N
O
NH
90% yield
O
12
9
10
O
CH3
O
CH3
O
O
OH
+
Ph3P, DEAD, THF
90% yield
O
N
NH
O
13
11
O
CH3
O
O
toluene
O
N
N
reflux, 91%yield
O
O
O
O
14
14
xylene
N
O
N
reflux, 92%yield
O
O
12
Scheme 3. Model reactions of 4-biphenylmethanol with protected maleimide and the corresponding retro Diels–Alder reaction.

Z. Lu et al. / Tetrahedron Letters 47 (2006) 7213–7217
7215
separation required for workup of these reactions. The
retro-D–A reaction of the 2-methylfuran adduct 14
was run in boiling toluene and the furan adduct was run in
xylene or anisole as reported by Clevenger and Turnbull.9
dures for the DCDHF precursors with hydroxyl func-
tionality (first column of Table 1) will be published
elsewhere. These hydroxyl functionalized DCDHF were
reacted with the 2-methylfuran protected maleimide to
obtain a protected DCDHF-maleimide. Either individ-
ual pure exo or endo isomers or a mixture of both iso-
mers of the 2-methylfuran protected maleimide are
useful for this purpose.
After examination of these model compounds, we
focused on the synthesis of the maleimide functionalized
DCDHF fluorophores. The detailed synthetic proce-
Table 1. Synthesis of maleimide functionalized DCDHFs via the 2-methylfuran protected maleimide and retro Diels–Alder reaction17
CH3
CH3
O
O
O
PPh3, DEAD (or DIAD)
THF, 0˚C to r.t.
toluene
reflux
DCDHF-OH +
O
HN
O
DCDHF
N
DCDHF
N
O
O
O
DCDHF-OH
DCDHF-protected maleimide
DCDHF-maleimide
CN
CN
CN
NC
NC
CN
CN
NC
CN
N
N
N
1
2
3
O
O
O
O
O
O
N
N
OH
22
15
O
O
CN
CN
CN
CN
NC
NC
CN
CN
NC
O
O
O
O
N
N
O
N
O
N
N
23
16
OH
O
O
CN
CN
CN
NC
NC
CN
CN
NC
CN
O
O
O
O
N
N
O
N
O
N
N
24
OH
17
O
O
CN
CN
NC
CN
CN
NC
CN
CN
NC
O
N
O
O
N
4
O
O
N
N
N
18
25
HO
O
O
O
CN
CN
NC
CN
CN
NC
CN
CN
NC
O
O
N
O
O
5
N
N
O
O
N
N
O
HO
26
27
O
19
O
CN
CN
NC
CN
CN
NC
CN
CN
NC
CH3
O
S
S
N
S
N
6
7
O
O
N
O
N
O
N
HO
20
O
O
O
CN
CN
NC
CN
CN
NC
CN
CN
NC
CH3
O
S
O
S
O
O
N
N
S
O
N
N
N
HO
28
O
O
21

7216
Z. Lu et al. / Tetrahedron Letters 47 (2006) 7213–7217
15. Representative method for prep of a furan-protected
maleimide 10: In a 5 mL vial were added pyrrole-2,5-
dione (98 mg, 1.0 mmol) and 3 mL diethyl ether. The
pyrrole-2,5-dione was dissolved with ultrasound and furan
(0.3 mL) was added. After 5 days the diethyl ether was
evaporated until only 1 mL, and white crystals were
filtered off (133 mg, ꢀ75% is endo and 25% exo). 1H NMR
(100 MHz, CDCl3) (mixture) 1H 400 Hz, 8.49 (s, br,
0.25H), 8.08 (s, br, 0.75H), 6.55–6.52 (m, 3H), 5.37–5.32
(m, 2H), 3.60–3.58 (m, 1.5H), 2.91 (s, 0.5H). 13C NMR
(100 MHz, CDCl3) (mixture) d 176.4, 175.0, 136.6, 134.6,
81.0, 79.4, 48.7, 47.4.
In contrast to the problems we encountered with the
Mitsunobu reaction of pyrrole-2,5-dione, the reactions
and purifications involving the protected maleimide
were straightforward. The reaction is not sensitive to
an excess of triphenylphosphine and DEAD or DIAD
and so typically 20–50% of excess of the reagents could
be used. Seven DCDHF-maleimides (22–28) have been
synthesized via their corresponding 2-methylfuran pro-
tected DCDHF-maleimides (15–21). The conjugation
linkages for DCDHFs include phenyl (22), naphthalene
(23–26), Th-Ph (27), and Th-V (28) and maleimide was
installed on either the donor side or acceptor side of
the molecule. The detailed photophysical properties of
these new DCDHF chromophores and their applica-
tions as biolabels will be described elsewhere.
Representative method for prep of a 2-methylfuran
protected maleimide 11: In a 500 mL flask were added
pyrrole-2,5-dione (5.82 g, 60 mmol) and 300 mL diethyl
ether. The pyrrole-2,5-dione was dissolved with ultra-
sound, and 2-methylfuran (7.38 g, 90 mmol) was added.
The mixture was permitted to stand for 3 days and diethyl
ether was removed by distillation until only 50 mL was
left. Crystals were filtered off to obtain 2.3 g white solid
(this sample is >98% endo isomer). The diethyl ether
solution was evaporated to dryness (this residue is >95%
Acknowledgment
Support from DOE (DG-FG02-04ER63777), NIH
(1P20HG003638-01), and the Ohio Board of Regents
is acknowledged.
1
exo isomer). endo isomer H NMR (400 Hz, CDCl3) 7.59
(s, br, 1H), 6.51 (dd, J = 1.7, 5.7 Hz, 1H), 6.34 (d,
J = 5.7 Hz, 1H), 5.24 (dd, J = 1.7, 5.6 Hz, 1H), 3.70
(ddd, J = 5.6, 7.6 Hz, 1H), 3.17 (d, J = 7.6 Hz, 1H), 1.84
1
(s, 3H). exo isomer H NMR (400 Hz, CDCl3) 8.85 (s, br,
References and notes
1H), 6.51 (dd, J = 1.5, 5.5 Hz, 1H), 6.31 (d, J = 5.7 Hz,
1H), 5.24 (d, J = 1.5 Hz, 1H), 3.02 (ddd, J = 6.4 Hz, 1H),
2.76 (d, J = 6.4 Hz, 1H), 1.75 (s, 3H).
1. (a) Moerner, W. E.; Orrit, M. Science 1999, 283, 1670–
1676; (b) Weiss, S. Science 1999, 283, 1676–1683.
2. Kim, S. Y.; Semyonov, A. N.; Twieg, R. J.; Horwich, A.
L.; Frydman, J.; Moerner, W. E. J. Phys. Chem. B 2005,
109, 24517–24525.
16. Hughes, D. L. Org. React. 1992, 42, 335–656.
17. General procedure (prep of 22 as an example): In a
100 mL dry round bottom flask were added 3-cyano-
2-dicyanomethylen-5-methyl-5-(3-hydroxypropyl)-4-(4-di-
3. (a) Willets, K. A.; Nishimura, S. Y.; Schuck, P. J.; Twieg,
R. J.; Moerner, W. E. Acc. Chem. Res. 2005, 38, 549–556;
(b) Willets, K. A.; Ostroverkhova, O.; He, M.; Twieg, R.
J.; Moerner, W. E. J. Am. Chem. Soc. 2003, 125, 1174–
1175; (c) Nishimura, S. Y.; Lord, S. J.; Klein, L. O.;
Willets, K. A.; He, M.; Lu, Z.; Twieg, R. J.; Moerner, W.
E. J. Phys. Chem. B 2006, 110, 8151–8157.
4. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.
Tetrahedron Lett. 1998, 39, 5077–5080.
5. Zhang, X.; Li, Z. C.; Li, K. B.; Du, F. S.; Li, F. M. J. Am.
Chem. Soc. 2004, 126, 12200–12201.
6. Girouard, S.; Houle, M. H.; Grandbois, A.; Keillor, J. W.;
Michnick, S. W. J. Am. Chem. Soc. 2005, 127, 559–566.
7. Corrie, J. E. T. J. Chem. Soc., Perkin Trans. 1 1994, 2975–
2982.
8. Reddy, P. Y.; Kondo, S.; Fujita, S.; Toru, T. Synthesis
1998, 999–1002.
hexylaminophenyl)-2,5-dihydrofuran
(100 mg,
0.20
mmol), 1-methyl-10-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-
dione (endo) (40.3 mg, 0.225 mmol) Ph3P (80.5 mg, 0.31
mmol), and 4 mL of anhydrous THF. The resulting
solution was cooled in an ice-water bath. DEAD (62 mg,
0.31 mmol) was added over 2 min. The reaction mixture
was stirred for 3 h and the bath was permitted to warm to
room temperature. THF was removed by rotary evapora-
tion and the residue was purified by flash column
chromatography eluting with dichloromethane and
dichloromethane/ethyl acetate (9:1) to give 128 mg (96%
1
yield) of 15 as an orange-red solid. H NMR (400 MHz,
CDCl3) d 7.95–7.92 (m, 2H), 6.70–6.68 (m, 2H), 6.39 (dd,
J = 1.7, 5.7 Hz, 0.5H), 6.32 (dd, J = 1.7, 5.7 Hz, 0.5H),
6.23 (d, J = 5.7 Hz, 0.5H), 6.15 (d, J = 5.7 Hz, 0.5H),
5.21–5.18 (m, 1H), 3.63–3.58 (1H), 3.40 (t, J = 7.7 Hz,
4H), 3.28 (t, J = 6.7 Hz, 2H), 3.11–3.07 (m, 1H), 2.15–1.97
(m, 2H), 1.82–1.81 (s, 3H), 1.79 (s, 3H), 1.69–1.57 (m, 4H),
1.45–1.18 (m, 14H), 0.92 (t, J = 6.8 Hz, 6H).
9. Clevenger, R. C.; Turnbull, K. D. Synth. Commun. 2000,
30, 1379–1388.
10. Walker, M. A. J. Org. Chem. 1995, 60, 5352–5355.
11. Farha, O. K.; Julius, R. L.; Hawthorne, M. F. Tetrahedron
Lett. 2006, 47, 2619–2622.
In a 100 mL long neck round bottom flask were placed
30 mL of toluene, the protected (endo) maleimide
DCDHF (128 mg, 0.197 mmol) and a stirbar. A nitrogen
flow was introduced to the surface of the solution through
a pipette and the system was heated to reflux. After
30 min, TLC indicated the retro Diels–Alder reaction was
complete and the system was cooled down with nitrogen
flow. The mixture was directly transferred onto a flash
silica gel column and the product was eluted with
dichloromethane containing 5% of ethyl acetate to obtain
100 mg (91% yield) of the maleimide DCDHF 22. 1H
NMR (400 MHz, CDCl3) d 7.95 (d, J = 9.5 Hz, 2H), 6.70
(s, 2H), 6.71 (d, J = 9.5 Hz, 2H), 3.51–3.47 (m, 2H), 3.42
(t, J = 7.9 Hz, 4H), 2.26–2.05 (m, 2H), 1.81 (s, 3H), 1.69–
1.60 (m, 4H), 1.60–1.40 (m, 14H), 0.94 (t, J = 6.9 Hz, 6H).
13C NMR (100 MHz, CDCl3) d 177.2, 171.7, 170.5, 152.9,
12. He, M.; Twieg, R. J.; Ostroverkhova, O.; Gubler, U.;
Wright, D.; Moerner, W. E. Proc. SPIE 2002, 4802, 9–20.
13. DCDHF 8: 1H NMR (400 MHz, CDCl3) d 8.01 (d,
J = 9.5 Hz, 2H), 6.73 (d, J = 9.5 Hz, 2H), 6.72 (s, 2H),
3.55 (t, J = 7.2 Hz, 2H), 3.45–3.37 (m, 4H), 1.85 (s, 6H),
1.73–1.59 (m, 6H), 1.40–1.37 (m, 4H), 1.01 (t, J = 7.4 Hz,
3H). 13C NMR (75 MHz, CDCl3) d 177.0, 173.6, 170.9,
152.8, 134.1, 132.5, 113.2, 113.0, 112.0, 97.2, 90.8, 54.1,
53.0, 51.3, 37.6, 28.3, 27.7, 27.1, 26.4, 26.3, 20.6, 11.3.
HRMS (m/z): [M+Na] calcd for C29H31N5O3Na,
520.2325; found, 520.2332.
14. Kwart, H.; Burchuk, I. J. Am. Chem. Soc. 1952, 74, 3094–
3097.

Z. Lu et al. / Tetrahedron Letters 47 (2006) 7213–7217
7217
134.2, 132.1, 113.11, 113.07, 112.8, 112.06, 111.9, 99.0,
91.5, 54.1, 51.4, 37.6, 37.0, 31.6, 27.3, 26.9, 26.7, 22.6, 14.2.
HRMS (m/z): [M+Na] calcd for C34H41N5O3Na,
590.3107; found, 590.3101.
24.4, 22.6, 14.0. HRMS (m/z): [M+Na] calcd for
C34H35N5O3Na, 584.2638; found, 586.2644.
Compound 26. 1H NMR (400 MHz, CDCl3) d 8.35 (d,
J = 2.1 Hz, 1H), 7.88 (dd, J = 9.1, 2.1 Hz, 1H), 7.82 (d,
J = 9.3 Hz, 1H), 7.72 (d, J = 9.1 Hz, 1H), 7.22 (dd, J =
9.2, 2.5 Hz, 1H), 7.00 (d, J = 2.5 Hz, 1H), 6.70 (s, 2H),
3.82 (t, J = 6.6 Hz, 2H), 3.69 (t, J = 7.6 Hz, 2H), 3.46 (t,
J = 7.6 Hz, 2H), 1.94 (s, 6H), 1.72–1.65 (m, 2H), 1.43–1.30
(m, 6H), 0.94 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3) d 176.2, 175.8, 170.5, 149.4, 138.3, 134.3, 132.0,
131.8, 127.4, 125.1, 124.5, 119.5, 116.6, 112.2, 112.1, 111.2,
105.2, 98.4, 96.5, 56.6, 51.0, 48.4, 34.6, 31.6, 27.4, 27.3,
26.6, 22.6, 14.0. HRMS (m/z): [M+Na] calcd for
C32H31N5O3Na, 556.2325; found, 556.2327.
Compound 23. 1H NMR (400 MHz, CDCl3) d 8.27 (d,
J = 2.0 Hz, 1H), 7.80–7.76 (m, 2H), 7.61 (d, J = 9.0 Hz,
1H), 7.06 (dd, J = 2.4, 9.0 Hz, 1H), 6.72 (d, J = 2.4 Hz,
1H), 6.53 (s, 2H), 3.53–3.45 (m, 6H), 2.30–2.23 (m, 1H),
2.17–2.09 (m,1H), 1.90 (s, 3H), 1.63–1.54 (m, 1H), 1.42–
1.36 (m, 1H). 13C NMR (100 MHz, CDCl3) d 176.5, 173.8,
170.5, 149.4, 138.5, 133.9, 132.1, 127.0, 124.9, 124.2, 118.6,
117.3, 112.3, 112.2, 111.3, 104.6, 100.2, 96.4, 56.2, 48.0,
37.1, 36.8, 26.8, 25.5, 22.6. HRMS (m/z): [M+Na] calcd
for C30H25N5O3Na, 526.1855; found, 526.1866.
Compound 24. 1H NMR (400 MHz, CDCl3) d 8.27 (d,
J = 2.1 Hz, 1H), 7.80 (dd, J = 9.0, 2.1 Hz, 1H), 7.78 (d,
J = 9.3 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.13 (dd,
J = 9.3, 2.5 Hz, 1H), 6.78 (d, J = 2.5 Hz, 1H), 6.57 (s,
2H), 3.55–3.40 (m, 6H), 2.33–2.10 (m, 2H), 1.92 (s, 3H),
1.73–1.55 (m, 6H), 1.45–1.33 (m, 12H), 0.94 (t, J = 7.0 Hz,
6H). 13C NMR (100 MHz, CDCl3) d 176.5, 173.8, 170.5,
150.0, 138.6, 134.0, 132.1, 131.8, 127.1, 124.7, 124.2, 118.7,
116.7, 112.2, 112.1, 111.2, 104.4, 100.1, 96.5, 56.3, 51.3,
37.1, 36.8, 31.7, 27.3, 26.81, 26.75, 22.7, 14.1. HRMS
(m/z): [M+Na] calcd for C38H43N5O3Na, 640.3264; found,
640.3283.
Compound 27. H NMR (400 MHz, CDCl3) d 7.91 (ddd,
1
J = 8.9, 2.4, 2.0 Hz, 2H), 7.54 (ddd, J = 8.9, 2.4, 2.0 Hz,
2H), 7.33 (d, J = 4.0 Hz, 1H), 6.71 (s, 2H), 6.02 (d,
J = 4.0 Hz, 1H), 3.82 (t, J = 6.6 Hz, 2H), 3.56 (t,
J = 6.6 Hz, 2H), 3.36 (t, J = 7.5 Hz, 2H), 1.88 (s, 6H),
1.72–1.64 (m, 2H), 1.43–1.37 (m, 2H), 1.00 (t, J = 7.4 Hz,
3H). 13C NMR (100 MHz, CDCl3) d 176.0, 174.5, 170.5,
160.5, 141.7, 134.3, 130.0, 128.5, 123.7, 123.6, 122.1, 112.1,
111.9, 111.1, 104.1, 98.2, 96.7, 56.9, 53.6, 50.5, 34.6, 29.1,
27.2, 20.1, 13.9. HRMS (m/z): [M+Na] calcd for
C30H27N5O3SNa, 560.1732; found, 560.1730.
1
Compound 28 H NMR (400 MHz, CDCl3) d 7.81 (d, br,
Compound 25. 1H NMR (400 MHz, CDCl3) d 8.35 (d,
J = 2.1 Hz, 1H), 7.88 (dd, J = 8.9, 2.1 Hz, 1H), 7.79 (d,
J = 9.2 Hz, 1H), 7.66 (d, J = 8.9 Hz, 1H), 7.13 (dd,
J = 9.2, 2.4 Hz, 1H), 6.81 (s, br, 1H), 6.73 (s, 2H), 3.61
(t, J = 6.4 Hz, 2H), 3.51 (t, J = 7.5 Hz, 2H), 3.46 (t,
J = 7.5 Hz, 2H), 1.93 (s, 6H), 1.75–1.63 (m, 6H), 1.43–1.32
(m, 6H), 0.94 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3) d 176.4, 175.6, 170.8, 149.7, 138.4, 134.2, 132.0,
127.1, 124.8, 124.5, 119.0, 116.7, 112.4, 112.3, 111.4, 104.7,
98.3, 95.7, 65.2, 51.3, 50.6, 35.4, 31.6, 27.5, 27.2, 26.7, 26.1,
J = 14.6 Hz, 1H), 7.35 (d, J = 4.6 Hz, 1H), 6.76 (s, 2H),
6.21 (d, J = 4.6 Hz, 1H), 6.00 (d, br, J = 14.6 Hz,
1H), 3.85 (t, J = 6.6 Hz, 2H), 3.67 (t, J = 6.6 Hz, 2H),
3.47 (t, J = 7.7 Hz, 2H), 1.78–1.67 (m, 8H), 1.49–1.39 (m,
2H), 1.03 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) d 176.9, 172.6, 170.2, 168.2, 143.0, 140.3, 134.5,
125.1, 113.7, 112.9, 112.8, 107.7, 105.0, 95.8, 54.7,
51.8, 50.7, 34.4, 29.7, 26.8, 20.1, 13.8. HRMS (m/z):
[M+Na] calcd for C26H25N5O3SNa, 510.1576; found,
510.1579.