Effect of indole-3-acetic acid analogs on the differentiation of HL-60 cells

Article abstract of DOI:10.1002/jccs.200800171

In continuing search for novel cell differentiation agents, a series of derivatives of indole-3-acetic acid and indole-3-carboxylic acid were prepared and tested against HL-60 cells for their differentiation and antiproliferation activities. Among them, N-ethyl-1-benzylindole-3-carboxamide (14) was the most potent, whereas N-methyl 1-benzylindole-3-acetamide (5) and N-methyl 1-benzylindole-3-carboxamide (13) synergistically potentiated with all-trans-retinoic acid to induce cell differentiation as well as antiproliferation. Our results indicate that these compounds are effective cell differentiation and antiproliferation agents in combination with retinoic acid.

Full text of DOI:10.1002/jccs.200800171

1160
Journal of the Chinese Chemical Society, 2008, 55, 1160-1165
Effect of Indole-3-acetic Acid Analogs on the Differentiation of HL-60 Cells
Jin-Cherng Lien* (
), Yu-Dong Jiang (
), Chun-Jen Chen (
)
),
Sheng-Chu Kuo (
) and Li-Jiau Huang* (
Graduate Institute of Pharmaceutical Chemistry, China Medical University,
91 Hsueh-Shin Road, Taichung, Taiwan, R.O.C.
In continuing search for novel cell differentiation agents, a series of derivatives of indole-3-acetic
acid and indole-3-carboxylic acid were prepared and tested against HL-60 cells for their differentiation
and antiproliferation activities. Among them, N-ethyl-1-benzylindole-3-carboxamide (14) was the most
potent, whereas N-methyl 1-benzylindole-3-acetamide (5) and N-methyl 1-benzylindole-3-carboxamide
(13) synergistically potentiated with all-trans-retinoic acid to induce cell differentiation as well as anti-
proliferation. Our results indicate that these compounds are effective cell differentiation and antiprolifera-
tion agents in combination with retinoic acid.
Keywords: Cell differentiation; Antiproliferation activity; Indole-3-acetic acids;
Indole-3-carboxylic acids.
INTRODUCTION
Leukemia is a cancer of the bone marrow or blood
novel differentiation agents and differentiation potentiators
which are able of reducing both the dosage and side effects
of ATRA and inducing complete differentiation is an im-
portant task for the improvement of cell differentiation
therapy.
In our previous work,7 we has been demonstrated that
indole-3-acetic acid (IAA) can act as ATRA to induce ter-
minal differentiation of HL-60. In the present work, a se-
ries of derivatives of indole-3-acetic acid and indole-3-car-
boxylic acid were prepared and tested against HL-60 cells
for their differentiation and antiproliferation activities.
and is characterized by an abnormal proliferation of blood
cells, usually leukocytes. It is part of the broad group of
diseases called hematological neoplasms. A trait of acute
leukemia cells is their ability to differentiate into functional
and mature cells. One possible tactic to the treatment of pa-
tients with acute leukemia is to induce differentiation of the
leukemia cells. Recent studies have been focused on the
human promyelocytic HL-60 leukemia cell line which pro-
vides a effective in vitro model system for studying the cel-
lular and molecular consequences involved in the differen-
tiation process.1 An effective inducer for the differentiation
of HL-60 cells is commonly recognized as having potential
therapeutic importance. Reliable compounds known as ef-
ficient anticancer agents, for instance, all-trans-retinoic
acid (ATRA), have been used to induce leukemia cell dif-
ferentiation and have resulted in almost complete remis-
sion in a high proportion of patients with acute promyelo-
cytic leukemia (APL).2-4 However, the efficient dose of
ATRA in clinical studies causes undesirable side effects,
e.g. retinoic acid syndrome.5 Besides, remission in APL pa-
tients resulting from ATRA treatment lasted for only a few
months before the disease in variably recurred. The reason
for recurrence is most likely because ATRA caused partial
differentiation of the cancer cells.6 Thus the search for
RESULTS AND DISCUSSION
In this study, the target compounds were synthesized
as shown in Scheme I. Briefly, indole-3-acetic acid (1) was
treated with benzyl chloride in the presence of NaH, the
corresponding compound 2 was obtained. Compound 3 ob-
tained by chlorination of compound 2 with SOCl2. Reac-
tion of compound 3 with ammonia or variety of alkylamine
afforded the target compounds 4-8. Replacing 1 with indole-
3-carboxylic acid (9), the target compounds 12-16 were
prepared by the same methods as described above.
The Effect of Compounds 4-8 and 12-16 on the Dif-
ferentiation of HL-60 Cells
The NBT cell differentiation assay was used to mea-
* Corresponding author. E-mail: jclien@mail.cmu.edu.tw

Indole-3-acetic Acid Analogs on the Cell Differentiation
J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 1161
compounds used in combination with RA, all of them even
though at a 10 mM concentration showed significant effi-
cacy in potentiating RA-induced differentiation.
Scheme I
The Effect of Compounds 4-8 and 12-16 on the Pro-
liferation of HL-60 Cells
The effect of compounds 4-8 and 12-16 alone on the
proliferation of HL-60 cells is summarized in Table 1. In
general, most of these compounds were nontoxic at low
concentrations (10 mM). While at 50 mM concentration,
compounds 13 and 15 suppressed proliferation about 50%.
In particular, the MTT reduction value of the most potent
compound 14 at 50 mM was about one-third less than con-
trol. There appeared to be correlation between their ability
to affect cell differentiation and their ability to affect cell
proliferation. When compounds 4-8 and 12-16 were used
in combination with RA, most of them suppressed prolifer-
ation at 10-50 mM. Among them, compounds 5 and 13 dis-
played the best inhibitory effect at 50 mM. In general, these
compounds combined with RA we found their ability to
promote differentiation seemed to correlate with their abil-
ity to suppress proliferation.
A series of derivatives of indole-3-acetic acid (4-8)
and indole-3-carboxylic acid (12-16) was synthesized and
examined for inducing the differentiation of HL-60 cells.
Among them, compound 14 was most potent. When used in
combination with RA, compounds 5 and 13 demonstrated
significant synergism in promoting cell differentiation. In
terms of their proliferation activities, we found that, when
used alone, compound 14 significantly suppressed prolifer-
ation. When used in combination with RA, most of these
amide derivatives inhibited cell proliferation. The ability of
these indole-3-carboxylic acid derivatives to affect prolif-
eration does seem to correlate with their ability to affect
cell differentiation. In general, the combinations that inhib-
ited proliferation to a greater extent were also more potent
inducers of cell differentiation. Our results indicate that
these compounds are probably reducing both the dosage
and side effects of ATRA for the improvement of cell dif-
ferentiation therapy.
Reagents and conditions: (i) benzyl chloride, NaH, DMF, 0 °C;
(ii) SOCl2, diethyl ether, reflux; (iii) RNH2, diethyl ether, 0 °C.
sure the potency of compounds 4-8 and 12-16 used alone or
in combination with 5 nM of RA, in inducing the differenti-
ation of HL-60 cells. As shown in Table 1, almost the tested
compounds only showed slightly activity at a concentration
range from 10 to 50 mM. In particular, the NBT reduction
value of the most potent compound 14 at 50 mM was about
100 fold greater than the control. In the derivatives of N-
substituted 1-benzylindole-3-acetamides (4-8), most com-
pounds when used in combination with RA demonstrated
potent activity in compared with control (RA). Among
them, significant synergism was particularly noticeable
with the N-methyl derivative (5) in which the NBT reduc-
tion value of RA was significant potentiated. To evaluation
the activity of N-substituted 1-benzylindole-3-carbox-
amides (12-16), they displayed potent synergistic effects
when combined with RA. Above all, the NBT reduction
value of the most potent compound 13 at 50 mM was about
2 fold greater than the control. As a whole, based on the
NBT assay, compounds 4-8 and 12-16 did not display sig-
nificant efficacy when used alone. Nevertheless, these
EXPERIMENTAL
Chemistry
IR spectra were recorded on a Nicolet Impact 400 FT-
IR spectrophotometer as KBr pellets. NMR spectra were
obtained on a Bruker Avance DPX-200 FT-NMR. MS were
measured with HP 5995 GC-MS and VG PLATFORM II

1162 J. Chin. Chem. Soc., Vol. 55, No. 5, 2008
Lien et al.
Table 1. Effects of N-substituted 1-benzylindole-3-acetamides (4-8) and N-substituted 1-benzyl-
indole-3-carboxamides (12-16) on differentiation and proliferation of HL-60 cells without
(or with) all-trans retinoic acid
NBT Assay (%)
MTT Assay (%)
Compounds
Conc.
– RA
+ RA
– RA
+ RA
(mM)
Control
--
0.3 ± 0.2
0.3 ± 0.2
0.4 ± 0.3
0.3 ± 0.2
0.3 ± 0.2
0.3 ± 0.2
0.7 ± 0.3
0.3 ± 0.5
0.3 ± 0.2
0.5 ± 0.4
0.3 ± 0.2
0.2 ± 0.3
0.3 ± 0.2
0.2 ± 0.3
0.3 ± 0.2
crystal out
0.3 ± 0.2
0.3 ± 0.2
0.4 ± 0.3
0
38.8 ± 2.6***
42.5 ± 1.9
100.0 ± 0.1
99.8 ± 1.6
66.6 ± 4.5***
112.1 ± 8.8**
55.2 ± 4.7*
53.0 ± 12.5***
53.6 ± 9.9*
23.6 ± 1.6***
15.5 ± 2.1***
57.8 ± 5.2
4
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
58.5 ± 4.7***
75.7 ± 3.8***
43.2 ± 1.4*
97.0 ± 6.8
78.9 ± 4.8***
94.6 ± 5.0
5
65.6 ± 1.3***
79.4 ± 0.8***
59.7 ± 3.4***
65.4 ± 0.1***
77.6 ± 3.7***
51.0 ± 1.4***
58.7 ± 1.6***
71.5 ± 2.2***
39.6 ± 4.1
88.6 ± 2.1***
75.1 ± 3.6***
85.0 ± 6.0**
95.5 ± 0.8***
94.3 ± 4.4*
105.1 ± 4.4
117.5 ± 5.6**
88.9 ± 2.0***
93.9 ± 4.6*
102.0 ± 8.0
crystal out
6
49.5 ± 4.2*
37.3 ± 2.4***
74.7 ± 1.8*
68.7 ± 5.7
7
55.6 ± 0.1*
50.5 ± 3.6**
49.5 ± 4.0**
crystal out
8
55.3 ± 5.2***
crystal out
12
13
14
15
16
40.5 ± 8.2
116.7 ± 2.1
108.6 ± 9.5
68.0 ± 1.3***
89.7 ± 2.7***
84.8 ± 3.7***
56.8 ± 4.1***
103.7 ± 3.8
79.6 ± 6.3***
29.7 ± 4.2***
110.1 ± 1.8***
84.5 ± 6.1**
51.9 ± 1.5***
106.5 ± 6.8
97.9 ± 3.7
88.6 ± 14.5
93.6 ± 9.6*
41.2 ± 3.2***
92.4 ± 3.4***
70.4 ± 4.8
40.9 ± 6.1
69.2 ± 4.6***
39.4 ± 1.9
1.0 ± 0.8
1.2 ± 0.8
0.3 ± 0.2
0.7 ± 0.6
0027.8 ± 1.7***
0
45.6 ± 2.1**
80.6 ± 0.3***
45.8 ± 6.7*
28.4 ± 5.6***
102.1 ± 3.7**
63.5 ± 4.9
55.0 ± 1.1***
67.8 ± 2.8***
39.7 ± 4.5
30.4 ± 3.8***
65.7 ± 2.2
0.2 ± 0.3
0.6 ± 0.3
0.6 ± 0.3
0.3 ± 0.2
crystal out
42.1 ± 1.4
57.2 ± 2.1*
34.0 ± 9.4***
69.6 ± 2.2
62.1 ± 2.1***
45.1 ± 2.1**
49.5 ± 2.3***
crystal out
59.7 ± 6.3
crystal out
crystal out
* p < 0.05 compared with control without (with) RA5 nM.
** p < 0.01 compared with control without (with) RA5 nM.
*** p < 0.001 compared with control without (with) RA5 nM.
Data was presented as mean ± SD from three separate experiments
GC-MS instruments. Elemental analyses of C, H, and N
were carried out on a Perkin-Elmer 2400 Series II CHNS/O
Analyzer and were accurate within ± 0.4% of theoretical
values.
thus obtained was further agitated for 1 h at room tempera-
ture. The mixture was pour into ice water, and acidifies
with conc. HCl. The resulting precipitate was filtered, dried
and crystallized from ethanol. Yield 81%; mp: 144-145 °C;
EIMS m/z: 265 (M+); 1H-NMR (DMSO-d6): d 7.50 (1H, dd,
J = 0.8, 7.8 Hz, H-5), 7.42-7.00 (1H, m, H-6, 7, 8, H-2¢, 3¢,
4¢, 5¢, 6¢), 6.96 (1H, s, H-2), 5.35 (2H, s, H-10), 3.65 (2H, s,
H-11). 13C-NMR (DMSO-d6, 50 MHz): d 173.34, 138.47,
136.15, 128.75 (2 ´ CH), 127.57 (C, 2 ´ CH), 127.30 (2 ´
CH), 121.59, 119.21, 119.03, 110.25, 107.90, 49.17, 31.10.
1-Benzylindole-3-acetic acid (2)
A mixture of indole-3-acetic acid (1.0 g, 5.7 mmol)
(1) and sodium hydride (0.2 g, 8.3 mmol) in N,N-dimethyl-
formamide (DMF, 10 mL) was stirred at ice bath (0 °C) for
about 30 min, and then benzyl chloride (1.4 g, 11.4 mmol)
was added dropwise with stirring. The reaction mixture

Indole-3-acetic Acid Analogs on the Cell Differentiation
J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 1163
Anal. Calcd for C17H15NO2: C, 76.96; H, 5.70; N, 5.28.
Found: C, 76.90; H, 5.72; N, 5.27.
122.15, 119.62, 118.80, 109.76, 108.12, 49.83, 34.14,
33.16, 14.54. Anal. Calcd for C19H20N2O: C, 78.05; H,
6.89; N, 9.58. Found: C, 78.06; H, 6.85; N, 9.55.
1-Benzylindole-3-acetic chloride (3)
A mixture of compound 2 (1.0 g, 3.8 mmol) and
thionyl chloride (20 mL) in anhydrous diethyl ether (100
mL) was reflux for 1 h. The solvent was evaporated to dry-
ness under reduced pressure to afforded 3.
N-n-Propyl-1-benzylindole-3-acetamide (7)
Compound 3 (1.0 g, 3.5 mmol) was reaction with
propylamine as described for the preparation of 4 to afford
7. Yield 53%; mp: 93-94 °C; EIMS m/z: 306 (M+); H-
1
1-Benzylindole-3-acetamide (4)
NMR (CDCl3): d 7.60 (1H, dd, J = 0.9, 7.4 Hz, H-5), 7.34-
7.11 (1H, m, H-6, 7, 8, H-2¢,3¢,4¢,5¢,6¢), 7.06 (1H, s, H-2),
5.73 (1H, s, -NH), 5.31 (2H, s, H-10), 3.72 (2H, s, H-11),
3.14 (2H, m, H-1¢¢), 1.47-1.21 (2H, m, H-2¢¢), 1.01 (3H, t, J
= 7.0, 14.6 Hz, H-3¢¢). 13C-NMR (CDCl3, 50 MHz): d
171.14, 136.93, 136.61, 128.61 (2 ´ CH), 127.58 (C, 2 ´
CH), 126.65 (2 ´ CH), 122.18, 119.64, 118.78, 109.74,
108.16, 49.83, 40.94 (C-1¢), 33.15, 22.47, 15.05. Anal.
Calcd for C17H16N2O: C, 78.40; H, 7.23; N, 9.14. Found: C,
78.35; H, 7.26; N, 9.16.
Ammonia gas was pass into the solution of the 1-ben-
zylindole-3-acetic chloride (3) (1.0 g, 3.5 mmol) in anhy-
drous diethyl ether (20 mL) at 0 °C for 1.5 h. The reaction
mixture was treated with ice water and extracted with ethyl
acetate. The extract was dried with magnesium sulfate and
evaporated to dryness. The residue was purified by column
chromatography (silica gel/ethyl acetate) to afforded 4.
Yield 24%; mp: 144-145 °C; EIMS m/z: 264 (M+); H-
1
NMR (CDCl3): d 7.61 (1H, d, J = 7.1 Hz, H-5), 7.29-7.12
(1H, m, H-6, 7, 8, H-2¢, 3¢, 4¢, 5¢, 6¢), 7.08 (1H, s, H-2), 5.80
(1H, s, -NHa), 5.69 (1H, s, -NHb), 5.29 (2H, s, H-10) , 3.72
(2H, s, H-11). 13C-NMR (CDCl3, 50 MHz): d 174.13, 136.90,
136.59, 128.63 (2 ´ CH), 127.59 (C, 2 ´ CH), 126.68 (2 ´
CH), 122.22, 119.67, 118.76, 109.78, 108.23, 49.84, 32.75.
Anal. Calcd for C17H16N2O: C, 77.25; H, 6.10; N, 10.60.
Found: C, 77.23; H, 6.11; N, 10.62.
N-n-Butyl-1-benzylindole-3-acetamide (8)
Compound 3 (1.0 g, 3.5 mmol) was reaction with
butylamine as described for the preparation of 4 to afford 8.
Yield 34%; mp: 76-77 °C; EIMS m/z: 320 (M+); 1H-NMR
(CDCl3): d 7.60 (1H, dd, J = 1.6, 7.4 Hz, H-5); 7.35-7.11
(1H, m, H-6, 7, 8, H-2¢,3¢,4¢,5¢,6¢), 7.06 (1H, s, H-2), 5.30
(2H, s, H-10), 5.80 (1H, s, -NH), 3.73 (2H, s, H-11), 3.23-
3.13 (2H, m, H-1¢¢), 1.40-1.18 (2H, m, H-2¢¢, 3¢¢), 0.84 (3H,
t, J = 7.2 Hz, H-4²). 13C-NMR (CDCl3, 50 MHz): d 171.15,
136.99, 136.61, 128.61 (2 ´ CH), 127.57 (C, 2 ´ CH),
126.66 (2 ´ CH), 122.17, 119.62, 118.79, 109.77, 108.18,
49.82, 39.03, 33.16, 31.31, 19.73, 13.49. Anal. Calcd for
C17H16N2O: C, 78.71; H, 7.55; N, 8.74. Found: C, 78.75; H,
7.57; N, 8.76.
N-Methyl-1-benzylindole-3-acetamide (5)
Compound 3 (1.0 g, 3.5 mmol) was reaction with
methylamine as described for the preparation of 4 to afford
5. Yield 49%; mp: 126-127 °C; EIMS m/z: 278 (M+); 1H-
NMR (CDCl3): d 7.58 (1H, d, J = 7.2 Hz, H-5), 7.34-7.12
(1H, m, H-6, 7, 8, H-2¢, 3¢, 4¢, 5¢, 6¢), 7.06 (1H, s, H-2), 5.85
(1H, s, -NH), 5.29 (2H, s, H-10), 3.75 (2H, s, H-11), 2.72
(3H, d, J = 4.8 Hz, H-1¢¢). 13C-NMR (CDCl3, 50 MHz): d
171.94, 136.90, 136.61, 128.63 (2 ´ CH), 127.55 (C, 2 ´
CH), 126.74 (2 ´ CH), 122.18, 119.65, 118.78, 109.79,
108.02, 49.86, 32.96, 26.16. Anal. Calcd for C18H18N2O:
C, 77.69; H, 6.52; N, 10.06. Found: C, 77.67; H, 6.49; N,
10.04.
1-Benzylindole-3-carboxylic acid (10)
Indole-3-carboxylic acid (1 g, 6.2 mmol) (9) was re-
action with benzyl chloride (1.6 g, 12.4 mmol) as described
for the preparation of 2 to afford 10. Yield 84%; mp:
195-196 °C; EIMS m/z: 251 (M+); 1H-NMR (DMSO-d6): d
8.22 (1H, s, H-2), 8.02 (1H, dd, J = 3.1, 5.1 Hz, H-5), 7.51
(1H, dd, J = 2.1, 5.1 Hz, H-8), 7.34-7.23 (1H, m, H-3¢, 4¢,
5¢, H-6, 7), 7.21-7.14 (1H, m, H-2¢, 6¢), 5.47 (2H, s, H-10).
13C-NMR (DMSO-d6, 50 MHz): d 165.93, 137.36, 136.54,
135.76, 128.87 (2 ´ CH), 127.84, 127.48 (2 ´ CH), 126.92,
122.61, 121.67, 121.18, 111.31, 107.19, 49.77. Anal. Calcd
for C16H13NO2: C, 76.47; H, 5.21; N, 5.57. Found: C,
76.49; H, 5.23; N, 5.59.
N-Ethyl-1-benzylindole-3-acetamide (6)
Compound 3 (1.0 g, 3.5 mmol) was reaction with
ethylamine as described for the preparation of 4 to afford 6.
Yield 53%; mp: 108-109 °C; EIMS m/z: 292 (M+); H-
1
NMR (CDCl3): d 7.60 (1H, dd, J = 1.5, 7.3 Hz, H-5), 7.34-
7.10 (1H, m, H-6, 7, 8, H-2¢, 3¢, 4¢, 5¢, 6¢), 7.07 (1H, s, H-2),
5.41 (1H, s, -NH), 5.30 (2H, s, H-10), 3.72 (2H, s, H-11),
3.29-3.16 (2H, m, H-1²), 1.01 (3H, t, J = 7.2 Hz, H-2²).
13C-NMR (CDCl3, 50 MHz): d 171.06, 136.96, 136.61,
128.61 (2 ´ CH), 127.57 (C, 2 ´ CH), 126.68 (2 ´ CH),
1-Benzylindole-3-carboxylic chloride (11)
Compound 10 (1 g, 4.0 mmol) was reaction with thio-
nyl chloride (20 mL) as described for the preparation of 3 to

1164 J. Chin. Chem. Soc., Vol. 55, No. 5, 2008
Lien et al.
afford 11.
H-10), 3.49-3.39 (2H, m, H-1²), 1.71-1.60 (2H, m, H-2²),
0.99 (3H, dt, J = 7.4, 15.4 Hz, H-3²). 13C-NMR (CDCl3, 50
MHz): d 165.04, 136.52, 135.99, 131.21, 128.66 (2 ´ CH),
127.75, 126.74 (2 ´ CH), 125.52, 122.42, 121.29, 120.16,
111.37, 110.34, 50.21, 41.02, 22.96, 11.36. Anal. Calcd for
C19H20N2O: C, 78.05; H, 6.89; N, 9.58. Found: C, 78.08; H,
6.91; N, 9.57.
1-Benzylindole-3-carboxamide (12)
Compound 11 (1 g, 3.7 mmol) was reaction with am-
monia gas as described for the preparation of 4 to afford 12.
Yield 63%; mp: 153-154 °C; EIMS m/z: 252 (M+); H-
1
NMR (DMSO-d6): d 8.00 (1H, dd, J = 0.7, 7.1 Hz, H-5),
7.75 (1H, s, H-2), 7.37-7.24 (1H, m, H-3¢, 4¢, 5¢, H-6, 7, 8),
7.11-7.13 (1H, m, H-2¢, 6¢), 5.74 (1H, s, -NH), 5.33 (2H, s,
H-10). 13C-NMR (DMSO-d6, 50 MHz): d 166.32, 137.68,
136.41, 131.99, 128.87 (2 ´ CH), 127.81, 127.35 (2 ´ CH),
126.97, 122.25, 121.58, 120.92, 110.78, 110.45, 49.64.
Anal. Calcd for C16H14N2O: C, 76.79; H, 5.64; N, 11.19.
Found: C, 76.77; H, 5.62; N, 11.21.
N-n-Butyl-1-benzylindole-3-carboxamide (16)
Compound 11 (1 g, 3.7 mmol) was reaction with bu-
tylamine as described for the preparation of 4 to afford 16.
1
Yield 53%; mp: 110-111 °C; EIMS m/z: 306 (M+); H-
NMR (CDCl3): d 7.97 (1H, dd, J = 0.6, 6.9 Hz, H-5); 7.71
(1H, s, H-2); 7.34-7.21 (1H, m, H-3¢, 4¢, 5¢, H-6, 7, 8);
7.14-7.09 (1H, m, H-2¢, 6¢), 6.06 (1H, s, -NH), 5.27 (2H, s,
H-10), 3.54-3.44 (2H, m, H-1²), 1.67-1.56 (2H, m, H-2²),
1.49-1.38 (2H, m, H-3²), 0.96 (3H, dt, J = 7.14, 14.36,
H-3²). 13C-NMR (CDCl3, 50 MHz): d 164.91, 136.54,
135.94, 131.25, 128.67 (2 ´ CH), 127.77, 126.75 (2 ´ CH),
125.36, 122.40, 121.27, 119.99, 111.46, 110.34, 50.25,
39.02, 31.81, 20.03, 13.62. Anal. Calcd for C20H22N2O: C,
78.40; H, 7.24; N, 9.14. Found: C, 78.43; H, 7.26; N, 9.12.
Cells and Culture8
N-Methyl-1-benzylindole-3-carboxamide (13)
Compound 11 (1 g, 3.7 mmol) was reaction with
methylamine gas as described for the preparation of 4 to af-
ford 13. Yield 68%; mp: 135-136 °C; EIMS m/z: 264 (M+);
1H-NMR (CDCl3): d 8.04 (1H, dd, J = 2.0, 7.8 Hz, H-5),
7.69 (1H, s, H-2), 7.31-7.20 (1H, m, H-3¢, 4¢, 5¢, H-6, 7, 8),
7.08-7.05 (1H, m, H-2¢, 6¢), 6.25 (1H, s, -NH), 5.19 (2H, s,
H-10), 2.99 (3H, d, J = 4.6 Hz, H-1²), 0.99 (3H, dt, J = 7.4,
15.38, H-3²). 13C-NMR (CDCl3, 50 MHz): d 165.74,
136.49, 135.97, 131.03, 128.66 (2 ´ CH), 127.75, 126.74
(2 ´ CH), 125.58, 122.43, 121.29, 120.30, 111.23, 110.26,
50.18, 26.09. Anal. Calcd for C17H16N2O: C, 77.25; H,
6.10; N, 10.60. Found: C, 77.23; H, 6.11; N, 10.62.
Human promyeloid leukemia HL-60 cells were ob-
tained from the American Type Culture Collection (Manassas,
VA, USA) and the Culture Collection and Research Center
(CCRC) (Taiwan, ROC). Human promyeloid leukemia
HL-60 cells were cultured in suspension in RPMI-1640
medium (GIBCO, Grand Is land, USA), containing 10% fe-
tal bovine serum (GIBCO, Grand Is land, USA), 100 unit/
mL penicillin, 100 mL/mL streptomycin and 1% L-gluta-
mine at 37 °C in a humidified atmosphere of 5% CO2 in air.
Cells were split every day to maintain the cell numbers be-
tween 2-5 ´ 105/mL. Cell numbers were assessed by the
standard procedure of leukocyte counting using a hemo-
cytometer and cell viability was checked by the ability of
cells to exclude Trypan blue.
N-Ethyl-1-benzylindole-3-carboxamide (14)
Compound 11 (1 g, 3.7 mmol) was reaction with eth-
ylamine gas as described for the preparation of 4 to afford
14. Yield 58%; mp: 131-132 °C; EIMS m/z: 278 (M+), 1H-
NMR (CDCl3): d 8.07 (1H, dd, J = 0.8, 5.9 Hz, H-5), 7.72
(1H, s, H-2), 7.36-7.19 (1H, m, H-3¢, 4¢, 5¢, H-6, 7, 8),
7.09-7.04 (1H, m, H-2¢, 6¢), 6.32 (1H, s, -NH), 5.16 (2H, s,
H-10), 3.57-3.43 (2H, m, H-1²), 1.24 (3H, dt, J = 7.2, 14.5
Hz, H-2²). 13C-NMR (CDCl3, 50 MHz): d 165.02, 136.49,
136.03, 131.08, 128.64 (2 ´ CH), 127.73, 126.73 (2 ´ CH),
125.69, 122.43, 121.28, 120.37, 111.27, 110.27, 50.16,
34.14, 14.54. Anal. Calcd for C18H18N2O: C, 77.67; H,
6.52; N, 10.06. Found: C, 77.69; H, 6.54; N, 10.66.
N-n-Propyl-1-benzylindole-3-carboxamide (15)
NBT Differentiation Assay8
Cellular differentiation was determined by the NBT
reduction assay. Briefly, cells were incubated in a 96-well
flat bottom plate (1 ´ 105 cells/well) for 4 days. The cells
were then harvested by centrifugation and washed twice
with HBSS (Hanks balanced salt solution). The cells were
resuspended in 200 mL HBSS containing 1 mg/mL of NBT
and 200 mg/mL of phorbol 12-myristate 13-acetate (PMA).
After incubation at 37 °C for 30 min, the cells were pelleted
and dissolved in 200 mL of DMSO, and their absorbance at
Compound 11 (1 g, 3.7 mmol) was reaction with pro-
pylamine as described for the preparation of 4 to afford 15.
Yield 46%; mp: 113-114 °C; EIMS m/z: 292 (M+); H-
1
NMR (CDCl3): d 8.04 (1H, dd, J = 0.8, 6.3, Hz, H-5), 7.72
(1H, s, H-2), 7.32-7.20 (1H, m, H-3¢, 4¢, 5¢, H-6, 7, 8),
7.11-7.06 (1H, m, H-2¢, 6¢), 6.23 (1H, s, -NH), 5.21 (2H, s,

Indole-3-acetic Acid Analogs on the Cell Differentiation
J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 1165
570 nm was determined.
MTT Proliferation Assay8
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ACKNOWLEDGMENTS
This work was financially supported by the research
grant CMU95-100, CMU96-049 and CMU96-230 from
China Medicinal University and by grant 96-2815-C-039-
049-B from National Science Council of Taiwan, R.O.C.
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Received June 4, 2008.