Syntheses and characterizations of novel pyrrolocoumarin probes for SNAP-tag labeling technology

Article abstract of DOI:10.1016/j.tet.2011.01.075

SNAP-tag technology is a revolutionary protein labeling technology employing in various biological studies. Since low signal/noise ratio and severe overlap between the FRET donors/acceptors often occurred in applying present fluorescent probes and thus limited the further applications, development of new fluorescent probes with excellent fluorescent properties is still of request by today's SNAP-tag technology. In this paper, a number of SNAP-tag protein probes have been developed by incorporating a novel pyrrolocoumarin fluorophore recently developed by our group. Examination of these novel synthetic compounds shows all these materials possess satisfactory fluorescent properties. Among these, probe 7 exhibits the most excellent characters, and its quantum yield, maximum emission wavelength and Stocks shift reach to 0.44, 534 nm and 112 nm, respectively. Further analysis of structure-property relationship indicates that the probes with a longer C3-substituted alkyl (such as pentyl) give stronger fluorescence.

Full text of DOI:10.1016/j.tet.2011.01.075

Tetrahedron 67 (2011) 2251e2259
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Syntheses and characterizations of novel pyrrolocoumarin probes for SNAP-tag
labeling technology
,
De-Sheng Mei a, Yi Qu b, Jin-Xiang He b, Lei Chen a, Zhu-Jun Yao a
*
a State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road,
Shanghai 200032, China
b Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
SNAP-tag technology is a revolutionary protein labeling technology employing in various biological
studies. Since low signal/noise ratio and severe overlap between the FRET donors/acceptors often occurred
in applying present fluorescent probes and thus limited the further applications, development of new
fluorescent probes with excellent fluorescent properties is still of request by today’s SNAP-tag technology.
In this paper, a number of SNAP-tag protein probes have been developed by incorporating a novel pyr-
rolocoumarin fluorophore recently developed by our group. Examination of these novel synthetic com-
pounds shows all these materials possess satisfactory fluorescent properties. Among these, probe 7
exhibits the most excellent characters, and its quantum yield, maximum emission wavelength and Stocks
shift reach to 0.44, 534 nm and 112 nm, respectively. Further analysis of structure-property relationship
indicates that the probes with a longer C3-substituted alkyl (such as pentyl) give stronger fluorescence.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 4 December 2010
Received in revised form 20 January 2011
Accepted 26 January 2011
Available online 2 February 2011
Keywords:
Pyrrolocoumarin
Fluorescent probe
SNAP-tag
Protein labeling
Synthesis
1. Introduction
fluorescence of cells and (ii) severe overlap between the FRET
(fluorescence resonance energy transfer) donors and acceptors in
Many real-time investigations of a target protein including its
position and evolution in the complicated situations require the use
of specific labeling techniques.1 In the last decade, the green fluo-
rescent protein (GFP) and its variants have emerged as the most
powerful tool in the optical research of protein functions due to
their bright clear fluorescence, which is detectable even in single
cells.2 However, the monotone fluorescent properties of GFPs hin-
ders them from further application in some cases.3
SNAP-tag technology is a different protein labeling technology
emerged for overcoming the above mentioned disadvantages.4
SNAP-tag can react with its ligand (O6-benzylguanine derivatives,
BG) specifically and rapidly in vivo and vitro. Because BG can carry
a variety of fluorescent groups, a wavelength variable protein la-
beling tag (SNAP-tag) with different fluorescent groups (as
requested) can be prepared. As a revolutionary self-labeled protein
technology, SNAP-tag has been successfully applied in the re-
searches of protein location, protein quantitative analysis and
proteineprotein interactions.5 However, current SNAP-tag tech-
nology is still far from perfection. Two major shortcomings existed:
(i) Low ratio of signal/noise, which results from the intrinsic
many proteineprotein interaction experiments.6 These problems
are generated by the poor fluorescent properties of many present
SNAP-tag probes. The low signal/noise ratio is due to the poor
fluorescence intensity of probes, and the spectral overlap between
FRET donors and acceptors is caused by small Stocks shifts of the
probes. The Stocks shifts of most commercially available probes are
usually below 30 nm. Therefore, development of small-molecule
fluorophores with intensive fluorescent and larger Stocks shift is of
urgency for ideal SNAP-tag probes.
2. Design of novel probes for SNAP-tag technology
Recently, we developed a novel series of pyrrolocoumarin
fluorescent probes with satisfactory fluorescent properties and
notable large Stocks shifts.7 As a representative, compound A
showed a large Stock shift (113 nm) and intense fluorescence
(
F
¼ 0.55, lem ¼ 523 nm). These properties indicate that A might be
able to apply into the fluorescent probes for those complicated
biological studies. In this study, as one application, fluorophore A
was successfully incorporated into a number of small-molecule
probes, which are designed for future protein function studies with
SNAP-tag technology (Fig. 1).
To achieve an ideal SNAP-tag probe, we examined the combi-
nations of couple easily available substituted sites and several
* Corresponding author. Tel.: þ86 21 5492 5133; fax: þ86 21 5492 5124; e-mail
address: yaoz@sioc.ac.cn (Z.-J. Yao).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.01.075

2252
D.-S. Mei et al. / Tetrahedron 67 (2011) 2251e2259
NH2
OH
NHCOCF3
CN
N
2
3
1
a
HN
b
+
O
A
O
CO2CH3
OH
11
9
10
Cl-
H
N
Cl
N
linker
HN
N
N
N
N
c
N
O
N
H
N
H
H2N
N
H2N
CF3
N
O
O
12
13
O
N
H
N
linkers: hydrocarbon chain
or PEG chain
NH2
N
N
O
N
H
H2N
Fig. 1. Design of novel protein probes based on SNAP-tag technology.
O
N
O
e
d
11
N
N
N
N
commonly used linker chains. We chose to alter pyrrole nitrogen
and C3 site of A as the anchors, and CH2, (CH2)3, (CH2)5 as the
linkers between the fluorescent core and BG moiety. Since many
reports used poly(glycol) chain as the linker, we also applied similar
linkages in this work. According to such design, we obtained two
groups of new probes, N1-substituted probes 1e4 (group I, Fig. 2)
and C3-substituted probes 5e8 (group II, Fig. 2).
N
H
N
H
H2N
H2N
N
15
14
Scheme 1. Synthesis of O6-[4-(aminomethyl)benzyl]guanine 15. Reagents and condi-
tions: (a) LiAlH4, THF, 65%; (b) (CF3CO)2O, Py, DCM, 81%; (c) N(CH3)3, DMF, 81%;
(d) t-BuOK, DMSO, 34%; (e) K2CO3, MeOH, 76%.
hydride to give amino alcohol 10, whose amino group was pro-
tected with trifluoroacetic anhydride (TFAA), affording the benzyl
alcohol 11. In parallel, ammonium salt 13 was prepared from the
reaction of 2-chloro-6-aminopurine (12) with trimethylamine.
Then, ammonium salt 13 reacted with 11 in the presence of t-BuOK
to yield the BG intermediate 14. Final deprotection of 14 with
potassium carbonate in methanol afforded the requested BG
derivative 15 in 76% yield.
1
Group I. N -Substituted probes 1-4
N
O
linker
N
O
N
NH
N
N
O
O
N
H
H2N
linker: CH2, (CH2)3, (CH2)5
or (CH2CH2O)4CH2CH2
3.2. Synthesis of N1-substituted pyrrolocoumarin carboxylic
acids
N
O
Synthesis of N1-substituted pyrrolocoumarin carboxylic acids
18aed is outlined in Scheme 2. Because the linker u-halo carboxylic
O
5-8
Group II. C3-Substituted probes
ester 16d is not commercially available, we prepared it prior to the
first step of N-alkylation. Reaction of tetraglycol reacted with tert-
butyl acrylate in NaH/DMF followed by iodination of the remaining
hydroxyl group afforded the iodide 16d in high yield. With all the
linkers in hand, the fluorescent core A reacted in parallel with
O
linker
O
N
NH
NH
N
N
linker: CH2, (CH2)3, (CH2)5
or (CH2CH2O)4CH2CH2
N
H
H2N
u-halo carboxylic esters 16aed in the presence of NaH, affording
the corresponding N1-substituted pyrrolocoumarin carboxylic es-
ters 17aed. Hydrolysis of esters 17aed in acid or base solution
yielded the acids 18aed in 53e93% yields.
Fig. 2. Novel protein probes based on SNAP-tag technology.
3.3. Synthesis of C3-substituted pyrrolocoumarin carboxylic
acids
3. Chemical synthesis of fluorescent probes
3.1. Synthesis of O6-[4-(aminomethyl)benzyl]guanine
Parallel syntheses of the C3-substituted pyrrolocoumarin car-
boxylic acids 24aec using linear hydrocarbon linkers 20aec are
shown in Scheme 3. Fischer indolization of aniline 19 with methyl
acetones 20aec via a two-step procedure afforded the corre-
sponding intermediates 21aec in acceptable yields. Suzuki cou-
plings of bromides 22aec (prepared from the corresponding acids
The common BG moiety used in this study, O6-[4-(amino-
methyl)benzyl]guanine (15), was synthesized with slight modifi-
cations of the known report (Scheme 1).8 The starting material,
methyl 4-cyanobenzoate (9) was reduced with lithium aluminum

D.-S. Mei et al. / Tetrahedron 67 (2011) 2251e2259
2253
O
alternative indirect strategy was adopted in the synthesis of the
C3-substituted pyrrolocoumarin carboxylic acid 24d (Scheme 4).
Oxa Michael addition of triglycol with tert-butyl acrylate using NaH
as a base followed by iodination afforded the linker iodide 20d in
a high yield. In a parallel experiment, Fischer indolization was
employed to construct the corestructure of 26, whoseindole NH was
thenprotectedwith Boc2O, yielding the requiredbuildingblock 27 in
78% yield. Suzuki reaction of bromide 27 with boric acid 23 afforded
fluorophore 28. Deprotection of the N-Boc functionality of 28 with
potassium carbonate gave alcohol 29. Finally, O-alkylation of com-
pound 29 with iodide 20d under basic conditions followed by
deprotection of N-Boc with TFA afforded 24d in 41% yield.
a
HO(C2H4O)3C2H4OH
+
OBut
HO(C2H4O)4C2H4CO2But
I(C2H4O)4C2H4CO2But
b
+
16d-1
16d
N
c
X
linker
CO2R
HN
X=Br, R=But
linker = CH2
16a
O
O
16b X=I, R=Et,
linker = (CH2)3
16c X=I, R=Et,
inker = (CH2)5
A
X=I, R=But,
O
a
HO(C2H4O)2C2H4CO2But
16d
HO(C2H4O)2C2H4OH
b
But
+
linker = (C2H4O)4C2H4
O
20d-1
N
I(C2H4O)2C2H4CO2But
N
linker
COOR
17a
N
d
linker
COOH
18a linker = CH2
18b linker = (CH2)3
18c
18d linker = (C2H4O)4C2H4
N
20d
OAc
O
O
R=But
linker = CH2,
17b R=Et
linker = (CH2)3
R=Et
linker = (CH2)5
17d R=But
linker = (C2H4O)4C2H4
O
O
O
H2N
Br
c
OH
HN
Br
O
+
O
O
linker = (CH2)5
O
17c
19
25
26
OAc
OAc
Br
O
N
Scheme 2. Synthesis of N1-substituted pyrrolocoumarin carboxylic acids 18aed.
(a) NaH, THF, 55%; (b) I2, PPh3, imidazole, 80%; (c) NaH, DMF, 34e67%; (d) deprotection,
53e93%. (See Experimental section for the details).
e
d
Boc
N
Boc
N
23
O
O
O
28
27
H2N
Br
O
O
a
O
O
+
linker COOH
linker = CH2
O
O
O
OH
OH
N
O
HN
g
20a
19
f
Boc
N
20b linker = (CH2)3
20c
20d
N
linker = (CH2)5
O
O
O
O
29
linker
COOH
Br
24d
linker
COOCH3
Br
Scheme 4. Synthesis of C3-substituted pyrrolocoumarin carboxylic acid 24d. (a) NaH,
THF, 62%; (b) I2, PPh3, imidazole, 80%; (c) (i) NaNO2, HCl; (ii) SnCl2, HCl; (iii) HOAc,
40%; (d) BOC2O, DMAP, 78%; (e) Pd(PPh3)4, K2CO3, 84%; (f) K2CO3, CH3OH, 86%; (g)
(i) t-BuOK, DMF; (ii) TFA, DCM, 41%.
HN
b
HN
O
O
O
O
21a linker = CH2
22a linker = CH2
3.4. Synthesis of small-molecule SNAP-tag probes
21b
linker = (CH2)3
linker = (CH2)5
22b
linker = (CH2)3
linker = (CH2)5
21c
22c
Parallel reactions of the pyrrolocoumarin carboxylic acids
18aed and 24aed with common BG derivative 15 were accom-
plished in the presence of EDCI/HOBt, providing the target SNAP-
tag probes 1e8 in 31e96% yields, respectively (Scheme 5).
OH
linker
COOH
N
B
OH
HN
23
N
4. Optical characterizations and discussion
c
O
O
24a
linker = CH2
24b linker = (CH2)3
Optical properties of all above synthesized probes 1e8 were
examined by standard characterizations with comparison of their
parent dye A (Table 1). To our delight, all probes exhibit satisfactory
quantum yields (0.26e0.44), and retain large Stocks shifts
(112e120 nm), as well as similar emission wavelengths
(533e538 nm). These data mention that linkage of the BG moiety
with the parent dye A in the final probes 1e8 does not severely
affect the original optical properties through either pyrrole N1- or
C3-substitution. Among these probes, probe 7 possesses the most
attractive characters. Its quantum yield, Stocks shift and maximum
emission wavelength reach to 0.44, 112 nm and 534 nm, re-
spectively. Probe 7 is thus an ideal tool potentially useful for
24c
linker = (CH2)5
Scheme 3. Synthesis of C3-substituted pyrrolocoumarin carboxylic acids 24aec. (a) (i)
NaNO2, HCl; (ii) SnCl2, HCl; (iii) HOAc, heating, 35e42%; (b) SOCl2, MeOH, 37e96%;
(c) Pd(PPh3)4, K2CO3, EtOH/toluene/H2O (1:1:2), 50e61%.
21aec) with boric acid 23 afforded the carboxylic acids 24aec in the
presence of catalytic amount of Pd(PPh3)4 under N2 atmosphere.
For the case using the polyether chain, strongly acidic conditions
are unsuitable for our synthesis. Instead of the above approach, an

2254
Gr oup 1
D.-S. Mei et al. / Tetrahedron 67 (2011) 2251e2259
N
a
15
HOOC
linker
N
18a linker = CH2
O
O
18b
linker = (CH2)3
18c linker = (CH2)5
18d
linker = (CH2CH2O)4CH2CH2
O
C
N
O
N
N
H
linker
N
N
N
O
O
N
H
N-substituted probes
H2N
1
linker = CH2
2
3
4
linker = (CH2)3
linker = ( CH2)5
linker = (CH2CH2O)4CH2CH2
Group 2
COOH
linker
N
a
HN
15
O
O
24a linker = CH2
Fig. 3. Fluorescence photograph of probe 7 in DMF (0.1 mM).
24b
linker = (CH2)3
24c linker = (CH2)5
24d linker = (CH2CH2O)4CH2CH2
Based on the fluorescent properties of these compounds with
substitution at different positions with different linkers, the
structureeproperty relationship is also analyzed and concluded.
It is found that the probes with a longer hydrocarbon or poly-
glycol linker gave higher quantum yields (3 vs 1 or 2, 7 vs 5 or 6).
This mentions that the longer linker may help to decrease the
unfavorable optical interactions between the newly introduced
BG group and the parent fluorescent core. By comparison to the
corresponding probes having a hydrocarbon chain (4 vs 3, 8 vs
7), introduction of the polyglycol chain seems to decrease
quantum yield. As compared to those probes branching a linker
at their pyrrole nitrogen, the corresponding C3-substituted
probes usually offer higher quantum yields (5 vs 1, 6 vs 2, 7 vs 3,
and 8 vs 4).
O
N
NH
C
O
N
N
linker
N
N
H
H2N
HN
C3-substituted probes
O
O
5
linker = CH2
6
7
8
linker = (CH2)3
linker = (CH2)5
linker = (CH2CH2O)4CH2CH2
Scheme 5. Synthesis of 1e8. (a) EDCI, HOBt, DIPEA, DMF, 31e95%. (See Experimental
section for details).
5. Conclusion
Table 1
Optical properties of parent compound A and novel probes 1e8a
Functional research of proteins has been keeping a hot field for
centuries, but it still confronts some most basic problems and
keeps challenging to us today. SNAP-tag technology enables the
specific, covalent attachment of reporters, including fluorescent
dyes, to a certain protein of interest in live cells, offering a powerful
tool for visualization of biological events inside live cells. Many
present problems in SNAP-tag technology are resulted from the
poor fluorescent properties of probes. In this study, we have syn-
thesized and characterized a series of novel pyrrolocoumarin de-
rivatives as potentially useful fluorescent probes for the protein
investigations with SNAP-tag technology. Examination of fluo-
rescent properties shows that all these probes possess good to
excellent fluorescent properties. Among these, probe 7 exhibits
the most promising optical characters. Structureeproperty re-
lationship of these compounds is also discussed in this work. To
achieve ideal fluorescent properties, it is suggested that the pyr-
role C3-position is the better anchor position for locating a rela-
tively longer hydrocarbon-linked BG moiety. These foundings will
be very helpful for future design of similar small-molecule probes
with excellent fluorescent properties.
Compound
lex (nm)
lem (nm)
F
Stocks
shift (nm)
A
1
2
3
4
5
6
7
8
421
421
421
422
421
419
422
422
422
537
536
533
534
535
538
537
534
538
0.53
0.26
0.29
0.34
0.30
0.38
0.30
0.44
0.38
116
112
120
112
114
119
115
112
116
a
All measurements were performed in DMF; quantum yields were measured and
calculated relative to 9,10-diphenylanthracene in cyclohexane as the standard
(excited at 372 nm).
labeling the functional proteins in future biological studies (Fig. 3).
Such results also indicate that our initial design of incorporating
pyrrolocoumarin dye A into the SNAP-tag probes is successful in the
optical property stage.

D.-S. Mei et al. / Tetrahedron 67 (2011) 2251e2259
2255
6. Experimental section
6.1. General methods
2H), 1.16 (t, J¼7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
d
(ppm)
172.3, 160.2, 150.2, 148.3, 135.5, 134.4, 131.9, 128.9 (2C), 124.0, 122.8,
122.0, 112.4, 111.8 (2C), 111.6, 108.1, 106.9. 59.9, 41.8, 40.1 (2C), 30.3,
25.0, 14.0, 11.5, 9.6. MS (ESI) m/z 447.1 [MþH]þ. HRMS (ESI) m/z
calcd for C27H31N2O4 [MþH]þ: 447.2290; found, 447.2278.
For thin-layer chromatography (TLC), silica gel plates GF254 were
used and compounds were visualized by irradiation with UV light
or iodine. FT-IR spectra were recorded on an AVATAR-360 spec-
trophotometer. 1H and 13C NMR spectra were recorded on Bruker
DRX-400, or Bruker DRX-300 spectrometers with TMS as the in-
ternal standard. HRMS were measured by using either FTMS-7 or
IonSpec 4.7 spectrometers. UVevis spectra were recorded with
a Varian Cary 500 spectrophotometer and fluorescence emission
spectra were measured on a Varian Cary Eclipse fluorescence
6.1.4. 4-[8-(4-Dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyr-
ano[3,2-e]indol-3-yl]-butyric acid (18b). To
a solution of 17b
(100 mg, 0.22 mmol) in THF (4.0 mL) and water (4.0 mL) was added
LiOH hydrate (24.0 mg, 0.28 mmol) at 0 ꢀC. The resulting solution
was stirred overnight. The mixture was adjusted to pH 7 with 2 M
HCl. The solvent were removed under reduced pressure. The resi-
due was purified by flash column chromatography (dichloro-
methane/methanol¼50/1) to afford 18b as a yellow solid (86 mg,
92%). Mp 228e230 ꢀC. IR (KBr) nmax 2916, 1701, 1607 cmꢁ1. 1H NMR
spectrophotometer. Reference compound
A was synthesized
according to our previous work,7 and the known intermediates 10,9
11,10 13,11 15,8 16b,12 16c,13 20c,15 and 2314 were synthesized
according to the corresponding reference procedures, respectively.
(400 MHz, DMSO-d6)
d (ppm) 12.20 (br, 1H), 8.42 (s, 1H), 7.68 (d,
J¼8.8 Hz, 2H), 7.64 (s, J¼8.8 Hz, 1H), 7.09 (s, J¼8.8 Hz, 1H), 6.80 (d,
J¼8.8 Hz, 2H), 4.18 (t, J¼6.8 Hz, 2H), 2.96 (s, 6H), 2.48 (s, 3H), 2.39 (s,
3H), 2.27 (t, J¼6.8 Hz, 2H), 1.86e1.83 (m, 2H). 13C NMR (75 MHz,
6.1.1. [8-(4-Dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyrano
[3,2-e]indol-3-yl]-acetic acid tert-butyl ester (17a). A solution of A
(200 mg, 0.6 mmol) in DMF (7.0 mL) at 0 ꢀC was treated with NaH
(60% dispersion in mineral oil, 33 mg, 0.8 mmol). After being stirred
at this temperature for additional 30 min, tert-butyl bromoacetate
16a (0.23 mL, 1.2 mmol) was added. The resulting solution was
warmed to room temperature and stirred for 12 h. The reaction
mixture was diluted with EtOAc and quenched with 1 M HCl. The
organic phase was separated, washed with brine, dried over an-
hydrous MgSO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography (petroleum
ether/EtOAc¼10/1) to afford 17a as a yellow solid (160 mg, 60%). Mp
195e197 ꢀC. IR (KBr) nmax 3314, 1715, 1165 cmꢁ1. 1H NMR (400 MHz,
DMSO-d6)
d (ppm) 173.9, 160.2, 150.2, 148.3, 135.6, 134.6, 131.9,
128.9 (2C), 124.1, 122.9, 122.1, 112.6, 111.9 (2C), 111.6, 108.2, 106.9,
41.9, 40.3 (2C), 30.4, 25.2, 11.6, 9.7. MS (ESI) m/z 419.0 [MþH]þ.
HRMS (MALDI) m/z calcd for C25H26N2O4Na [MþNa]þ: 441.1790;
found, 441.1785.
6.1.5. 6-[8-(4-Dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyr-
ano[3,2-e]indol-3-yl]-hexanoic acid ethyl ester (17c). Compound 17c
was prepared using the above procedure for 17a. Column chro-
matography mobile phase: petroleum ether/EtOAc¼3/1. Yellow
solid (67%). Mp 150e152 ꢀC. IR (KBr) nmax 3436, 2923, 1729, 1698,
1609 cmꢁ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm) 8.35 (s, 1H), 7.65
DMSO-d6)
d
(ppm) 8.41 (s, 1H), 7.67 (d, J¼8.8 Hz, 2H), 7.58 (d,
(d, J¼8.8 Hz, 2H), 7.55 (d, J¼8.8 Hz, 1H), 7.03 (d, J¼8.8 Hz, 1H), 6.77
(d, J¼8.8 Hz, 2H), 4.08 (t, J¼7.2 Hz, 2H), 4.00 (q, J¼7.2 Hz, 2H), 2.94
(s, 6H), 2.42 (s, 3H), 2.34 (s, 3H), 2.23 (t, J¼7.2 Hz, 2H), 1.61e1.48 (m,
4H), 1.29e1.24 (m, 2H), 1.13 (t, J¼7.2 Hz, 3H). 13C NMR (100 MHz,
J¼9.2 Hz, 1H), 7.08 (d, J¼9.2 Hz, 1H), 6.79 (d, J¼8.8 Hz, 2H), 4.99 (s,
2H), 2.95 (s, 6H), 2.47 (s, 3H), 2.28 (s, 3H), 1.42 (s, 9H). 13C NMR
(100 MHz, DMSO-d6) d (ppm) 168.0, 160.2, 150.2, 148.4, 136.1, 134.5,
132.7, 128.9 (2C), 124.3, 122.8, 122.3, 112.4, 111.8 (2C), 111.7, 108.4,
107.3, 81.6, 45.2, 40.1 (2C), 27.6 (3C), 11.5, 9.6. MS (ESI) m/z 447.2
[MþH]þ. HRMS (ESI) m/z calcd for C27H31N2O4 [MþH]þ: 447.2289;
found, 447.2278.
DMSO-d6) d (ppm) 172.7, 160.2, 150.2, 148.3, 135.5, 134.5, 131.9,
128.9 (2C), 123.9, 122.8, 121.9, 112.5, 111.7 (2C), 111.6, 108.0, 106.7,
59.6, 42.5, 40.3 (2C), 33.3, 29.6, 25.7, 24.1, 14.0, 11.5, 9.7. MS (ESI) m/z
475.1 [MþH]þ. HRMS (ESI) m/z calcd for C29H35N2O4 [MþH]þ:
475.2609; found, 475.2591.
6.1.2. [8-(4-Dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyrano
[3,2-e]indol-3-yl]-acetic acid (18a). A solution of 17a (130 mg,
0.29 mmol) in CH2Cl2 (20 mL) was cooled to 0 ꢀC and treated with
TFA (0.85 mL). The reaction was warmed to room temperature and
stirred overnight. The volatiles were removed under reduced
pressure. The residue was purified by flash column chromatogra-
phy (dichloromethane/methanol¼25/1) to afford 18a as a yellow
solid (100 mg, 96%). Mp: 145e147 ꢀC. IR (KBr) nmax 3430, 2918,1704,
6.1.6. 6-[8-(4-Dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyr-
ano[3,2-e]indol-3-yl]-hexanoic acid (18c). Compound 18c was pre-
pared as the above procedure for 18b. Column chromatography
mobile phase: dichloromethane/methanol¼50/1. Yellow solid
(53%). Mp 142e144 ꢀC. IR (KBr) nmax 3434, 2919,1704,1609 cmꢁ1. 1H
NMR (400 MHz, DMSO-d6)
d (ppm) 11.99 (br, 1H), 8.39 (s, 1H), 7.67
(d, J¼8.8 Hz, 2H), 7.59 (d, J¼8.8 Hz, 1H), 7.06 (d, J¼8.8 Hz, 1H), 6.79
(d, J¼8.8 Hz, 2H), 4.12 (t, J¼7.2 Hz, 2H), 2.95 (s, 6H), 2.46 (s, 3H), 2.37
(s, 3H), 2.19 (t, J¼7.2 Hz, 2H), 1.65e1.48 (m, 4H), 1.33e1.23 (m, 2H).
1612, 1186 cmꢁ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm) 8.36 (s, 1H),
7.66 (d, J¼8.8 Hz, 2H), 7.56 (d, J¼8.8 Hz, 1H), 7.05 (d, J¼8.8 Hz, 1H),
6.78 (d, J¼8.8 Hz, 2H), 4.98 (s, 2H), 2.94 (s, 6H), 2.44 (s, 3H), 2.29 (s,
13C NMR (100 MHz, DMSO-d6)
d (ppm) 174.3, 160.2, 150.2, 148.3,
3H). 13C NMR (100 MHz, DMSO-d6)
d
(ppm) 170.4, 160.2, 150.1,
135.6, 134.5, 131.9, 128.9 (2C), 124.0, 122.9, 122.0, 112.6, 111.8 (2C),
111.6, 108.1, 106.7, 42.6, 40.3 (2C), 33.5, 29.7, 25.8, 24.2, 11.6, 9.7. MS
(ESI) m/z 445.1 [MþH]þ. HRMS (MALDI) m/z calcd for C27H30N2O4
Na [MþNa]þ: 469.2101; found, 469.2098.
148.5, 136.2, 134.5, 132.7, 128.9 (2C), 124.1, 123.0, 122.3, 112.4, 111.9
(2C), 111.6, 108.3, 107.2, 44.3, 40.1 (2C), 11.6, 9.6. MS (ESI) m/z 391.0
[MþH]þ. HRMS (ESI) m/z calcd for C23H21N2O4 [MdH]ꢁ: 389.1492;
found, 389.1506.
6.1.7. tert-Butyl
1-hydroxy-3,6,9,12-tetraoxapentadecan-15-oate
6.1.3. 4-[8-(4-Dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyr-
ano[3,2-e]indol-3-yl]-butyric acid ethyl ester (17b). Compound 17b
was prepared using the above procedure for 17a. Column chro-
matography mobile phase: petroleum ether/EtOAc¼3/1. Yellow
solid (67%). Mp: 150e152 ꢀC. IR (KBr) nmax 2916, 1736, 1697,
(16de1). To a solution of tetraethylene glycol (20.0 g, 103 mol) in
anhydrous THF (54.0 mL) were added NaH (60% dispersion in
mineral oil, 42 mg, 1.05 mmol) and tert-butyl acrylate (5.2 mL,
36.0 mol). The resulting solution was stirred for 20 h at room
temperature. The solvent was removed under reduced pressure.
The residue was purified by flash column chromatography (petro-
leum ether/EtOAc¼1/1) to afford 16de1 as a colorless oil (9.3 g,
1607 cmꢁ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm) 8.42 (s, 1H), 7.68
(d, J¼8.8 Hz, 2H), 7.64 (d, J¼8.8 Hz, 1H), 7.10 (d, J¼8.8 Hz, 1H), 6.80
(d, J¼8.8 Hz, 2H), 4.18 (t, J¼7.2 Hz, 2H), 4.04 (q, J¼7.2 Hz, 2H), 2.96 (s,
6H), 2.48 (s, 3H), 2.39 (s, 3H), 2.34 (t, J¼7.2 Hz, 2H), 1.90e1.86 (m,
80%). IR (KBr) nmax 3445, 2873, 1729 cmꢁ1 1H NMR (400 MHz,
.
CDCl3)
d
(ppm) 3.65e3.53 (m, 18H), 2.43 (t, J¼8.4 Hz, 2H), 1.37

2256
D.-S. Mei et al. / Tetrahedron 67 (2011) 2251e2259
(s, 9H). 13C NMR (100 MHz, CDCl3)
(2C), 70.3, 70.2, 70.1, 70.0, 66.7, 61.3, 36.0, 27.9 (3C). MS (ESI) m/z
340.2 [MþNH4]þ. HRMS (ESI) m/z calcd for C15H30O7Na [MþNa]þ:
345.1883; found, 345.1884.
d
(ppm) 170.7, 80.3, 72.6, 70.4
mixture was adjusted to pH 8 by the addition of saturated NaHCO3
solution. The organic phase was separated and washed with brine,
dried over anhydrous Na2SO4, and concentrated. The residue was
purified by flash column chromatography (dichloromethane/
methanol¼25/1) to afford 21a as a yellow solid (117 mg, 35%). Mp
6.1.8. tert-Butyl 1-iodo-3,6,9,12-tetraoxapentadecan-15-oate(16d). To
a solution of 16de1 (1.2 g, 3.6 mmol) in THF (30 mL) were added
PPh3 (2.8 g, 10.8 mmol) and imidazole (780 mg, 11.5 mmol) slowly at
0 ꢀC. After the mixture was stirred at same temperature for 2 min, I2
(2.9 g, 11.5 mmol) was added. The resulting brown-black slurry was
stirred overnight at 0 ꢀC. Saturated aqueous Na2S2O3 was added to
quench the reaction. The resultant mixture was filtered, and the
filtrate was concentrated under reduced pressure. The residue was
extracted with ethyl acetate. The extract was successively washed
with 1 M HCl and brine, and concentrated. The residue was purified
by flash column chromatography (petroleum ether/EtOAc¼10/1) to
afford 16d as a colorless oil (1.2 g, 82%). IR (KBr) nmax 2870,
238e240 ꢀC. IR (KBr) nmax 3292, 1708 cmꢁ1 1H NMR (400 MHz,
.
DMSO-d6)
d (ppm) 12.40 (s, 1H), 11.54 (s, 1H), 8.84 (s, 1H), 7.59 (d,
J¼8.8 Hz, 1H), 7.09 (d, J¼8.8 Hz, 1H), 3.83 (s, 2H), 2.41 (s, 3H). 13C
NMR (75 MHz, DMSO-d6)
d (ppm) 173.2, 156.8, 149.1, 143.0, 137.5,
131.6, 122.0, 115.7, 110.2, 108.6, 107.5, 105.4, 31.1, 11.3. MS (ESI) m/z
335.9 [MþH]þ. HRMS (ESI) m/z calcd for C14H10BrNO4 Na [MþNa]þ:
357.9687; found, 357.9685.
6.1.12. Methyl 2-(8-bromo-2-methyl-7-oxo-3,7-dihydropyrano[3,2-
e]indol-1-yl)acetate (22a). To a solution of 21a (100 mg, 0.3 mmol)
in dry MeOH (2.1 mL) was added SOCl2 (34 006DL, 0.47 mmol).
The resulting mixture was refluxed for 1.5 h, and then cooled
down to room temperature. The solvent was removed under re-
duced pressure. The residue was purified by flash column chro-
matography (petroleum ether/EtOAc¼3/1) to afford 22a as
a yellow solid (100 mg, 96%). Mp 269e270 ꢀC. IR (KBr) nmax 3438,
1731 cmꢁ1. 1H NMR (400 MHz, CDCl3)
d (ppm) 3.70e3.55 (m, 16H),
3.19 (t, J¼7.2 Hz, 2H), 2.42 (t, J¼6.4 Hz, 2H), 1.38 (s, 9H). 13C NMR
(75 MHz, CDCl3)
d (ppm) 170.5, 80.0, 71.7, 70.4 (2C), 70.3 (2C),
70.2 (2C), 70.0, 66.7, 36.1, 27.9 (3C). MS (ESI) m/z 450.0 [MþNH4]þ.
HRMS (ESI) m/z calcd for C15H29IO6Na [MþNa]þ: 455.0897;
found,455.0901.
1695 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
. d (ppm) 11.58 (s, 1H),
8.79 (s, 1H), 7.59 (d, J¼8.8 Hz, 1H), 7.08 (d, J¼8.8 Hz, 1H), 3.94 (s,
2H), 3.61 (s, 3H), 2.41 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
6.1.9. tert-Butyl-1-(8-(4-(dimethylamino)phenyl)-1,2-dimethyl-7-
oxopyrano[3,2-e]indol-3(7H)-yl)-3,6,9,12-tetraoxapentadecan-15-
oate (17d). Compound 17d was prepared as the above procedure
for 17a. Column chromatography mobile phase: petroleum ether/
EtOAc¼2/1. Yellow oil (34%). IR (KBr) nmax 2867, 1709, 1610 cmꢁ1. 1H
d (ppm) 172.1, 156.8, 149.1, 142.9, 137.7, 131.7, 121.9, 115.8, 110.2,
108.8, 107.6, 104.6, 51.8, 30.7, 11.3. MS (MALDI) m/z 350.0 [MþH]þ.
HRMS (MALDI) m/z calcd for C15H12BrNO4 [MþH]þ 350.0034;
found, 350.0025.
NMR (400 MHz, CDCl3)
d
(ppm) 8.47 (s, 1H), 7.71 (d, J¼8.8 Hz, 2H),
6.1.13. 2-(8-(4-(Dimethylamino)phenyl)-2-methyl-7-oxo-3,7-dihy-
dropyrano[3,2-e]indol-1-yl)acetic acid (24a). To a mixture of 22a
(240 mg, 0.69 mmol), 23 (230 mg, 1.35 mmol), K2CO3 (470 mg,
2.06 mmol), and Pd(PPh3)4 (40 mg, 0.034 mmol) was added the
mixture solvent EtOH/toluene/H2O (1.7 mL, 1.7 mL, 3.4 mL) under
nitrogen. The reaction was heated at 90 ꢀC for 6 h. The solvents
were evaporated under reduced pressure, the residue was diluted
with H2O (10 mL) and EtOAc (50 mL). The organic solution was
washed with saturated brine, dried over anhydrous Na2SO4, and
concentrated. The residue was purified by flash column chroma-
tography (dichloromethane/methanol¼10/1) to afford 24a as
a yellow solid (130 mg, 50%). Mp 180e181 ꢀC. IR (KBr) nmax 3399,
7.38 (d, J¼8.8 Hz, 1H), 7.10 (d, J¼8.8 Hz, 1H), 6.80 (d, J¼8.8 Hz, 2H),
4.29 (t, J¼6.0 Hz, 2H), 3.74e3.49 (m, 16H), 3.00 (s, 6H), 2.50e2.40
(m, 5H), 2.40 (s, 3H), 1.44 (s, 9H). 13C NMR (75 MHz, CDCl3)
d (ppm)
171.0, 161.8, 150.7, 149.4, 135.8, 135.3, 132.6, 129.4 (2C), 125.6, 123.9,
122.7, 112.5, 112.2 (2C), 111.9, 109.1, 107.5, 80.4, 70.8, 70.5 (2C), 70.4
(2C), 70.3 (2C), 70.1, 66.8, 43.5, 40.4, 36.2, 28.0 (2C), 12.1, 10.2. MS
(MALDI) m/z 637.3 [MþH]þ. HRMS (MALDI) m/z calcd for
C36H48N2O6Na [MþNa]þ: 659.3318; found, 659.3302.
6.1.10. 1-(8-(4-(Dimethylamino)phenyl)-1,2-dimethyl-7-oxopyrano
[3,2-e]indol-3(7H)-yl)-3,6,9,12-tetraoxapentadecan-15-oic acid (18d).
Compound 18d was prepared as the above procedure for 18a. Col-
umn chromatography mobile phase: dichloromethane/meth-
anol¼50/1. Yellow oil (80%). IR (KBr) nmax 2919, 1707, 1609 cmꢁ1. 1H
1678 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
. d (ppm) 12.36 (s, 1H),
11.41 (s, 1H), 8.49 (s, 1H), 7.72 (d, J¼8.8 Hz, 2H), 7.49 (d, J¼8.8 Hz,
1H), 7.06 (d, J¼8.8 Hz, 1H), 6.78 (d, J¼8.8 Hz, 2H), 3.87 (s, 2H), 2.97
NMR(300 MHz, CDCl3)
d
(ppm) 8.47 (s, 1H), 7.71 (d, J¼8.7 Hz, 2H),
(s, 6H), 2.41 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
d (ppm) 173.4,
7.40 (d, J¼8.7 Hz, 1H), 7.10 (d, J¼8.7 Hz, 1H), 6.82 (d, J¼8.7 Hz, 2H),
4.29 (t, J¼4.8 Hz, 2H), 3.75e3.56 (m, 16H), 3.01 (s, 6H), 2.63 (t,
J¼7.6 Hz, 2H), 2.50 (s, 3H), 2.40 (s, 3H). 13C NMR (75 MHz, CDCl3)
160.2, 150.2, 148.2, 136.5, 134.9, 131.4, 128.9 (2C), 123.7, 122.7, 122.5,
114.1, 111.7 (2C), 111.0, 108.4, 105.4, 31.5, 11.2. MS (ESI) m/z 377.1
[MþH]þ. HRMS (ESI) m/z calcd for C22H21N2O4 [MþH]þ: 377.1491;
found, 377.1496.
d
(ppm) 175.2,161.9,150.6,149.4,135.8,135.5,132.7,129.4 (2C),125.5,
124.0, 122.9, 112.8, 112.5 (2C), 112.3, 109.4, 107.7, 71.1, 70.8 (2C), 70.7
(2C), 70.6, 70.4, 70.3, 66.6, 43.7, 40.7 (2C), 35.1, 29.9, 12.4, 10.4. MS
(ESI) m/z 579.2 [MꢁH]ꢁ. HRMS (MALDI) m/z calcd for C32H40N2O8Na
[MþNa]þ: 603.2688; found, 603.2677.
6.1.14. 4-(8-Bromo-2-methyl-7-oxo-3,7-dihydropyrano[3,2-e]indol-
1-yl)butanoic acid (21b). Compound 21b was prepared as the above
procedure for 21a. Column chromatography mobile phase:
dichloromethane/methanol¼25/1. Yellow solid (35%). Mp
6.1.11. 2-(8-Bromo-2-methyl-7-oxo-3,7-dihydropyrano[3,2-e]indol-
1-yl)acetic acid (21a). To a solution of the 6-amino-3-bromocou-
marin (19) (239 mg, 1.0 mmol) in HCl (37%, 1.0 mL) was added
NaNO2 (83 mg, 1.20 mmol) in H2O (0.5 mL) at ꢁ5 ꢀC. After being
stirred at this temperature for 30 min, the resulting mixture was
treated with a pre-cooled (ꢁ10 to ꢁ5 ꢀC) solution of stannous
chloride dihydrate (452 mg, 2.0 mmol) in HCl (37%, 0.5 mL). The
mixture was stirred at ꢁ5 ꢀC for an additional 5 h. The reaction
mixture was filtered. The solid was added into a solution of 20a
(278 mg, 2.4 mmol) in HOAc (5 mL). The mixture was then heated
to 80 ꢀC for 4 h. The solvent was evaporated in vacuo, and the
residue was diluted with H2O (10 mL) and EtOAc (50 mL). The
169e171 ꢀC. IR (KBr) nmax 3418, 1729 cmꢁ1 1H NMR (300 MHz,
.
DMSO-d6)
d (ppm) 12.19 (s, 1H), 11.39 (s, 1H), 8.75 (s, 1H), 7.53 (d,
J¼8.4 Hz, 1H), 7.00 (d, J¼8.4 Hz, 1H), 2.79 (t, J¼6.9 Hz, 2H),
2.35e2.29 (m, 5H), 1.73e1.69 (m, 2H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm) 174.9, 157.3, 149.4, 142.9, 136.7, 132.2, 121.8, 116.1, 111.7,
110.7, 108.8, 108.5, 33.3, 25.6, 24.3, 11.7. MS (ESI) m/z 362.0 [MꢁH]ꢁ.
HRMS (MALDI) m/z calcd for C16H14BrNO4 Na [MþNa]þ: 385.9999;
found, 385.9998.
6.1.15. Methyl 4-(8-bromo-2-methyl-7-oxo-3,7-dihydropyrano[3,2-
e]indol-1-yl)butanoate (22b). Compound 22b was prepared as the
above procedure for 22a. Column chromatography mobile phase:

D.-S. Mei et al. / Tetrahedron 67 (2011) 2251e2259
2257
petroleum ether/EtOAc¼3/1. Yellow solid (85%). Mp 202e204 ꢀC. IR
petroleum ether/EtOAc¼1/2. Colorless oil (62%). IR (KBr) nmax 3444,
(KBr) nmax 3272, 1732, 1689 cmꢁ1
.
1H NMR (400 MHz, DMSO-d6)
2873, 1728 cmꢁ1. 1H NMR (400 MHz, CDCl3)
d (ppm) 3.66e3.56 (m,
d
(ppm) 11.41 (s, 1H), 8.74 (s, 1H), 7.56 (d, J¼8.8 Hz, 1H), 7.05 (d,
J¼8.8 Hz, 1H), 3.59 (s, 3H), 2.84 (t, J¼7.2Hz, 2H), 2.42e2.36 (m, 5H),
1.80e1.76 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
(ppm) 173.2,
14H), 2.62 (br, 1H), 2.44 (t, J¼5.2 Hz, 2H), 1.38 (s, 9H). 13C NMR
(100 MHz, CDCl3)
d (ppm) 170.8, 80.3, 72.5, 70.5, 70.4, 70.2 (2C),
d
66.8, 61.5, 36.1, 28.0 (3C). MS (ESI) m/z 301.0 [MþNa]þ. HRMS (ESI)
156.8, 148.9, 142.3, 136.3, 131.7, 121.2, 115.6, 111.0, 110.1, 108.3, 107.9,
51.2, 32.5, 25.1, 23.7, 11.2. MS (ESI) m/z 399.8 [MþNa]þ. HRMS (ESI)
m/z calcd for C17H17BrNO4 [MþH]þ: 378.0346; found, 378.0336.
m/z calcd for C13H26O6Na [MþNa]þ: 301.1619; found, 301.1622.
6.1.21. tert-Butyl 3-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)propanoate
(20d). Compound 20d was prepared as the above procedure for
16d. Column chromatography mobile phase: petroleum ether/
EtOAc¼20/1. Colorless oil (79%). IR (KBr) nmax 2870, 1729 cmꢁ1. 1H
6.1.16. 4-(8-(4-(Dimethylamino)phenyl)-2-methyl-7-oxo-3,7-dihy-
dropyrano[3,2-e]indol-1-yl)butanoic acid (24b). Compound 24b was
prepared as the above procedure for 24a. Column chromatography
mobile phase: dichloromethane/methanol¼50/1. Yellow solid
(55%). Mp 204e205 ꢀC. IR (KBr) nmax 3314, 1685, 1608 cmꢁ1. 1H NMR
NMR (400 MHz, CDCl3)
d (ppm) 3.77e3.60 (m, 12H), 3.26
(t, J¼6.8 Hz, 2H), 2.51 (t, J¼6.4 Hz, 2H), 1.45 (s, 9H). 13C NMR
(100 MHz, CDCl3)
d (ppm) 170.8, 80.4, 71.9, 70.6 (2C), 70.5, 70.4,
(400 MHz, DMSO-d6)
d
(ppm) 12.06 (s,1H),11.29 (s,1H), 8.33 (s,1H),
70.2, 66.9, 36.3, 28.1 (3C). MS (ESI) m/z 410.9 [MþNa]þ. HRMS (ESI)
7.67 (d, J¼8.8 Hz, 2H), 7.47 (d, J¼8.8 Hz, 1H), 7.04 (d, J¼8.8 Hz, 1H),
6.80 (d, J¼8.8 Hz, 2H). 2.97 (s, 6H), 2.92 (t, J¼7.2 Hz, 2H), 2.38 (s, 3H),
2.30 (t, J¼7.2 Hz, 2H), 1.86 (t, J¼7.2 Hz, 2H). 13C NMR (75 MHz,
m/z calcd for C13H25IO5Na [MþNa]þ: 411.0635; found, 411.0639.
6.1.22. 2-(8-Bromo-2-methyl-7-oxo-3,7-dihydropyrano[3,2-e]indol-
1-yl)ethyl acetate (26). Compound 26 was prepared as the above
procedure for 21a. Column chromatography mobile phase: petro-
leum ether/EtOAc¼4/1. Yellow solid (20%). Mp 211e212 ꢀC. IR (KBr)
DMSO-d6) d (ppm) 174.8,160.7,150.7,148.9,135.9,134.9,132.1,129.4
(2C), 124.7, 123.2, 122.4, 114.7, 112.3 (2C), 111.6, 111.4, 108.7, 40.9
(2C), 33.6, 26.0, 24.7,11.7. MS (ESI) m/z 403.1[MꢁH]ꢁ. HRMS (ESI) m/
z calcd for C24H23N2O4 [MꢁH]ꢁ: 403.1671; found, 403.1663.
nmax 3350, 1736, 1700 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
d (ppm)
11.53 (s, 1H), 8.93 (s, 1H), 7.57 (d, J¼8.8 Hz, 1H), 7.07 (d, J¼8.8 Hz,
6.1.17. 6-(8-Bromo-2-methyl-7-oxo-3,7-dihydro-pyrano[3,2-e]indol-
1-yl)-hexanoic acid (21c). Compound 21c was prepared as the
above procedure for 21a. Column chromatography mobile phase:
dichloromethane/methanol¼25/1. Yellow solid (35%). Mp
1H), 4.17 (t, J¼7.2 Hz, 2H), 3.16 (t, J¼7.2 Hz, 2H), 2.40 (s, 3H), 1.98 (s,
3H). 13C NMR (75 MHz, DMSO-d6)
d (ppm) 190.5, 176.8, 169.0, 162.5,
157.3, 151.6, 141.7, 135.7, 130.2, 128.5, 127.9, 127.1, 83.8, 44.2, 40.6,
31.2. MS (ESI) m/z 385.9 [MþNa]þ. HRMS (MALDI) m/z calcd for
C16H14NO4 BrNa [MþNa]þ: 386.0004; found, 385.9998.
169e171 ꢀC. IR (KBr) nmax 3312, 1717 cmꢁ1 1H NMR (400 MHz,
.
DMSO-d6)
d (ppm) 11.96 (s, 1H), 11.38 (s, 1H), 8.58 (s, 1H), 7.56 (d,
J¼8.8 Hz, 1H), 7.05 (d, J¼8.8 Hz,1H), 2.80 (s, 2H), 2.37 (s, 3H), 2.21 (t,
6.1.23. tert-Butyl 1-(2-acetoxyethyl)-8-bromo-2-methyl-7-oxopyr-
ano[3,2-e]indole-3(7H)-carboxylate (27). To a solution of 26 (66 mg,
0.18 mmol) in THF (3.0 mL) was added di-tert-butyl dicarbonate
(0.09 mL, 0.42 mmol) and 4-N,N-dimethylaminopyridine (33 mg,
0.27 mmol) at 0 ꢀC. The resulting mixture was stirred at 0 ꢀC for
further 5 min. Then, 1 M HCl (0.22 mL) was added to quench the
reaction. The solvent was removed under reduced pressure. Water
was added, and the mixture was extracted with ethyl acetate. The
extract was dried over Na2SO4, filtered, and concentrated to dry-
ness under reduced pressure. The residue was purified by flash
column chromatography (petroleum ether/EtOAc¼10/1) to afford
27 as a white solid (69 mg, 82%). Mp 178e179 ꢀC. IR (KBr) nmax 2978,
J¼7.2 Hz, 2H), 1.59e1.36 (m, 6H). 13C NMR (75 MHz, DMSO-d6)
d
(ppm) 174.3, 156.7, 148.9, 142.3, 135.9, 131.6, 121.2, 115.5, 111.8,
110.0, 108.1, 107.6, 40.3, 33.7, 29.5, 28.4, 24.3, 11.3. MS (ESI) m/z
392.0 [MþH]þ. HRMS (MALDI) m/z calcd for C18H19NO4Br [MþH]þ:
392.0491; found, 392.0492.
6.1.18. Methy 6-(8-bromo-2-methyl-7-oxo-3,7-dihydropyrano[3,2-e]
indol-1-yl)hexanoate (22c). Compound 22c was prepared as the
above procedure for 22a. Column chromatography mobile phase:
petroleum ether/EtOAc¼6/1. Yellow solid (55%). Mp 141e142 ꢀC. IR
(KBr) nmax 3330, 1694 cmꢁ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm)
11.39 (s, 1H), 8.58 (s, 1H), 7.56 (d, J¼8.8 Hz, 1H), 7.05 (d, J¼8.8 Hz,
1728 cmꢁ1. 1H NMR (400 MHz, CDCl3)
d (ppm) 8.92 (s, 1H), 8.38 (d,
1H), 3.56 (s, 3H), 2.80 (t, J¼7.2 Hz, 2H), 2.37 (s, 3H), 2.30 (t, J¼7.2 Hz,
J¼8.8 Hz, 1H), 7.20 (d, J¼8.8 Hz, 1H), 4.26 (t, J¼7.2 Hz, 2H), 3.22 (t,
2H), 1.60e1.36 (m, 6H). 13C NMR (100 MHz, DMSO-d6)
d (ppm)
J¼7.2 Hz, 2H), 2.61 (s, 3H), 2.06 (s, 3H), 1.70 (s, 9H). 13C NMR
173.2, 156.8, 149.0, 142.4, 136.0, 131.7, 121.3, 115.6, 111.7, 110.1, 108.2,
107.7, 51.1, 40.1, 33.3, 29.5, 28.2, 24.3, 11.3. MS (ESI) m/z 406.0
[MþH]þ. HRMS (ESI) m/z calcd for C19H20BrNO4Na [MþNa]þ:
428.0484; found, 428.0468.
(75 MHz, CDCl3) d (ppm) 170.9,157.2,150.9,149.8,141.6,138.1,132.4,
124.1, 119.3, 113.5, 112.0, 111.1, 110.6, 85.1, 63.4, 28.2 (3C), 25.1, 20.9,
13.8. MS (MALDI) m/z 464.1[MþH]þ.
6.1.24. tert-Butyl 1-(2-acetoxyethyl)-8-(4-(dimethylamino)phenyl)-2-
methyl-7-oxopyrano [3,2-e]indole-3(7H)-carboxylate(28). Compound
28 was prepared as the above procedure for 24a. Column chroma-
tography mobile phase: petroleum ether/EtOAc¼8/1. Yellow solid
(88%). Mp 173e174 ꢀC. IR (KBr) nmax 3446, 1717, 1609 cmꢁ1. 1H NMR
6.1.19. 6-(8-(4-(Dimethylamino)phenyl)-2-methyl-7-oxo-3,7-dihy-
dropyrano[3,2-e]indol-1-yl)hexanoic acid (24c). Compound 24c was
prepared as the above procedure for 24a. Column chromatography
mobile phase: dichloromethane/methanol¼50/1. Yellow solid
(61%). Mp 194e195 ꢀC. IR (KBr) nmax 3258, 2924,1685 cmꢁ1. 1H NMR
(400 MHz, DMSO-d6)
d
(ppm) 8.40 (s, 1H), 8.25 (d, J¼9.2 Hz, 1H), 7.68
(400 MHz, DMSO-d6)
d
(ppm) 11.96 (br, 1H), 11.25 (s, 1H), 8.28 (s,
(d, J¼8.8 Hz, 2H), 7.28 (d, J¼9.2 Hz,1H), 6.80 (d, J¼8.8 Hz, 2H), 4.26 (t,
1H), 7.64 (d, J¼8.4 Hz, 2H), 7.46 (d, J¼8.4 Hz, 1H), 7.03 (d, J¼8.4 Hz,
1H), 6.81 (d, J¼8.4 Hz, 2H), 2.97 (s, 6H), 2.86 (t, J¼6.8 Hz, 2H), 2.38
(s, 3H), 2.20 (t, J¼6.8 Hz, 2H), 1.64e1.40 (m, 6H). 13C NMR (100 MHz,
J¼6.4 Hz, 2H), 3.30 (t, J¼6.8 Hz, 2H), 2.97 (s, 6H), 2.56 (s, 3H), 1.91 (s,
3H),1.66 (s, 9H). 13C NMR (75 MHz, CDCl3)
d (ppm) 170.8,160.9,150.5
(2C), 150.1, 137.3, 133.6, 132.3, 129.4 (2C), 126.8, 124.5, 117.8, 114.0,
112.3, 112.2, 112.0, 111.9 (2C), 84.7, 63.5, 40.5, 28.3 (3C), 25.4, 20.9,
13.8. MS (MALDI) m/z 505.2 [MþH]þ. HRMS (MALDI) m/z calcd for
C29H33N2O6 [MþH]þ: 505.2336; found, 505.2333.
DMSO-d6)
d (ppm) 174.3, 160.2, 150.2, 148.4, 135.0, 134.6, 131.5,
128.8 (2C),124.1,122.8, 121.9, 114.1, 111.8 (3C),110.9,108.0, 40.3 (2C),
33.8, 30.1, 28.7, 24.9, 24.5, 11.2. MS (ESI) m/z 433.0 [MþH]þ. HRMS
(ESI) m/z calcd for C26H29N2O4 [MþH]þ: 433.2126; found, 433.2122.
6.1.25. tert-Butyl 8-(4-(dimethylamino)phenyl)-1-(2-hydroxyethyl)-
6.1.20. tert-Butyl
3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)prop-
2-methyl-7-oxopyrano[3,2-e]
indole-3(7H)-carboxylate
(29). A
anoate (20de1). Compound 20de1 was prepared as the above
procedure for 16de1. Column chromatography mobile phase:
mixture of 28 (145 mg, 0.29 mmol) and K2CO3 (80 mg, 0.58 mmol)
in MeOH (14.0 mL) and CH2Cl2 (14.0 mL) was stirred at room

2258
D.-S. Mei et al. / Tetrahedron 67 (2011) 2251e2259
temperature for 2 h. Water (60 mL) was added. The resulting so-
lution was extracted with CH2Cl2 (20 mLꢂ3). The organic phase was
dried over Na2SO4 and concentrated. The residue was purified by
flash column chromatography (petroleum ether/EtOAc¼4/1) to af-
ford 29 as a yellow solid (114 mg, 86%). Mp 155e157 ꢀC. IR (KBr)
procedure for 1. Column chromatography mobile phase: DCM/
MeOH¼50/1. Yellow solid (85%). Mp 262e263 ꢀC. IR (KBr) nmax
3274, 1610 cmꢁ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm) 12.41 (s,
1H), 8.45 (s, 1H), 8.36 (br, 1H), 7.80 (s, 1H), 7.69 (d, J¼8.8 Hz, 2H),
7.65 (d, J¼8.8 Hz, 2H), 7.45 (d, J¼7.6 Hz, 2H), 7.27 (d, J¼7.6 Hz, 2H),
7.09 (d, J¼8.8 Hz, 1H), 6.81 (d, J¼8.8 Hz, 2H), 6.28 (s, 2H), 5.44 (s,
2H), 4.27 (d, J¼5.2 Hz, 2H), 4.18 (t, J¼6.8 Hz, 2H), 2.96 (s, 6H), 2.38
(s, 3H), 2.20 (t, J¼6.4 Hz, 2H), 1.90e1.87 (m, 2H). 13C NMR (75 MHz,
nmax 3291, 1678 cmꢁ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm) 8.47 (s,
1H), 8.24 (d, J¼9.2 Hz, 1H), 7.69 (d, J¼8.8 Hz, 2H), 7.26 (d, J¼9.2 Hz,
1H), 6.80 (d, J¼8.8 Hz, 2H), 4.87 (br, 1H), 3.65 (s, 2H), 3.10 (t,
J¼6.8 Hz, 2H), 2.97 (s, 6H), 2.54 (s, 3H), 1.65 (s, 9H). 13C NMR
DMSO-d6) d (ppm) 171.3, 160.2, 159.5 (2C), 150.3 (2C), 148.4, 139.5,
(75 MHz, DMSO-d6)
d
(ppm) 159.6, 150.4, 149.6, 149.5, 136.4, 133.4,
138.2, 135.7, 135.1, 134.6, 131.9, 128.9 (2C), 128.5 (2C), 127.3 (2C),
124.1, 122.8, 122.1, 112.7, 111.8 (3C), 111.6, 108.2, 106.8, 66.58, 42.27,
41.90, 40.1 (2C), 31.8, 25.8, 11.6, 9.7. MS (MALDI) m/z 671.7 [MþH]þ.
HRMS (ESI) m/z calcd for C38H38N8O4Na [MþNa]þ: 693.2889;
found, 693.2908.
131.5, 129.2 (2C), 129.1, 125.3, 124.4, 122.2, 117.2, 115.4, 111.7 (2C),
111.4,110.9, 84.4, 60.5, 40.1 (2C), 28.7, 27.6 (3C),13.5. MS (MALDI) m/
z 463.2 [MþH]þ. HRMS (MALDI) m/z calcd for C27H31N2O5 [MþH]þ:
463.2235; found, 463.2228.
6.1.26. 3-{2-[2-(2-{2-[8-(4-Dimethylamino-phenyl)-2-methyl-7-
oxo-3,7-dihydro-pyrano[3,2-e]indol-1-yl]-ethoxy}-ethoxy)-ethoxy]-
ethoxy}-propionic acid (24d). To a solution of 29 (60 mg, 0.13 mmol)
in anhydrous DMF (10 mL) was added t-BuOK (19 mg, 0.20mmol) at
room temperature. After being stirred for 30 min, compound 20d
(85 mg, 0.26 mmol) was added. The reaction mixture was stirred for
4 h. The solvent was evaporated under reduced pressure. The res-
idue was diluted with H2O (5 mL) and EtOAc (10 mL), and the or-
ganic phase was separated and concentrated. TFA (0.2 mL) and THF
(2.0 mL) was added to the residue at 0 ꢀC, and the resulting solution
was warmed to room temperature and stirred overnight. The re-
action mixture was concentrated. The residue was diluted with H2O
(5 mL) and EtOAc (10 mL), the mixture was adjusted to pH 7 with
saturated aqueous NaHCO3. The organic phase was separated and
concentrated. The residue was purified by flash column chroma-
tography (dichloromethane/methanol¼20/1) to afford 24d as
a yellow oil (30 mg, 41%). IR (KBr) nmax 3361, 2920, 1706 cmꢁ1. 1H
6.1.29. 6-[8-(4-Dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyr-
ano[3,2-e]indol-3-yl]-hexanoic acid 4-(2-amino-9H-purin-6-yloxy-
methyl)-benzylamide (3). Probe 3 was prepared as the above
procedure for 1. Column chromatography mobile phase: DCM/
MeOH¼50/1. Yellow solid (46%). Mp 195e198 ꢀC. IR (KBr) nmax 3391,
2922,1609 cmꢁ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm) 8.44 (s,1H),
8.29 (br, 1H), 7.93 (s, 1H), 7.68 (d, J¼8.4 Hz, 2H), 7.61 (d, J¼8.8 Hz,
1H), 7.45 (d, J¼8.0 Hz, 2H), 7.25 (d, J¼8.0 Hz, 2H), 7.08 (d, J¼8.8 Hz,
1H), 6.79 (d, J¼8.4 Hz, 2H), 6.44 (s, 2H), 5.45 (s, 2H), 4.24 (d,
J¼5.2 Hz, 2H), 4.14 (t, J¼6.8 Hz, 2H), 2.96 (s, 6H),2.46 (s, 3H), 2.38 (s,
3H), 2.19 (t, J¼7.2 Hz, 2H), 1.65e1.53 (m, 4H), 1.29e1.27 (m, 2H). 13C
NMR (75 MHz, DMSO-d6)
d (ppm) 171.9, 160.2, 159.3 (2C), 155.6,
150.2, 148.3, 139.6, 138.7, 135.7, 135.0, 134.6, 131.9, 128.9 (2C), 128.5
(2C), 127.3 (2C), 124.1, 122.8, 122.0, 112.7, 111.8 (3C), 111.6, 108.1,
106.7, 66.7, 42.6, 41.7, 40.3 (2C), 35.1, 29.7, 25.9, 24.9, 11.6, 9.8. MS
(MALDI) m/z 721.6 [MþNa]þ. HRMS (MALDI) m/z calcd for
C40H42N8O4Na [MþNa]þ: 721.3215; found, 721.3221.
NMR (400 MHz, CDCl3)
d
(ppm) 8.36 (s, 1H, 8.36), 7.67 (d, J¼8.4 Hz,
2H), 7.34 (d, J¼8.8 Hz, 1H), 7.04 (d, J¼8.4 Hz, 1H), 6.81 (d, J¼8.4 Hz,
2H), 4.26e3.45 (m, 16H), 3.18 (t, J¼7.6 Hz, 2H), 2.98 (s, 6H), 2.55 (t,
6.1.30. N-[4-(2-Amino-9H-purin-6-yloxymethyl)-benzyl]-3-{2-[2-(2-
{2-[8-(4-dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyrano[3,2-
e]indol-3-yl]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionamide
(4). Probe 4 was prepared as the above procedure for 1. Column
chromatography mobile phase: DCM/MeOH¼50/1. Yellow solid
(54%). Mp 142e143 ꢀC. IR (KBr) nmax 3411, 2864, 1609 cmꢁ1. 1H NMR
J¼6.0 Hz, 2H), 2.43 (s, 3H). 13C NMR (100 MHz, CDCl3)
d (ppm) 174.9,
161.5, 149.3 (2C), 137.7, 134.9, 132.6, 129.3 (2C), 125.3, 122.2 (2C),
112.6, 112.3 (2C), 112.1, 109.3, 108.3, 70.8, 70.6 (2C), 70.4, 70.3, 70.0,
66.4, 62.5, 43.6, 40.6, 34.8, 29.3, 10.6. MS (ESI) m/z 565.2[MꢁH]ꢁ.
HRMS (MALDI) m/z calcd for C26H29N2O4 [MþH]þ: 566.2612;
found, 566.2628.
(400 MHz, DMSO-d6)
d
(ppm) 8.45 (s, 1H), 8.32 (t, J¼6.0 Hz, 1H),
7.83 (s, 1H), 7.68 (d, J¼8.8 Hz, 2H), 7.64 (d, J¼8.8 Hz, 1H), 7.43 (d,
J¼8.0 Hz, 2H), 7.25 (d, J¼8.0 Hz, 2H), 7.08 (d, J¼8.8 Hz, 1H), 6.80 (d,
J¼8.8 Hz, 2H), 6.27 (s, 2H), 5.45 (s, 2H), 4.35 (t, J¼5.2 Hz, 2H), 4.25
(d, J¼5.6 Hz, 2H), 3.66e3.32 (m, 18H), 2.95 (s, 6H), 2.40 (s, 3H), 2.36
6.1.27. N-[4-(2-Amino-9H-purin-6-yloxymethyl)-benzyl]-2-[8-(4-di-
methylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyrano[3,2-e]indol-3-
yl]-acetamide (1). To a solution of compound 15 (52 mg, 0.19 mmol),
compound 18a (75 mg, 0.19 mmol), EDC$HCl (55 mg, 0.29 mmol),
and HOBt (38 mg, 029 mmol) in anhydrous DMF (3 mL) was added
(t, J¼6.4 Hz, 2H). 13C NMR (75 MHz, DMSO-d6)
d (ppm) 170.1, 160.3,
159.6 (2C), 150.3 (2C), 148.5, 139.4 (2C), 136.5, 135.2, 134.7, 132.3,
128.9 (2C), 128.5 (2C), 127.3 (2C), 124.2, 122.9, 122.3, 113.0, 111.9
(3C), 111.6, 108.1, 106.8, 70.1, 69.8 (2C), 69.7 (2C), 69.5 (2C), 66.9,
66.2, 43.1, 41.9, 40.4 (2C), 36.2, 11.7, 9.99. MS (MALDI) m/z 855.4
[MþNa]þ. HRMS (MALDI) m/z calcd for C45H53N8O8 [MþH]þ:
833.3977; found, 833.3981.
DIPEA (67 mL, 0.40 mmol) at room temperature. The reaction was
stirred overnight. The solvent was removed in vacuo. The residue
was purified by flash column chromatography (dichloromethane/
methanol¼25/1) to afford 1 as a yellow solid (85 mg, 69%). Mp
287e290 ꢀC. IR (KBr) nmax 3392, 1610 cmꢁ1 1H NMR (400 MHz,
.
DMSO-d6)
d
(ppm) 12.41 (s,1H), 8.69 (br,1H), 8.67 (s,1H), 7.81 (s,1H),
6.1.31. N-[4-(2-Amino-9H-purin-6-yloxymethyl)-benzyl]-2-[8-(4-di-
methylamino-phenyl)-2-methyl-7-oxo-3,7-dihydro-pyrano[3,2-e]in-
7.69 (d, J¼8.8 Hz, 2H), 7.60 (d, J¼8.8 Hz, 1H), 7.46 (d, J¼7.6 Hz, 2H),
7.29 (d, J¼7.6 Hz, 2H), 7.11 (d, J¼8.8 Hz, 1H), 6.81 (d, J¼8.8 Hz, 2H),
6.29 (s, 2H), 5.46 (s, 2H), 4.93 (s, 2H), 4.31 (d, J¼5.2 Hz, 1H), 2.96 (s,
dol-1-yl]-acetamide (5). Probe
5 was prepared as the above
procedure for 1. Column chromatography mobile phase: DCM/
6H),2.35 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
d (ppm) 167.9, 160.7,
MeOH¼20/1. Yellow solid (31%). Mp 172e173 ꢀC. IR (KBr) nmax 3409,
160.3, 160.1, 155.7, 150.8, 149.0, 139.3, 138.3, 137.1, 135.9, 135.1, 133.3,
129.5 (2C),129.0 (2C),127.9 (2C),124.7,123.2,122.8,114.0,113.0,112.3
(2C), 112.2, 108.8, 107.6. 66.9, 46.4, 41.1, 40.8 (2C), 12.2, 10.4. MS (ESI)
m/z 640.8 [MdH]ꢁ. HRMS (MALDI) m/z calcd for C36H35N8O4
[MþH]þ: 643.2783; found, 643.2775.
1642 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
. d (ppm) 12.41 (s, 1H),
11.39 (s, 1H), 8.52 (s, 1H), 8.26 (t, J¼5.6 Hz, 1H), 7.80 (s, 1H), 7.66 (d,
J¼8.8 Hz, 2H), 7.48 (d, J¼8.8 Hz, 1H), 7.29 (d, J¼8.0 Hz, 2H), 7.16 (d,
J¼8.0 Hz, 2H), 7.05 (d, J¼8.8 Hz, 1H), 6.73 (d, J¼8.8 Hz, 2H), 6.72 (s,
2H), 5.39 (s, 2H), 4.25 (d, J¼5.6 Hz, 1H), 3.83 (s, 2H), 2.93 (s, 6H),
2.43 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
d (ppm) 170.9, 160.3,
6.1.28. N-[4-(2-Amino-9H-purin-6-yloxymethyl)-benzyl]-4-[8-(4-
dimethylamino-phenyl)-1,2-dimethyl-7-oxo-7H-pyrano[3,2-e]in-
dol-3-yl]-butyramide (2). Probe 2 was prepared as the above
159.6 (2C), 150.1, 148.4, 139.3, 136.8, 135.2, 135.1, 131.6, 129.0 (2C),
128.2 (2C), 127.1 (2C), 124.4, 123.7, 122.9, 122.8, 119.0, 114.0, 111.7
(2C), 111.3, 109.6, 108.3, 105.9, 66.5, 42.1, 40.3 (2C), 32.8, 11.4. MS

D.-S. Mei et al. / Tetrahedron 67 (2011) 2251e2259
2259
(MALDI) m/z 629.3 [MþH]þ. HRMS (MALDI) m/z calcd for
C35H33N8O4 [MþH]þ: 629.2617; found, 629.2619.
d (ppm) 170.5, 160.7, 160.1 (2C), 150.7, 149.1, 139.8 (2C), 137.9, 135.6,
135.0, 132.7, 129.5 (2C), 128.9 (2C), 127.7 (2C), 124.7 (2C), 123.2,
122.3, 113.6, 112.3 (3C), 111.6, 109.2, 108.6, 70.5, 70.2 (2C), 70.1, 69.9,
67.3, 67.0, 61.9, 43.5, 42.3, 41.2 (2C), 36.6, 29.8, 10.5. MS (MALDI) m/z
841.4 [MþNa]þ. HRMS (MALDI) m/z calcd for C44H51N8O8 [MþH]þ:
819.3807; found, 819.3824.
6.1.32. N-[4-(2-Amino-9H-purin-6-yloxymethyl)-benzyl]-4-[2-
methyl-8-(4-dimethylamino-phenyl)-7-oxo-3,7-dihydro-pyrano[3,2-e]
indol-1-yl]-butyramide (6). Probe 6 was prepared as the above pro-
cedure for 1. Column chromatography mobile phase: DCM/
MeOH¼20/1. Yellow solid (96%). Mp 210e211 ꢀC. IR (KBr) nmax 3450,
Acknowledgements
1610 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
. d (ppm) 11.24 (s, 1H),
8.35e8.31 (m, 2H), 7.92 (s, 1H), 7.69 (d, J¼8.0 Hz, 2H), 7.48e7.42 (m,
3H), 7.25 (d, J¼7.6 Hz, 2H), 7.04 (d, J¼8.4 Hz, 1H), 6.77 (d, J¼8.0 Hz,
2H), 6.39 (s, 2H), 5.48 (s, 2H), 4.25 (d, J¼5.6 Hz, 1H), 2.92e2.90 (m,
8H), 2.36 (s, 3H), 2.23 (t, J¼3.6 Hz, 2H), 1.88e1.86 (m, 2H). 13C NMR
Financially support from Ministry of Science and Technology of
China (2010CB833200, 2009ZX09103-101), and the National Nat-
ural Sciences Foundation of China (21032002, 90713044,
20921091) is greatly appreciated.
(75 MHz, DMSO-d6)
d (ppm) 172.3, 160.8, 159.8 (2C), 156.0, 150.7,
148.9, 140.2 (2C), 135.9, 135.4, 135.0, 132.1, 129.5 (2C), 129.0 (2C),
127.8 (2C),124.7,123.3,122.4,114.7,112.3 (3C),111.9,111.5,108.6, 67.3,
42.3, 41.3 (2C), 39.2, 26.7, 25.0, 11.8. MS (MALDI) m/z 657.3 [MþH]þ.
Supplementary data
1H and 13C NMR copies of new compounds (PDF). This material
is available free of charge via the internet at http://www.else-
vier.com or from the authors. Supplementary data associated with
this article can be found in online version at doi:10.1016/
j.tet.2011.01.075. These data include MOL files and InChIKeys of the
most important compounds described in this article.
6.1.33. 6-[8-(4-Dimethylamino-phenyl)-2-methyl-7-oxo-3,7-dihydro-
pyrano[3,2-e]indol-1-yl]-hexanoic acid 4-(2-amino-9H-purin-6-yloxy-
methyl)-benzylamide (7). Probe
7 was prepared as the above
procedure for 1. Column chromatography mobile phase: DCM/
MeOH¼20/1. Yellow solid (45%). Mp 170e171 ꢀC. IR (KBr) nmax 3407,
1610 cmꢁ1. 1H NMR (400 MHz, DMSO-d6)
d (ppm) 12.42 (s, br, 1H),
References and notes
11.26 (s, 1H), 8.29 (s, 1H), 8.25 (t, J¼5.6 Hz, 1H), 7.82 (s, 1H), 7.64 (d,
J¼8.8 Hz, 2H), 7.47e7.41 (m, 3H), 7.22 (d, J¼8.0 Hz, 2H), 7.03 (d,
J¼8.8 Hz, 1H), 6.82 (d, J¼8.8 Hz, 2H), 6.27 (s, 2H), 5.44 (s, 2H), 4.23 (d,
J¼6.0 Hz, 1H), 2.94 (s, 6H), 2.86 (t, J¼7.2 Hz, 3H), 2.37 (s, 3H), 2.14 (t,
J¼7.2 Hz, 3H), 1.66e1.43 (m, 6H). 13C NMR (75 MHz, DMSO-d6)
1. Nicolle, O.; Rouillon, A.; Guyodo, H.; Tamanai-Shacoori, Z.; Chandad, F.; Meuric,
V.; Bonnaure-Mallet, M. FEMS Immunol. Med. Microbiol. 2010, 59, 357.
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d
(ppm) 172.0,160.2,159.6 (2C),150.2,148.4,139.5,135.1,135.0,134.6,
131.5, 128.8 (2C), 128.5 (2C), 127.2 (2C), 124.4, 124.1, 122.7, 121.9, 119.1,
114.1, 111.9 (3C), 110.9, 109.6, 108.1, 66.5, 41.7, 40.1 (2C), 35.4, 30.1,
28.7, 25.3, 24.9, 11.3. MS (MALDI) m/z 685.3 [MþH]þ. HRMS (MALDI)
m/z calcd for C39H41N8O4 [MþH]þ: 685.3247; found, 685.3245.
6. Maurel, D.; Comps-Agrar, L.; Brock, C.; Rives, M. L.; Bourrier, E.; Ayoub, M. A.;
Bazin, H.; Tinel, N.; Durroux, T.; Prezeau, L.; Trinquet, E.; Pin, J. P. Nat. Methods.
ꢀ
2008, 5, 561.
6.1.34. N-[4-(2-Amino-9H-purin-6-yloxymethyl)-benzyl]-3-{2-[2-(2-
{2-[8-(4-dimethylamino-phenyl)-2-methyl-7-oxo-3,7-dihydro-pyr-
ano[3,2-e]indol-1-yl]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propiona-
mide (8). Probe 8 was prepared as the above procedure for 1.
Column chromatography mobile phase: DCM/MeOH¼20/1. Yellow
solid (76%). Mp 155e156 ꢀC. IR (KBr) nmax 2922, 1609 cmꢁ1. 1H NMR
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Soc. 2004, 126, 4762.
€
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3456.
€
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(400 MHz, DMSO-d6)
d
(ppm) 8.49 (s,1H), 8.31 (t, J¼5.6 Hz,1H), 7.94
(s, 1H), 7.68 (d, J¼8.8 Hz, 2H), 7.64 (d, J¼8.8 Hz, 1H), 7.44 (d,
J¼8.0 Hz, 2H), 7.25 (d, J¼8.0 Hz, 2H), 7.08 (d, J¼8.8 Hz, 1H), 6.80 (d,
J¼8.8 Hz, 2H), 6.42 (s, 2H), 5.46 (s, 2H), 4.34 (t, J¼5.2 Hz, 2H), 4.25
(d, J¼6.0 Hz, 1H), 3.65e3.40 (m, 14H), 3.08 (t, J¼4.8 Hz, 2H), 2.96 (s,
6H), 2.40 (s, 3H), 2.35 (t, J¼6.0 Hz, 2H). 13C NMR (75 MHz, DMSO-d6)