Enhancement of chemically-induced HL-60 cell differentiation by 3,3′-diindolylmethane derivatives

Article abstract of DOI:10.1248/cpb.57.536

3,3′-Diindolylmethane (DIM, 1) and its derivatives have been prepared, and their enhancing effects on chemically-induced HL-60 cell differentiation were analyzed. Among the prepared compounds, IndDIM (12) showed the most potent enhancing effect on HL-60 cell differentiation induced by chemicals, including retinoids, 1,25-dihydroxyvitamin D₃, 12-O-tetradecanoyl phorbol-13-acetate and dimethyl sulfoxide.

Full text of DOI:10.1248/cpb.57.536

536
Notes
Chem. Pharm. Bull. 57(5) 536—540 (2009)
Vol. 57, No. 5
Enhancement of Chemically-Induced HL-60 Cell Differentiation by
3,3ꢀ-Diindolylmethane Derivatives
Tomomi NOGUCHI-YACHIDE,* Masashi TETSUHASHI, Hiroshi AOYAMA, and Yuichi HASHIMOTO
Institute of Molecular & Cellular Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan.
Received January 8, 2009; accepted February 19, 2009; published online February 24, 2009
3,3ꢀ-Diindolylmethane (DIM, 1) and its derivatives have been prepared, and their enhancing effects on
chemically-induced HL-60 cell differentiation were analyzed. Among the prepared compounds, IndDIM (12)
showed the most potent enhancing effect on HL-60 cell differentiation induced by chemicals, including retinoids,
1,25-dihydroxyvitamin D3, 12-O-tetradecanoyl phorbol-13-acetate and dimethyl sulfoxide.
Key words differentiation; diindolylmethane; tamibarotene
3,3ꢀ-Diindolylmethane (DIM, 1) is a major metabolite (APL) and induce differentiation of HL-60 cells to mature
(spontaneously formed product in intragastric acid condition) granulocytes.8) On the other hand, 1,25-VD3 and TPA have
of indole-3-carbinol (I3C, 2) which is a phytochemical ex- been established to induce differentiation of HL-60 cells to
pressed in brassica vegetables, including broccoli, brussels mature monocytes.9) DMSO is another typical cell differenti-
sprouts, kales and the cabbages, and has been thought to be ation inducer.10)
associated with the anticancer/cancer chemopreventive activ-
ities of vegetable consumption.1) DIM (1) controls prolifera- Results and Discussion
tion of various tumor cells, including cells of breast cancer,
Chemistry DIM (1), its derivatives (4—17), and isomers
prostate cancer, ovary cancer, lung cancer and pancreatic of DIM [2,2ꢀ-DIM (22) and 2,3ꢀ-DIM (25)] were synthesized
cancer, by inducing G1/S arrest of the cell cycle and apopto- using the methods reported by Nagarajan and Perumal11) and
sis.2) In addition, DIM (1) modulates metabolism of estrogen
and testosterone,3,4) which act as indigenous tumor promoters
for several hormone-dependent cancers. DIM (1) is also re-
ported to be a weak agonist for the arylhydrocarbon receptor
(AhR) and blocks the effects of estrogens via inhibition of
AhR-estrogen receptor cross talk.5) In addition, DIM (1) pos-
sesses other various activities, including immunomodulatory
effect.6) All the experimental studies suggest that DIM (1)
has incredible potential both for prevention and for treatment
of cancer.
Although the molecular basis of cancer chemopreventive
and cancer chemotherapic effects of DIM (1) is unclear, ef-
fects of DIM (1) on cell differentiation would be one of
candidate mechanisms underlying the biological activities
elicited by DIM (1). In fact, Kim et al. recently reported that
indirubin, another AhR agonist with a structure related to
Chart 1. Synthesis of DIM (1) and Its Derivatives (4—17)
that of DIM (1), and its derivatives show enhancing effect on
1,25-dihydroxyvitamin D3 (1,25-VD3)- and all-trans retinoic
acid (ATRA)-induced cell differentiation of HL-60 leukemia
cells.7)
Under such circumstances, we investigated the enhancing
effect on chemically-induced HL-60 cell differentiation of
DIM (1) and its derivatives in combination with various
cell differentiation inducers, including ATRA, tamibarotene
(Am80, 3), 1,25-VD3, 12-O-tetradecanoyl phorbol-13-acetate
(TPA) and dimethyl sulfoxide (DMSO). All of these chemi-
cals are known to induce terminal differentiation in leukemic
cell lines, including HL-60 and U937 cells. ATRA and Am80
are therapeutic agents for acute promyelocytic leukemia
Fig. 1. Structures of DIM (1), I3C (2) and Am80 (3)
Chart 2. Synthesis of 2,2ꢀ-DIM (22) and 2,3ꢀ-DIM (25)
© 2009 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: noguchi@iam.u-tokyo.ac.jp

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Table 1. Effect of 3 mM DIM Derivatives (4—17) on 2 nM Am80 (3)-In-
duced HL-60 Cell Differentiation
Compound
treated
with Am80
Relative population
of NBT-positive
cellsa)
R1
—
R2
None
MeDIM (4)
EtDIM (5)
—
H
H
H
H
H
H
H
H
100
103
175
164
146
136
185
176
251
168
134
128
188
140
Me
Et
Fig. 2. Effects of DIM (1), Its Positional Isomers (22, 25) and Its Deriva-
tive (6) on 2 nM Am80 (3)-Induced HL-60 Cell Differentiation
PrDIM (7)
n-Pr
n-Bu
All the compounds were dissolved in DMSO, and the concentration of DMSO was
adjusted to be 0.5% (v/v) in all cases.
BuDIM (8)
PhDIM (9)
BnDIM (10)
PheDIM (11)
IndDIM (12)
diMeDIM (13)
diEtDIM (14)
diPrDIM (15)
c5DIM (16)
c6DIM (17)
Ph
Bn
–(CH2)2Ph
Indol-3-yl
Me
usual organic synthetic methods as shown in Charts 1 and 2.
Briefly, DIM (1) and its derivatives (4—17) were obtained by
the reaction of indole or methylindole with various aldehydes
or ketones (Chart 1). Bis(1-benzenesulfonyl-1H-indol-2-
yl)methanol (20) and (1-benzenesulfonyl-1H-indol-2-yl)(1-
benzenesulfonyl-1H-indol-3-yl)methanol (24) was obtained
by the reaction of 2- (19) or 3-formyl-1-benzenesulfonyl-1H-
indole (23) with 1-benzenesulfonyl-1H-indole (18), followed
by reduction and desulfonylation to give 2,2ꢀ-DIM (22) or
2,3ꢀ-DIM (25) (Chart 2).
Me
Et
Pr
Et
n-Pr
–(CH2)4–
–(CH2)5–
a) The population of NBT-positive cells treated with 2 nM Am80 alone was defined
as 100%. Test compounds were added at the concentration of 3 mM.
experiment was shown in Table 1. As shown in Table 1, the
Effects on HL-60 Cell Differentiation At first, we in- enhancing effect of DIM derivatives 4—8 [MeDIM (4),
vestigated the enhancing effect on Am80-induced HL-60 cell EtDIM (5), PrDIM (7), BuDIM (8)] seems to depend on the
differentiation of DIM (1), its derivative 6 and positional iso- methylene-length of the introduced alkyl group. Although a
mers of DIM (22, 25). HL-60 cell differentiation was assayed methyl substitution [MeDIM (4)] caused almost no effect on
by the use of nitroblue tetrazolium (NBT) reduction assay, as the activity (103%), introduction of an alkyl group with a
described previously.12) Of course, the population of NBT chain length longer than a methyl group, i.e., EtDIM (5),
positive cells (%) deviates from experiments and experi- PrDIM (7), and BuDIM (8), apparently caused increase of
ments. However, the deviation of the value in one experiment the activity (146—175%). Among the compounds 4—8,
(performed in duplicate or triplicate) is less than 5%, and the EtDIM (5) seemed to be the most potent in the enhancing ef-
order of the activity of the test compounds was reproducible fect (175%) on Am80 (3)-induced HL-60 cell differentiation.
(repeated at least three times). The typical set of data ob-
Dialkyl substitution at the methylene carbon of DIM
tained from one experiment performed in side-by-side was [diMeDIM (13), diEtDIM (14), diPrDIM (15)] also increased
presented in Fig. 2. As expected, DIM (1) was revealed to the activity, with the dimethyl derivative, diMeDIM (13),
show enhancing effect on 2 nM Am80 (3)-induced HL-60 cell seemed to be the most potent (168%). The cycloalkyl deriva-
differentiation (Fig. 2). Under our experimental conditions, tives, c5DIM (16) and c6DIM (17), also showed rather potent
the population of NBT-positive cells in untreated HL-60 cells enhancing activity, with the former (16) possessing the
is generally less than 5% (data not shown). Treatment of the higher activity (188%) than the latter (17: 140%). These re-
cells with 2 nM Am80 (3) caused increase of the NBT-posi- sults suggest that appropriate size of hydrophobic group is
tive cells. Although the %-values of NBT-positive cell popu- critical for the enhancing effect on Am80 (3)-induced HL-60
lation of HL-60 cells treated with 2 nM Am80 (3) deviate cell differentiation.
among experiment to experiment, the %-values are generally
The effect of a substituent with an aromatic group [PhDIM
between 15 to 30%. In Fig. 2, a typical set of data performed (9), BnDIM (10), PheDIM (11), IndDIM (12)] was also in-
in one side-by-side experiment is shown. In this case, 2 nM vestigated. All of aromatic group-bearing DIM derivatives
Am80 (3)-treatment caused increase of the NBT-positive (9—12) showed potent enhancing effect (136—251%) on
cells to approximately 25% (Fig. 2). But co-treatment of the Am80 (3)-induced HL-60 cell differentiation induction. The
cells with 0.3 mM and 3 mM DIM (1) caused drastic increase order of the activity of the compounds bearing a phenyl
of the NBT-positive cells to approximately 30% and 37%, re- group was: PhDIM (9)ꢁPheDIM (11)ꢁBnDIM (10).
spectively. Its isomers, 2,2ꢀ-DIM (22) and 2,3ꢀ-DIM (25) Among the compounds we prepared, IndDIM (12) showed
also showed the enhancing effect, but were less potent than the most potent enhancing effect on Am80 (3)-induced HL-
DIM (1). 5,5ꢀ-DiMe-EtDIM (6) showed the enhancing activ- 60 cell differentiation (251% at 3 mM and 186% at 0.3 mM).
ity which is compatible to that of DIM (1).
To examine whether the HL-60 cell differentiation-en-
Based on the results, we analyzed the effects of an hancing activity of DIM derivatives is specific to the cell dif-
alkyl/aryl substituent(s) introduced into the methylene car- ferentiation induction by Am80 (3), the effects of typical
bon of DIM on the Am80 (3)-induced cell differentiation-en- DIM derivatives [EtDIM (5), BnDIM (10), PheDIM (11), In-
hancing effect. Also, the data obtained by one-side-by-side dDIM (12) and c5DIM (16), all of which showed rather po-

538
Vol. 57, No. 5
Table 2. Cell Differentiation-Enhancing Effect of DIM Derivatives
interpreted by the difference of cell differentiation direction
induced by the chemicals, i.e., granulocytic or monocytic. In-
terestingly, AhR has been reported to be expressed in the
monocyte, whereas it is not expressed in the granulocyte.13)
Moreover, the expression of AhR in HL-60 cells was po-
tently-induced by TPA, moderately-induced by 1,25-VD3,
and not induced by ATRA. In addition, 1,25-VD3-induced
AhR expression was strongly enhanced by transforming
growth factor (TGF)-b1. Because our synthesized DIM de-
rivatives possess AhR-agonistic feature (details will be pub-
lished elsewhere), induction and/or function of AhR might
underlie the above-mentioned difference, at least in part.
Ratio of cell differentiation induction (%) at 0.3 mM
Compound
2 nM ATRA 4 nM 1,25-VD3 500 pM TPA
DMSO
None
31.3
40.7
40.2
44.1
43.7
34.7
28.7
31.0
35.7
22.4
46.0
28.2
12.7
10.7
13.7
16.8
14.3
12.3
7.4
8.1
EtDIM (5)
BnDIM (10)
PheDIM (11)
IndDIM (12)
c5DIM (16)
15.4
16.5
20.5
16.2
tent Am80 (3)-induced HL-60 cell differentiation-enhancing Concerning the effect of AhR agonists on cell differentiation,
activity] were investigated in combination with other typical it has been reported that AhR agonists inhibit differentiation
cell differentiation inducers, including ATRA, 1,25-VD3, of monocyte into macrophage.14) Therefore, different effects
TPA and DMSO.
of our synthesized DIM derivatives on HL-60 cell differenti-
As shown in Table 2, all of the compounds examined ation among those induced by Am80/ATRA/DMSO, 1,25-
showed enhancing effect on ATRA-induced HL-60 cell dif- VD3 and TPA might be attributed to the difference of AhR
ferentiation. Although all the test compounds did not possess agonistic feature of our DIM derivatives and AhR induction
HL-60 cell differentiation-inducing activity by themselves, manner of the employed chemical cell differentiation induc-
coexistence of the physiological level concentration (2 nM) ers.
of ATRA caused cell differentiation, suggesting that these
In conclusion, we synthesized DIM and its derivatives. Al-
compounds are able to act as cell differentiation inducers in most all of these compounds enhance chemically induced
our body/under the physiological conditions where 1—3 nM HL-60 cell differentiation induction. IndDIM (12) seemed to
ATRA exists. Especially, PheDIM (11) and IndDIM (12) be the most potent enhancer in our derivatives and enhances
showed potent enhancing effect on ATRA-induced HL-60 HL-60 cell differentiation induced by Am80 (3), ATRA,
cell differentiation.
1,25-VD3 and DMSO. c5DIM (16) might be a selective en-
Concerning other cell differentiation inducers, there seems hancer of granulocytic HL-60 cell differentiation, because it
to be a tendency that all of the examined compounds enhance was potent in differentiation-inducing system using Am80
the cell differentiation, though the structure–activity relation- (3), ATRA and DMSO, while it did not enhance HL-60 cell
ships are different among the employed inducers. IndDIM differentiation induced by 1,25-VD3 and TPA which cause
(12) showed the most potent enhancing effect on 4 nM 1,25- monocytic differentiation.
VD3-induced HL-60 cell differentiation, but it seems to be
not so effective toward TPA-induced HL-60 cell differentia-
tion. PheDIM (11) and c5DIM (16) seem to be negative to-
ward 1,25-VD3-induced HL-60 cell differentiation, though
they are effective toward ATRA-induced cell differentiation.
On the other hand, PheDIM (11) showed the most potent en-
hancing effect on 500 pM TPA-induced HL-60 cell differenti-
ation. BnDIM (10) and IndDIM (12) showed weak activity,
and EtDIM (5) and c5DIM (16) did not show the activity to-
Experimental
Chemicals. General 1H-NMR spectra were recorded on a JEOL JNM-
GX500 (500 MHz) spectrometer. Chemical shifts are expressed in d (ppm)
values with tetramethylsilane (TMS) as an internal reference. High-resolu-
tion mass spectra (HR-MS) and mass spectra (MS) were recorded on a
JEOL JMS-DX303 spectrometer with m-nitrobenzyl alcohol. Flash column
chromatography was performed on silica gel 60 (Kanto Kagaku, 40—
100 mm).
3,3ꢀ-Diindolylmethane (DIM: 1)11,15): General Procedure for the Syn-
thesis of Compounds 1, 4—17 To a stirred solution of indole (351 mg,
ward TPA-induced HL-60 cell differentiation. Concerning 3.00 mmol) in a mixture of MeOH (10 ml) and H2O (0.5 ml) were added
paraformaldehyde (90% purity, 45.4 mg, 1.36 mmol) and KHSO4 (204 mg,
1.50 mmol) at the room temperature, and the reaction mixture was stirred for
40 h at the same temperature. The volatile solvents were removed under re-
duced pressure. The resulting residue was added to H2O, and then extracted
DMSO-induced HL-60 cell differentiation, IndDIM (12)
showed the most potent enhancing activity. BnDIM (10),
PheDIM (11) and c5DIM (16) showed moderate activity, and
EtDIM (5) showed weak activity.
with CH2Cl2. The organic layer was dried over MgSO4, and concentrated in
vacuo. The resulting residue was purified by flash column chromatography
(n-hexane/AcOEtꢂ4/1) to give 1 (201 mg, 818 mmol, 60%) as colorless
Although we could not extract clear structure–activity re-
lationships in each chemically induced HL-60 differentiation
system, our results suggest that the HL-60 cell differentia-
tion-inducing systems employed seem to be classified into
two groups. One group is cell differentiation system induced
by ATRA or DMSO, both of which have been known as
granulocytic differentiation inducers for HL-60 cells, in
which almost all the examined compounds showed cell dif-
ferentiation-enhancing activity. Another group is cell differ-
entiation system induced by 1,25-VD3 or TPA, both of which
have been known as monocytic differentiation inducers, in
which each examined compounds showed different effects
from those observed in ATRA/DMSO-induced granulocytic
differentiation system. This difference of structure–activity
relationships between above-mentioned two groups might be
1
crystal; mp 168—170 °C (lit.15) 164 °C). H-NMR (CDCl3) d: 4.23 (2H, s),
6.929 (1H, s), 6.931 (1H, s), 7.08 (2H, dd, Jꢂ6.7, 8.0 Hz), 7.17 (2H, dd,
Jꢂ6.7, 8.0 Hz), 7.34 (2H, d, Jꢂ8.0 Hz), 7.61 (2H, d, Jꢂ8.0 Hz), 7.89 (2H,
br s). FAB-MS m/z: 246.1143 (Calcd for C17H14N2: 246.1157).
1,1-(3,3ꢀ-Diindolyl)ethane (4)11,15) Yield 24%, white solid; mp 165—
1
166 °C (lit.15) 156 °C). H-NMR (CDCl3) d: 1.81 (3H, d, Jꢂ7.3 Hz), 4.69
(1H, q, Jꢂ7.3 Hz), 6.94 (2H, d, Jꢂ1.8 Hz), 7.04 (2H, dd, Jꢂ7.3, 7.9 Hz),
7.16 (2H, dd, Jꢂ7.3, 7.9 Hz), 7.36 (2H, d, Jꢂ7.9 Hz), 7.58 (2H, d,
Jꢂ7.9 Hz), 7.89 (2H, br s). FAB-MS m/z: 260.1343 (Calcd for C18H16N2:
260.1313).
1,1-(3,3ꢀ-Diindolyl)propane (5)16) Yield 16%, white solid; mp 128—
1
130 °C. H-NMR (CDCl3) d: 1.02 (3H, t, Jꢂ7.3 Hz), 2.26 (2H, qd, Jꢂ7.3,
7.3 Hz), 4.39 (1H, t, Jꢂ7.3 Hz), 7.05—7.01 (4H, m), 7.15 (2H, dd, Jꢂ7.9,
8.5 Hz), 7.34 (2H, d, Jꢂ7.9 Hz), 7.59 (2H, d, Jꢂ7.9 Hz), 7.90 (2H br s). EI-
MS m/z 274.1472 (Calcd for C19H18N2: 274.1470).
1-(5-methylindol-3-yl)-1-(5ꢀ-methylindol-3ꢀ-yl)propane (6) Yield

May 2009
539
1
1H-NMR (CDCl3) d: 6.67 (1H, d, Jꢂ3.7 Hz), 7.23 (1H, dd, Jꢂ7.3, 7.3 Hz),
7.31 (1H, dd, Jꢂ7.3, 7.3 Hz), 7.43 (2H, t, Jꢂ7.6 Hz), 7.51—7.54 (2H, m),
7.57 (1H, d, Jꢂ3.7 Hz), 7.87—7.89 (2H, m), 8.00 (1H, d, Jꢂ8.5 Hz). FAB-
MS m/z: 257 (MꢃHꢃ).
12%, colorless crystal; mp 132—133 °C. H-NMR (CDCl3) d: 1.01 (3H, t,
Jꢂ7.3 Hz), 2.23 (2H, qd, Jꢂ7.3, 7.3 Hz), 2.41 (6H, s), 4.33 (2H, t,
Jꢂ7.3 Hz), 6.95 (2H, d, Jꢂ1.8 Hz), 6.98 (2H, dd, Jꢂ7.9, 1.8 Hz), 7.23 (2H,
d, Jꢂ7.9 Hz), 7.40 (2H, s), 7.80 (2H, br s). FAB-MS m/z: 302.1757 (Calcd
for C21H22N2: 302.1783).
2-Formyl-1-benzenesulfonyl-1H-indole (19)23) To a stirred solution of
18 (1.50 g, 5.83 mmol) in dry THF (40 ml) was added n-BuLi (1.55 M in
hexane, 4.52 ml, 7.00 mmol) at ꢄ78 °C. After stirring for 30 min at ꢄ78 °C
and then for 1 h at room temperature, the solution was cooled again to
ꢄ78 °C and DMF (1.00 ml, 12.9 mmol) was added dropwise, and the reac-
tion mixture was stirred for 4 h at room temperature. The reaction was
quenched by adding saturated aqueous NH4Cl, and then extracted with
AcOEt. The organic layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. The resulting residue was purified by flash column
chromatography (n-hexane/AcOEtꢂ10/1) to give 19 (1.07 g, 3.75 mmol,
1,1-(3,3ꢀ-Diindolyl)butane (7)11,16) Yield 40%, pale yellow solid; mp
154—156 °C. 1H-NMR (CDCl3) d: 0.96 (3H, t, Jꢂ7.3 Hz), 1.44 (2H, qt,
Jꢂ7.3, 7.3 Hz), 2.21 (2H, td, Jꢂ7.3, 7.3 Hz), 4.50 (1H, t, Jꢂ7.3 Hz), 7.01
(2H, d, Jꢂ1.2 Hz), 7.04 (2H, dd, Jꢂ7.9, 7.9 Hz), 7.15 (2H, dd, Jꢂ7.9,
7.9 Hz), 7.33 (2H, d, Jꢂ7.9 Hz), 7.60 (2H, d, Jꢂ7.9 Hz), 7.89 (2H, br s).
FAB-MS m/z: 288.1636 (Calcd for C20H20N2: 288.1626).
1,1-(3,3ꢀ-Diindolyl)pentane (8)17) Yield 26%, white solid; mp 97—
99 °C (lit.17) 80—82 °C). 1H-NMR (CDCl3) d: 0.87 (3H, t, Jꢂ6.7 Hz),
1.42—1.36 (4H, m), 2.22 (2H, td, Jꢂ7.3, 7.3 Hz), 4.48 (1H, t, Jꢂ7.3 Hz),
7.05—7.01 (4H, m), 7.15 (2H, dd, Jꢂ7.9, 8.5 Hz), 7.34 (2H, d, Jꢂ7.9 Hz),
7.60 (2H, d, Jꢂ7.9 Hz), 7.90 (2H, br s). FAB-MS m/z: 302.1803 (Calcd for
C21H22N2: 302.1783).
1
64%) as a white solid: H-NMR (CDCl3) d: 7.32 (1H, dd, Jꢂ7.3, 7.9 Hz),
7.41 (2H, dd, Jꢂ7.3, 7.3 Hz), 7.48 (1H, s), 7.52—7.56 (2H, m), 7.63 (1H, d,
Jꢂ7.9 Hz), 7.78 (2H, dd, Jꢂ8.5, 1.2 Hz), 8.24 (1H, d, Jꢂ8.5 Hz), 10.53 (1H,
s). FAB-MS m/z: 286 (Mꢃ).
3,3ꢀ-Diindolyl(phenyl)methane (9)11) Yield 98%, pink foam; H-NMR
1
Bis(1-benzenesulfonyl-1H-indol-2-yl)methanol (20)24) To a stirred so-
lution of 18 (613 mg, 2.38 mmol) in dry THF (15 ml) was added n-BuLi
(1.66 M in hexane, 1.58 ml, 2.62 mmol) at ꢄ78 °C. After stirring for 1 h at
room temperature, the solution was cooled again to ꢄ78 °C and 19 (680 mg,
2.38 mmol) in dry THF (5 ml) was added dropwise, and then the reaction
mixture was stirred for 18 h at room temperature. The reaction was
quenched by adding 1 N HCl, and then extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and concentrated in vacuo.
The resulting residue was purified by flash column chromatography (n-
hexane/AcOEtꢂ4/1) to give 20 (965 mg, 1.78 mmol, 75%) as a yellow foam:
1H-NMR (CDCl3) d: 3.97 (1H, d, Jꢂ4.9 Hz), 6.54 (2H, s), 7.21—7.24 (3H,
m), 7.30—7.37 (6H, m), 7.40 (2H, d, Jꢂ7.9 Hz), 7.47 (2H, dd, Jꢂ7.3,
7.9 Hz), 7.84 (4H, d, Jꢂ7.9 Hz), 8.10 (2H, d, Jꢂ8.5 Hz). FAB-MS m/z: 542
(Mꢃ).
(CDCl3) d: 5.89 (1H, s), 6.67 (2H, d, Jꢂ1.2 Hz), 7.00 (2H, t, Jꢂ7.3 Hz),
7.17 (2H, t, Jꢂ8.5 Hz), 7.21 (1H, t, Jꢂ7.3 Hz), 7.28 (2H, t, Jꢂ7.3 Hz),
7.34—7.37 (4H, m), 7.39 (2H, d, Jꢂ8.5 Hz), 7.91 (2H, br s). FAB-MS m/z
322.1455 (Calcd for C23H18N2: 322.1470).
1
1,1-(3,3ꢀ-Diindolyl)-2-phenylethane (10) Yield 55%, white foam; H-
NMR (CDCl3) d: 3.55 (2H, t, Jꢂ7.9 Hz), 4.80 (1H, d, Jꢂ7.9 Hz), 6.95 (2H,
d, Jꢂ1.8 Hz), 7.02 (2H, dd, Jꢂ7.9, 7.9 Hz), 7.09—7.17 (7H, m), 7.33 (2H, d,
Jꢂ7.9 Hz), 7.57 (2H, d, Jꢂ7.9 Hz), 7.87 (2H, br s). FAB-MS m/z: 335.1563
(Calcd for C24H19N2: 335.1548).
1,1-(3,3ꢀ-Diindolyl)-3-phenylpropane (11)18) Yield 27%, white solid;
mp 158—159 °C. 1H-NMR (CDCl3) d: 2.54—2.59 (2H, m), 2.73 (2H, t,
Jꢂ8.5 Hz), 4.52 (1H, t, Jꢂ7.3 Hz), 7.01—7.05 (4H, m), 7.14—7.19 (5H,
m), 7.27 (2H, dd, Jꢂ7.9, 7.9 Hz), 7.35 (2H, d, Jꢂ8.5 Hz), 7.55 (2H, d,
Jꢂ7.9 Hz), 7.92 (2H, br s). FAB-MS m/z: 350.1783 (Calcd for C25H22N2:
350.1783).
Bis(1-benzenesulfonyl-1H-indol-2-yl)methane (21)24) To a solution of
20 (303 mg, 0.558 mmol) in dry dichloromethane (DCM) (5.0 ml) was added
Et3SiH (2.00 ml, 12.5 mmol) followed by TFA (0.1 ml), and the reaction
mixture was stirred for 18 h. The reaction was quenched by adding water,
and then extracted with DCM. The organic layer was washed with brine,
dried over MgSO4, and concentrated in vacuo. The resulting residue was pu-
rified by flash column chromatography (n-hexane/AcOEtꢂ4/1) to give 21
(230 mg, 0.436 mmol, 78%) as a white foam: 1H-NMR (CDCl3) d: 4.85 (2H,
s), 6.22 (2H, s), 7.20 (2H, dd, Jꢂ7.3, 7.3 Hz), 7.29 (2H, dd, Jꢂ7.9, 8.5 Hz),
7.32—7.37 (6H, m), 7.47 (2H, dd, Jꢂ7.3, 7.3 Hz), 7.77 (4H, d, Jꢂ8.5 Hz),
8.16 (2H, d, Jꢂ7.9 Hz). FAB-MS m/z: 527 (MꢃHꢃ).
3,3ꢀ,3ꢁ-Triindolylmethane (12)18,19) Yield 69%, beige solid; mp 251—
253 °C (lit.19) 245—247 °C). 1H-NMR (DMSO-d6) d: 6.03 (1H, s), 6.83 (3H,
dd, Jꢂ7.9, 7.9 Hz), 6.91 (3H, d, Jꢂ2.4 Hz), 6.99 (3H, dd, Jꢂ7.9, 7.9 Hz),
7.31 (3H, d, Jꢂ7.9 Hz), 7.37 (3H, d, Jꢂ7.9 Hz), 10.69 (3H, br s). FAB-MS
m/z: 361.1546 (Calcd for C25H19N3: 361.1579).
1
2,2-(3,3ꢀ-Diindolyl)propane (13)16) Yield 47%, pale yellow foam; H-
NMR (CDCl3) d: 1.90 (6H, s), 6.87 (2H, dd, Jꢂ7.5, 7.5 Hz), 7.05 (2H, d,
Jꢂ2.0 Hz), 7.06 (2H, dd, Jꢂ7.5, 7.5 Hz), 7.30 (2H, d, Jꢂ8.5 Hz), 7.40 (2H,
d, Jꢂ8.5 Hz), 7.89 (2H, br s). FAB-MS m/z: 274.1476 (Calcd for C19H18N2:
274.1470).
3,3-(3,3ꢀ-Diindolyl)pentane (14)20) Yield 91%, white foam; 1H-NMR
(CDCl3) d: 0.66 (6H, t, Jꢂ7.5 Hz), 2.29 (4H, q, Jꢂ7.5 Hz), 6.73 (2H, dd,
Jꢂ7.5, 7.5 Hz), 6.99 (2H, dd, Jꢂ7.5, 7.5 Hz), 7.175 (2H, s), 7.180 (2H, d,
Jꢂ8.5 Hz), 7.27 (2H, d, Jꢂ8.5 Hz), 7.95 (2H, br s). FAB-MS m/z: 302.1831
(Calcd for C21H22N2: 302.1783).
2,2ꢀ-Diindolylmethane (22)24) To a stirred solution of 21 (60.0 mg,
0.114 mmol) in MeOH (3.0 ml) was added Cs2CO3 (190 mg, 0.570 mmol),
and the reaction mixture was refluxed for 15 h. The reaction was quenched
by adding water, and then extracted with AcOEt. The organic layer was
washed with brine, dried over MgSO4, and concentrated in vacuo. The
resulting residue was purified by flash column chromatography (n-hexane/
AcOEtꢂ6/1) and recrystallized from n-hexane/AcOEt to give 22 (6.7 mg,
27 mmol, 24%) as a colorless crystal: mp 168—169 °C (lit.24) 168 °C).
1H-NMR (CDCl3) d: 4.32 (2H, s), 6.45 (2H, s), 7.09 (2H, dd, Jꢂ7.6,
8.5 Hz), 7.14 (2H, dd, Jꢂ7.6, 7.9 Hz), 7.25 (2H, d, Jꢂ8.5 Hz), 7.57 (2H, d,
Jꢂ7.9 Hz), 7.90 (2H, br s). FAB-MS m/z: 246.1203 (Calcd for C17H14N2:
246.1157).
4,4-(3,3ꢀ-Diindolyl)heptane (15) Yield 17%, white solid; mp 158—
1
160 °C. H-NMR (CDCl3) d: 0.82 (6H, t, Jꢂ7.3 Hz), 1.05—1.13 (4H, m),
2.24—2.27 (4H, m), 6.75 (2H, dd, Jꢂ7.9, 7.3 Hz), 7.01 (2H, dd, Jꢂ7.9,
7.3 Hz), 7.16 (2H, d, Jꢂ2.4 Hz), 7.22 (2H, d, Jꢂ7.9 Hz), 7.28 (2H, d,
Jꢂ7.9 Hz), 7.94 (2H, br s). FAB-MS m/z: 330.2137 (Calcd for C23H26N2:
330.2096).
1,1-(3,3ꢀ-Diindolyl)cyclopentane (16)21) Yield 24%, white solid; mp
167—169 °C. 1H-NMR (CDCl3) d: 1.82—1.85 (4H, m), 2.50—2.53 (4H,
m), 6.90 (2H, dd, Jꢂ7.3, 7.9 Hz), 7.07 (2H, dd, Jꢂ7.3, 7.9 Hz), 7.10 (2H, d,
Jꢂ2.4 Hz), 7.30 (2H, d, Jꢂ7.9 Hz), 7.51 (2H, d, Jꢂ7.9 Hz), 7.88 (2H, br s).
FAB-MS m/z: 300.1621 (Calcd for C21H20N2: 300.1626).
3-Formyl-1-benzenesulfonyl-1H-indole (23)25) To a stirred solution of
3-formylindole (435 mg, 3.00 mmol) in dry THF (15 ml) was added NaH
(55% in oil suspension, 160 mg, 3.67 mmol) at 0 °C. After stirring at 0 °C
for 30 min, benzenesulfonyl chloride (0.430 ml, 3.36 mmol) was added
slowly. The reaction mixture was stirred for 22 h at room temperature. The
reaction was quenched by adding water, and then extracted with AcOEt. The
organic layer was washed with brine, dried over MgSO4, and concentrated in
vacuo. The resulting residue was purified by flash column chromatography
(n-hexane/AcOEtꢂ6/1 to 4/1) to give 23 (670 g, 2.35 mmol, 78%) as a pale
1,1-(3,3ꢀ-Diindolyl)cyclohexane (17)21) Yield 74%, white foam; 1H-
NMR (CDCl3) d: 1.57 (2H, quintet, Jꢂ6.0 Hz), 1.64 (4H, td, Jꢂ5.5, 6.0 Hz),
2.53 (4H, dd, Jꢂ5.5, 6.0 Hz), 6.87 (2H, dd, Jꢂ7.5, 8.0 Hz), 7.04 (2H, dd,
Jꢂ7.5, 7.5 Hz), 7.09 (1H, s), 7.10 (1H, s), 7.28 (2H, d, Jꢂ8.0 Hz), 7.54 (2H,
d, Jꢂ7.5 Hz), 7.90 (2H, br s). FAB-MS m/z: 314.1809 (Calcd for C22H22N2:
314.1783).
1
yellow solid: H-NMR (CDCl3) d: 7.35 (1H, dd, Jꢂ7.3, 7.9 Hz), 7.40 (2H,
1-Benzenesulfonyl-1H-indole (18)22) To a stirred solution of indole
(3.50 g, 29.9 mmol) in dry THF (100 ml) was added NaH (55% in oil sus-
pension, 1.44 g, 33.0 mmol) at 0 °C. After stirring at 0 °C for 30 min, ben-
zenesulfonyl chloride (4.2 ml, 33 mmol) was added slowly and the reaction
mixture was stirred for 18 h at room temperature. The reaction was
quenched by adding water, and then extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and concentrated in vacuo.
The resulting residue was purified by flash column chromatography (n-
hexane/AcOEtꢂ9/1) to give 18 (6.80 g, 26.4 mmol, 88%) as a white solid:
dd, Jꢂ7.3, 7.3 Hz), 7.50 (2H, t, Jꢂ7.9 Hz), 7.60 (1H, t, Jꢂ7.3 Hz), 7.93—
7.96 (3H, m), 8.21 (1H, s), 8.24 (1H, d, Jꢂ7.9 Hz), 10.09 (1H, s). FAB-MS
m/z: 286 (Mꢃ).
(1-Benzenesulfonyl-1H-indol-2-yl)(1-benzenesulfonyl-1H-indol-3-
yl)methanol (24)26) To a stirred solution of 18 (386 mg, 1.50 mmol) in dry
THF (10 ml) was added n-BuLi (1.55 M in hexane, 1.20 ml, 1.86 mmol) at
ꢄ78 °C. After stirring for 1 h at room temperature, the solution was cooled
again to ꢄ78 °C and 23 (427 mg, 1.50 mmol) in dry THF (5.0 ml) was added
dropwise, and then the reaction mixture was stirred for 5 h at room tempera-

540
Vol. 57, No. 5
ture. The reaction was quenched by adding 1 N HCl, and then extracted with
AcOEt. The organic layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. The resulting residue was purified by flash column
chromatography (n-hexane/AcOEtꢂ4/1) to give 24 (224 mg, 0.413 mmol,
4) Jellinck P. H., Forkert P. G., Riddick D. S., Okey A. B., Michnovicz J.
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1
28%) as a yellow foam: H-NMR (CDCl3) d: 3.69 (1H, d, Jꢂ4.8 Hz), 6.20
(1H, s), 6.52 (1H, d, Jꢂ4.8 Hz), 6.92 (1H, d, Jꢂ7.9 Hz), 7.04 (1H, dd,
Jꢂ7.3, 7.9 Hz), 7.21 (1H, dd, Jꢂ6.7, 7.9 Hz), 7.28—7.34 (3H, m), 7.41—
7.48 (4H, m), 7.55—7.59 (2H, m), 7.72 (1H, s), 7.80 (2H, dd, Jꢂ1.2,
8.5 Hz), 7.92 (2H, dd, Jꢂ1.2, 8.5 Hz), 8.01 (1H, d, Jꢂ8.5 Hz), 8.15 (1H, d,
Jꢂ9.1 Hz). FAB-MS m/z: 542 (Mꢃ).
2,3ꢀ-Diindolylmethane (25)27) To a stirred suspension of lithium alu-
minum hydride (LAH) (500 mg, 13.1 mmol) in dry THF (10 ml) was added
24 (225 mg, 0.415 mmol) slowly at ꢄ20 °C. After the addition, the suspen-
10) Collins S. J., Ruscetti F. W., Gallagher R. E., Gallo R. C., Proc. Natl.
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adding saturated aqueous potassium sodium tartrate (50 ml). After stirring
for 1 h, the reaction mixture was extracted with AcOEt. The organic layer
was washed with brine, dried over MgSO4, and concentrated in vacuo. The
resulting residue was purified by flash column chromatography (n-
hexane/AcOEtꢂ4/1) and recrystallized from n-hexane/AcOEt to give 25
(88.0 mg, 0.357 mmol, 86%) as a colorless needle: mp 139—140 °C (lit.27)
12) Noguchi T., Shinji C., Kobayashi H., Makishima M., Miyachi H.,
Hashimoto Y., Biol. Pharm. Bull., 28, 563—564 (2005).
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1
141—143 °C). H-NMR (CDCl3) d: 4.15 (2H, s), 6.13 (1H, d, Jꢂ1.2 Hz),
6.96—6.86 (3H, m), 7.03 (1H, dd, Jꢂ6.7, 7.3 Hz), 7.20 (1H, d, Jꢂ2.4 Hz),
7.23 (1H, d, Jꢂ8.5 Hz), 7.33 (1H, d, Jꢂ7.9 Hz), 7.36 (1H, d, Jꢂ7.3 Hz),
7.46 (1H, d, Jꢂ7.9 Hz), 10.85 (2H, br s). FAB-MS m/z: 246.1193 (Calcd for
C17H14N2: 246.1157).
Assay of Cell Differentiation-Inducting Activity Human leukemia 18) Ramesh C., Banerjee J., Pal R., Das B., Adv. Synth. Catal., 345, 557—
cells, HL-60, were cultured in RPMI1640 medium containing 5% fetal 559 (2003).
bovine serum at 37 °C in a humidified atmosphere of 5% CO2 in the air. Cell 19) Nair V., Abhilash K. G., Vidya N., J. Chem. Res., 2003, S, 72—74
differentiation was performed by the nitro blue tetrazolium (NBT) reduction (2003).
method. For this assay, HL-60 cells in 1ꢅ105 cells/ml were incubated in 20) Shokoufeh M., Najmoddin A., Mohammad R. S., Lett. Org. Chem., 3,
RPMI1640 medium in the presence or absence of test compounds (test com-
pounds were added as a DMSO solution with the final concentration of
DMSO in the incubation mixture being adjusted to be 0.5% (v/v)) for 3 d.
161—164 (2006).
21) Yadav J. S., Reddy B. V. S., Murthy Ch. V. S. R., Kumar G. M., Madan
Ch., Synthesis, 2001, 783—787 (2001).
Treated HL-60 cells were harvested via centrifugation and incubated with an 22) Naka H., Akagi Y., Yamada K., Imahori T., Kasahara T., Kondo Y.,
equal volume of 0.2% (w/v) NBT dissolved in a phosphate-buffered saline
(PBS), containing 20 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) at
37 °C for 30 min. NBT positivity was measured by counting at least 200
cells and the results were expressed as the percentage of NBT-positive cells.
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Acknowledgments The work described in this paper was partially sup- 24) Mahboobi S., Burgemeister T., Dove S., Kuhr S., Popp A., J. Org.
ported by Grants-in-Aid for Scientific Research from The Ministry of Edu-
cation, Culture, Sports, Science and Technology-Japan, and the Japan Soci-
ety for the Promotion of Science.
Chem., 64, 8130—8137 (1999).
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