Design, synthesis and activity evaluation of some novel indole derivatives

Article abstract of DOI:10.14233/ajchem.2015.17583

A series of novel indole derivatives as CDK4 inhibitors were designed and synthesized though the condensation reaction between the indolic acid and the corresponding substituted amine. The key step of our synthetic process is the efficient condensation reaction conducted by two different methods. The MTT and kinase assays were conducted used to assess the antitumor activity and cyclin-dependent kinases (CDKs) inhibitory activity. The most active compound 8b has an IC₅₀ of 10-25 μM for the inhibition of four different tumor cells and CDK4. The higher activities of 8b were influenced by more conformational freedom resulted form the non-planar structure and by the stronger hydrogen bonding capability. Thus, the strategy we adapt to design potent, non-toxic CDK4 inhibitors is successful.

Full text of DOI:10.14233/ajchem.2015.17583

Asian Journal of Chemistry; Vol. 27, No. 5 (2015), 1729-1734
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2015.17583
Design, Synthesis and Activity Evaluation of Some Novel Indole Derivatives
*
DIAN HE , ZHU-QING YANG and MENG HOU
Institute of Medicinal Chemistry, School of Pharmaceutical Science, Lanzhou University, Lanzhou 730000, P.R. China
*Corresponding author: Fax: +86 931 8915686; Tel: +86 13008738922; E-mail: hed@lzu.edu.cn
Received: 11 March 2014;
Accepted: 23 May 2014;
Published online: 20 February 2015;
AJC-16880
A series of novel indole derivatives as CDK4 inhibitors were designed and synthesized though the condensation reaction between the
indolic acid and the corresponding substituted amine. The key step of our synthetic process is the efficient condensation reaction conducted
by two different methods. The MTT and kinase assays were conducted used to assess the antitumor activity and cyclin-dependent kinases
(CDKs) inhibitory activity. The most active compound 8b has an IC50 of 10-25 µM for the inhibition of four different tumor cells and
CDK4. The higher activities of 8b were influenced by more conformational freedom resulted form the non-planar structure and by the
stronger hydrogen bonding capability. Thus, the strategy we adapt to design potent, non-toxic CDK4 inhibitors is successful.
Keywords: Indole derivatives, Condensation reaction, Activity evaluation.
activity14. Taking into account that fascaplysin actions on
INTRODUCTION
CDK4 through competing the hydrogen binding sites withATP,
Searching for new small molecule inhibition of cyclin-
reduced activity of the intermediate may be due to the lack of
dependent kinases (CDKs) is an area of major current interest
hydrogen bonding capability caused by changes in the spatial
in the antitumor field1. CDKs perform a vital role in the diffe-
structure. We consider that whether the following measures
rent checkpoints of the eukaryotic cells division cycle. In parti-
can be taken to prevent the reduction, such as flipping the
cular, CDK4/cyclin D1 controls the restriction point, a keeper
amide bond, extending carbon chain, changing the hydrogen
governing the G1-to-S transition. At this checkpoint, CDK4-
type from donor to acceptor. Hence, a series of indole deriv-
cyclin D1phosphorylatesthe retinoblastoma protein (pRB).
atives based on fascaplysin were synthesized and the synthesis
Phosphorylation leads to the release of E2F, which stimulates
and biological activity were reported in this paper. These
the G1-to-S transition across the restriction point where the
compounds are not planar and then the intercalation in DNA
cell is committed to division2-5. Recent researches have found
is very unlikely.
that deregulation of CDK4-cyclin D1 kinase may play a parti-
We chose the inexpensive and commercially available
cularly important role in tumorigenesis6. Cyclin D1 and Cdk4
indolic acid as the raw material. The target compounds were
genes are often amplified or overexpressed in many types of
prepared by the treatment ofthe corresponding substituted
cancers7,8, intimating that CDK4-cyclin D1 kinase would be a
good breakthrough point in the cancer treatment.
amine with acylated indolic acid, or with indolic acid in the
presence of CDI through the condensation reaction. The
The natural pigment fascaplysin 1 (Fig. 1) isolated from
synthetic route ofnovel CDK4-cyclin D1 inhibitors (4a-12b)
the Fijian sponge Fascaplysinopsis Bergquist sp. in 1988 is a
is shown in Scheme-I.
famous CDK4 specific inhibitor9,10. Importantly, it also actives
on cancer cell lines11. However, fascaplysin is highly toxic due
EXPERIMENTAL
to intercalating with DNA resulting from its planar structure12.
Thus, our study is aimming at devising a potent, non-toxic
(non-planar) fascaplysin-based CDK4 inhibitor. Our design
strategy is to open rings of fascaplysin to seek analogues that
have more conformational freedom. Intermediate is a good
pointcut. Compound 2 (Fig. 1) is one intermediate in the
pathway of fascaplysin synthesis by Radchenko in 199713. It
has been reported the weaker CDK4-cyclin D1 inhibitory
Melting points were determined on anYRT-3 digital melting
point apparatus and without correction. 1H NMR and 13C NMR
spectra were recorded in DMSO-d6 or CDCl3 solution on a
Brucker DRX-300M spectrometer operating at 400 MHz.
Chemical shifts were reported in δ (ppm) units relative to the
internal standard tetramethylsilane (TMS) via the solvent signal
(2.49 for 1H or 39.78 for 13C). Mass spectra was measured on

1730 He et al.
Asian J. Chem.
Cl
R
POCl3
N
i
N
n
NH
n1
N
N
H
O
H
ii
N
H
O
O
Intermediate 2
n=1, 2, 3; n1=0,1,2; R=H, CH3
Fascaplysin 1
4a-12b
Fig. 1. Strategy adopted to convert intermediate 2 into the 4a-12b. i:flip the of the intermediate 2; ii:extend carbon chain of the products flipped
R
N
a
b
COOH
n
n
n
COCl
n1
n
(A)
O
N
N
H
N
H
H
4a-12b
R
N
c
COOH
n1
n
NHR
n1
(B)
+
O
N
H
N
H
n=1, 2, 3; n1=0,1,2; R=H, CH3
4a-12b
Scheme-1:Synthetic route to target compounds 4a-12b; Reagents and conditions: Method A, a) Dry THF, (COCl)2, 50 °C, 1h; b) Amine, Dry THF, rt, 4 h;
Method B, c) CDI, rt
a HP-5988 mass spectrometer and optical rotation was detected
by WXG-4 polarimeters.All chemicals and solvents used were
of reagent grade purified and dried by standard methods
before use. TLC monitoring all the reaction was performed
on precoated silica gel G plates at 254/365 nm under a UV
lamp, with petrol ether/ethyl acetate/triethylamine as the
mobile phase.
bottomed flask. The oxalyl chloride was then evaporated and
the resulting solid was dissolved in anhydrous THF (5 mL).
The reaction was cooled in an ice bath and aniline (1 mmol)
in the presenceof TEA (3 mL) was added dropwise. After
addition, the mixture was warmed to room temperature and
stirred for 4 h. After the completion of reaction, the product
was extracted with ethyl acetate. The organic layer was washed
with 5 % NaHCO3 and 10 % HCl followed by distilled water.
Finally the organic layer was dried over anhydrous MgSO4.
The product was purified by chromatography on silica gel to
give compound 4a-12b .
Method B: Acid (1 mmol) and carbonyl diimidazole
(CDI) (1 mmol) were dissolved in 3 mL of THF and stirred at
room temperature. After 0.5 h, amine was added and the
reaction mixture stirred overnight (monitored by TLC). Then
the mixture was poured into water (20 mL), extracted by ethyl
acetate and the combined organic solvent was dried over
MgSO4, filtered and concentrated in vacumn. The crude
product was purified by recrystallization in petrol ether and
ethyl acetate to give compound 4a-12b.
General procedure for the synthesis of the compounds
of series N-substituted amines: To a solution of corresponding
amine (5 mmol) and triethylamine (4 mL) in CH2Cl2 (30 mL)
cooled to 0 °C, ethyl chloroformate (0.75 mL, 6.25 mmol)
was added dropwise over10 min with stirring. The reaction
mixture was stirred for an additional 0.5 h at 0 °C, then allowed
to warm to room temperature and stirred for an additional 1.5
h. The solvent was removed in vacuo. Afterwards, a solution
of the product above (3.2 mmol) in THF (8 mL) was slowly
added to a suspension of LiAlH4 (35.1 mmol) in THF (30 mL)
cooled to 0°C under argon. After the addition was completed,
the reaction mixture was allowed to warm to room temperature,
then refluxed for 3 h. After allowing to cool to room tempe-
rature, the reaction was quenched by the addition of water
(5 mL) and 15 % aqueous KOH (w/v, 1 mL). The precipitate
was removed by filtration and washed with ether (90 mL),
then with MeOH (16 mL). The combined filtrates were dried
over Na2SO4, then the solvent removed in vacuo and the
residues purified by flash chromatography (EtOAc/CH2Cl2,
1:1) to give N-substituted amines.
2-(1H-Indol-3-yl)-N-phenyl acetamide(4a):White solid;
1
81 % yield; m.p. 156-157 °C; H NMR (400 MHz CDCl3):
δ: 8.48 (1H, s, NH), 7.61 (1H, d, J = 8.0 Hz ), 7.47 (1H, s,
CONH), 7.42 (1H, d, J = 8.0 Hz), 7.34 (2H, d, J = 8.0 Hz),
7.27 ( 1H, d, J = 7.6 Hz), 7.23 ( 2H, t, J1 = 7.6 Hz, J2 = 8.0 Hz),
7.17 ( 2H, t, J1 = 6.4 Hz, J2 = 7.2 Hz), 7.05 (1H, t, J1 = 7.2 Hz,
J2 = 7.2 Hz), 3.90 (2H, s, CH2); 13C NMR (100 MHz, CDCl3)
δ: 165.4, 141.8, 136.0, 131.6, 128.7, 125.9, 122.8, 121.7,
120.9, 120.8, 118.4, 117.4, 111.8, 111.6, 104.1, 55.3; H RMS
(ESI): (M + H) 251.1179 (calculated 251.3031 ), error = 1.2
ppm.
General Procedure for the synthesis of compounds 4a-12b:
MethodA: The corresponding acid (2 mmol) was heated
at 50 °C in oxalyl chloride (1 mL) for 1 h in a 25 mL round-

Vol. 27, No. 5 (2015)
Design, Synthesis and Activity Evaluation of Some Novel Indole Derivatives 1731
2-(1H-Indol-3-yl)-N-methyl-N-phenyl acetamide (4b):
White solid; 73 % yield; m.p. 172-173 °C; 1H NMR (400 MHz
CDCl3): δ:8.61 (1H, s, NH), 7.66 (1H, d, J = 7.6 Hz ), 7.42
(1H, d, J = 7.2 Hz), 7.31 (2H, d, J = 7.6 Hz), 7.27 ( 1H, d, J =
7.6 Hz), 7.19 ( 2H, t, J1 = 7.2 Hz, J2 = 7.4 Hz), 7.11 ( 2H, t, J1
= 7.6 Hz, J2 = 7.2 Hz), 7.05 (1H, t, J1 = 7.2 Hz, J2 = 7.2 Hz),
3.89 (2H, s, CH2), 2.81 (3H, s, CH3); 13C NMR (100 MHz,
CDCl3) δ:163.7, 142.1, 136.2, 131.5, 128.7, 125.9, 122.8,
121.1, 120.8, 120.8, 118.4, 118.4, 111.8, 111.6, 104.2, 47.5,
34.3 H RMS (ESI): (M + H) 265.2736 (calculated 265.3296),
error = 0.8 ppm.
J1 = 7.6 Hz, J2 = 7.2 Hz), 7.06 (1H, t, J1 = 7.2 Hz, J2 = 7.2 Hz),
6.94 (1H, s, J = CONH), 3.17 (2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz,
CH2), 2.70 (2H, t, J1 =7.2 Hz, J2 = 7.2 Hz, CH2); 13C NMR
(100 MHz, CDCl3) δ: 171.0, 139.4, 136.1, 131.6, 127.7, 127.7,
125.1, 122.3, 121.7,120.9, 120.9, 120.5 119.4, 113.2, 111.0,
34.1, 26.1; H RMS(ESI): (M + H) 265.1335 (calculated
265.3296), error = 1.1 ppm.
3-(1H-Indol-3-yl)-N-methyl-N-phenyl propionamide
(7b): Light yellow powder; 83 % yield; m.p. 143-144 °C; 1H
NMR (400MHz CDCl3): δ: 8.04 (1H, s, NH), 7.63 (2H, d, J =
7.2 Hz), 7.44 (1H, d, J = 8.0 Hz), 7.36 (3H, t, J1 = 7.2 Hz, J2 =
7.6 Hz), 7.31 (1H, d, J = 7.2 Hz), 7.25 (1H, d, J = 8.0 Hz),
7.11 (1H, t, J1 = 7.6 Hz, J2 = 7.6 Hz), 7.04 (1H, t, J1 = 7.6 Hz,
J2 = 7.2 Hz), 3.18 (2H, t, J1 = 7.6 Hz, J2 = 7.2 Hz, CH2), 2.82
(3H, s, CH3); 2.64 (2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz, CH2); 13C
NMR (100 MHz, CDCl3) δ: 170.1, 139.4, 136.0, 131.6, 127.8,
127.7, 125.2, 122.3, 121.6, 120.9, 120.9, 120.4, 119.6, 113.1,
111.2, 35.2, 32.1, 26.6; H RMS(ESI): (M + H) 279.3538
(calculated 279.3562), error = 1.1 ppm.
2-(1H-Indol-3-yl)-N-benzyl acetamide (5a): Yellow
1
solid; 65 % yield; m.p. 154-155 °C; H NMR (400 MHz
CDCl3): δ:10.9 (1H, s, NH), 8.4 (1H, s, CONH), 7.54 (1H, d,
J = 7.6 Hz), 7.33 (1H, d, J = 8.0 Hz), 7.26 (2H, d, J = 6.0 Hz),
7.21 (4H, t, J1 = 7.6 Hz, J2 = 6.4 Hz), 7.06 (1H, t, J1 = 7.6 Hz,
J2 = 7.2 Hz), 6.95 (1H, d, J = 7.2 Hz), 4.39 (2H, d, J = 4.8 Hz,
CH2), 3.81 (2H, s, CH2); 13C NMR (100 MHz, CDCl3) δ: 176.3,
140.1, 138.5, 132.1, 129.8, 129.8, 127.7, 127.7, 126.5, 123.8,
121.5, 120.7, 119.6, 117.4, 112.8, 52.3, 30.9 , H RMS(ESI):
(M + H) 265.1335 (calculated 265.3296 ), error = 0.8 ppm.
2-(1H-Indol-3-yl)-N-methyl-N-benzyl acetamide (5b):
Yellow solid; 51 % yield; m.p.160-162 °C; 1H NMR (400 MHz
CDCl3): δ:10.9 (1H, s, NH), 7.56 (1H, d, J = 7.2 Hz), 7.34
(1H, d, J = 7.6 Hz), 7.25 (2H, d, J = 6.0 Hz ), 7.22 ( 4H, t, J1 =
7.6 Hz, J2 = 7.2 Hz), 7.11 (1H, t, J1 = 7.2 Hz, J2 = 7.2 Hz), 6.90
(1H, d, J = 7.2 Hz), 4.40 (2H, d, J = 6.4 Hz, CH2), 3.82 (2H, s,
CH2), 2.97 (3H, s, CH3); 13C NMR (100 MHz, CDCl3) δ: 175.1,
139.3, 138.5, 131.1, 129.8, 129.8, 128.1, 127.7, 126.5, 123.8,
122.5, 120.6, 119.1, 117.4, 112.8, 55.3, 32.6, 28.5 , H
RMS(ESI): (M + H) 279.1600 (calculated 279.3562), error =
0.8 ppm.
3-(1H-Indol-3-yl)-N-benzyl propionamide (8a): White
1
solid; 66 % yield; m.p. 119-120 °C; H NMR (400 MHz
CDCl3): δ: 8.04 (1H, s, NH), 7.60 (1H, d, J = 8.0 Hz), 7.36
(1H, d, J = 8.4 Hz), 7.20 (2H, t, J1 = 7.2 Hz, J2 = 7.6 Hz), 7.12
(2H, d, J = 7.6 Hz), 7.08 (3H, t, J1 = 7.6 Hz, J2 = 5.6 Hz), 6.96
(1H, d, J = 7.6 Hz), 5.90 (1H, s, CONH), 4.36 (2H, d, J = 8.0
Hz, CH2 ), 3.14 (2H, t, J1 = 6.4 Hz, J2 = 6.8 Hz, CH2), 2.61
(2H, t, J1 = 6.8 Hz, J2 = 7.2 Hz, CH2); 13C NMR (100 MHz,
CDCl3) δ: 171.9, 139.6, 136.3, 131.6, 128.2, 128.2, 127.1,
127.1, 126.6,122.2, 120.9, 118.1, 113.7, 112.1, 111.3, 41.2,
36.3, 21.1; H RMS (ESI): (M + H) 279.1492 (calculated
279.3562), error = 0.7 ppm.
3-(1H-Indol-3-yl)-N-methyl-N-benzyl propionamide
1
2-(1H-Indol-3-yl)-N-phenethyl acetamide (6a): White
(8b): White solid; 59 % yield; m.p. 123-125 °C; H NMR
1
solid; 89 % yield; m.p. 101-102 °C; H NMR (400 MHz
(400 MHz CDCl3): δ: 8.11 (1H, s, NH), 7.57 (1H, d, J = 7.6
Hz), 7.26 (1H, d, J = 8.0 Hz), 7.19 (2H, t, J1 = 7.6 Hz, J2 = 7.6
Hz), 7.11 (2H, d, J = 7.2 Hz), 7.04 (3H, t, J1 = 7.2 Hz, J2 = 7.2
Hz), 6.96 (1H, d, J = 7.6 Hz), 4.44 (2H, d, J = 7.2 Hz, CH2),
3.12 (2H, t, J1 = 7.2 Hz, J2 = 6.8 Hz, CH2), 3.02 (3H, s, CH3);
2.65 (2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz, CH2); 13C NMR (100
MHz, CDCl3) δ: 171.2, 137.6, 136.3, 131.6, 129.2, 129.2,
127.5, 127.5, 126.6, 122.2, 120.8, 118.0, 113.7, 112.3, 111.1,
47.2, 35.3, 32.2, 21.5; H RMS(ESI): (M + H) 293.3757
(calculated 293.3828), error = 0.7 ppm.
3-(1H-Indol-3-yl)-N-phenethyl propionamide (9a):
White solid; 91 % yield; m.p. 156-158 °C; 1H NMR (400 MHz
CDCl3): δ: 10.8 (1H, s, NH),7.99 (1H, t, J1 = 7.6 Hz, J2 = 7.2
Hz, CONH), 7.53 (2H, d, J = 7.6 Hz), 7.33 (2H, d, J = 8.0 Hz),
7.11 (1H, d, J = 8.8 Hz), 7.06 (3H, t, J1 = 6.8 Hz, J2 = 6.0 Hz),
6.97 (1H, t, J1 = 7.2 Hz, J2 = 7.6 Hz), 3.33 (2H, q, J1 = 7.2 Hz,
J2 = 6.0 Hz, J3 = 6.8 Hz, CH2), 2.92 (2H, t, J1 = 7.6 Hz, J2 = 7.2
Hz, CH2), 2.80 (2H, t, J1 = 7.2 Hz, J2 = 6.8 Hz, CH2 ), 2.44
(2H, t, J1 = 7.6 Hz, J2 = 7.6 Hz, CH2 ); 13C NMR (100 MHz,
CDCl3) δ: 171.7, 140.6, 136.2, 130.1, 128.7, 128.7, 127.2,
127.1, 122.5,122.0, 118.2, 118.1, 113.9, 111.9, 111.3, 41.2,
36.4, 25.3, 21.0; H RMS (ESI): (M + H) 293.3525 (calculated
293.3828 ), error = 0.9 ppm.
CDCl3): δ:10.8 (1H, s, NH), 8.7 (1H, t, J1= 4.8 Hz, J2 = 5.6
Hz, CONH), 7.52 (2H, d, J = 7.6 Hz ), 7.34 (2H, d, J = 8.0
Hz), 7.17 (1H, d, J = 7.6 Hz), 7.06 (3H, t, J1 = 6.8 Hz, J2 = 6.0
Hz), 6.96 (2H, t, J1 = 7.6 Hz, J2 = 7.2 Hz), 3.33 (2H, q, J1 = 7.2
Hz, J2 = 6.8 Hz, J3 = 6.8 Hz, CH2), 2.81 (2H, t, J1 = 7.6 Hz, J2
= 7.2 Hz, CH2); 13C NMR (100 MHz, CDCl3) δ: 170.6, 136.2,
136.1, 131.6, 128.5, 128.5, 127.9, 127.9, 126.7, 122.8, 122.7,
120.5, 118.6, 111.4, 108.9, 50.3, 47.2, 33.9. H RMS(ESI): (M
+ H) 279.3532 (calculated 279.3562), error = 0.6 ppm.
2-(1H-Indol-3-yl)-N-methyl-N-phenethyl acetamide
1
(6b): White solid; 76 % yield; m.p. 145-146 °C; H NMR
(400 MHz CDCl3): δ: 10.8 (1H, s, NH), 7.61 (2H, d, J = 7.2
Hz ), 7.43 (2H, d, J = 7.6 Hz), 7.20 (1H, d, J = 7.6 Hz ), 7.05
(3H, t, J1 = 6.8 Hz, J2 = 7.2 Hz), 6.92 (2H, t, J1 = 7.2 Hz, J2 =
7.2 Hz), 3.31 (2H, q, J1 = 7.6 Hz, J2 = 7.2 Hz, J3 = 6.8 Hz,
CH2), 3.01 (3H, s, CH3), 2.81 (2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz,
CH2); 13C NMR (100 MHz, CDCl3) δ: 169.6, 136.2, 136.1,
131.6, 128.3, 128.3, 127.7, 127.6, 126.1, 122.8, 122.8, 120.5,
118.6, 111.4, 108.7, 52.1, 49.3, 33.3, 32.1. H RMS(ESI): (M
+ H) 293.3797 (calculated 293.3828), error = 0.6 ppm.
3-(1H-Indol-3-yl)-N-phenyl propionamide (7a): Light
yellow powder; 87 % yield; m.p. 135-136°C; 1H NMR (400
MHz CDCl3): δ: 8.04 (1H, s, NH), 7.60 (1H, d, J = 8.0 Hz),
7.36 (3H, t, J1 = 8.0 Hz, J2 = 7.6 Hz), 7.35 (1H, d, J = 7.6 Hz),
7.30 (1H, d, J = 8.0 Hz), 7.19 (2H, d, J = 6.8 Hz), 7.12 (1H, t,
3-(1H-Indol-3-yl)-N-methyl-N-phenethyl propion-
1
amide (9b): White solid; 88 % yield; m.p. 175-176 °C; H
NMR (400 MHz CDCl3): δ: 10.8 (1H, s, NH), 7.67 (2H, d, J =

1732 He et al.
Asian J. Chem.
7.2 Hz), 7.31 (2H, d, J = 7.6 Hz), 7.13 (1 H, d, J = 8.0 Hz),
7.01 (3H, t, J1 = 7.2 Hz, J2 = 7.2 Hz), 6.89 (1H, t, J1 = 7.6 Hz,
J2 = 7.6 Hz), 3.54 (2H, q, J1 = 7.2 Hz, J2 = 7.2 Hz, J3 = 6.8 Hz,
CH2), 3.01 (2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz, CH2 ), 2.96 (3H, s,
CH3), 2.77 (2H, t, J1 = 7.6 Hz, J2 = 7.2 Hz, CH2), 2.41 (2H, t,
J1 = 7.2 Hz, J2 = 7.6 Hz, CH2 ); 13C NMR (100 MHz, CDCl3)
δ: 170.3, 140.6, 136.2, 130.1, 127.9, 127., 127.3, 127.2, 122.1,
122.1, 118.3, 118.1, 112.9, 111.3, 111.0, 51.4, 35.3, 33.1, 25.3,
21.7; H RMS (ESI): (M + H) 307.3790 (calculated 307.4093),
error = 0.9 ppm.
137.3, 131.7, 128.2, 128.2, 127.2, 126.7, 121.3,120.1, 120.1,
118.7, 118.3, 115.6, 111.3, 41.9, 35.3, 32.4, 27.2, 24.3; H RMS
(ESI):(M + H) 307.4313 (calculated 307.4094 ), error = 0.3
ppm.
4-(1H-Indol-3-yl)-N-phenethyl butyramide (12a):
White solid; 95 % yield; m.p.124-125°C; 1H NMR (400 MHz
CDCl3): δ: 10.8 (1H, s, NH), 7.92 (1H, t, J1 = 5.2 Hz, J2 = 4.8
Hz, CONH), 7.53 (1H, d, J = 8.0 Hz), 7.49 (1H, d, J = 7.6 Hz),
7.32 (2H, d, J = 8.0 Hz), 7.14 (1H, d, J = 7.2 Hz), 7.06 (3H, t,
J1 = 7.6 Hz, J2 = 7.6 Hz), 6.97 (2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz),
3.33 (2H, dd, J1 = 6.8 Hz, J2 = 6.4 Hz, CH2), 2.82 (2H, t, J1 =
7.6 Hz, J2 = 7.2 Hz, CH2), 2.66 (2H, t, J1 = 7.2 Hz, J2 = 7.6 Hz,
CH2), 2.15 (2H, q, J1 = 6.8 Hz, J2 = 7.2 Hz, J3 = 7.6 Hz, CH2),
1.91 (2H, t, J1 = 7.6 Hz, J2 = 7.6 Hz, CH2); 13C NMR (100
MHz, CDCl3) δ: 171.2, 136.3, 128.7, 128.3, 127.2, 122.5,
122.2, 120.9, 120.8, 118.2, 118.2, 118.1, 114.2, 111.9, 111.3,
40.6, 35.4, 26.1, 25.3, 24.3; H RMS (ESI): (M + H) 307.3983
(calculated 307.4094), error = 0.6 ppm.
4-(1H-Indol-3-yl)-N-phenyl butyramide (10a): Yellow
1
powder; 93 % yield; m.p.102-103 °C; H NMR (400 MHz
CDCl3): δ: 8.05 (1H, s, NH), 7.59 (1H, d, J = 7.6 Hz), 7.45
(2H, d, J = 7.2 Hz ), 7.33 (2H, d, J = 8.4 Hz), 7.29 (2H, t, J1 =
7.6 Hz, J2 = 7.2 Hz), 7.19 (1H, t, J1 = 6.8 Hz, J2 = 7.6 Hz), 7.10
(2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz), 6.93 (1H, s, CONH), 2.84
(2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz, CH2), 2.37 (2H, q, J1 = 7.2 Hz,
J2 = 7.6 Hz, J3 = 6.8 Hz, CH2), 2.13 (2H, t, J1 = 7.2 Hz, J2 = 7.6
Hz, CH2); 13C NMR (100MHz, CDCl3) δ: 171.2, 139.4, 136.3,
128.6, 128.6, 127.2, 127.1, 122.8,122.3, 119.1, 118.3, 118.1,
114.0, 111.3, 111.3, 40.1, 25.9, 24.3; H RMS (ESI): (M + K)
317.1051 (calculated 317.4826 ), error = 4.1 ppm.
4-(1H-Indol-3-yl)-N-methyl-N-phenyl butyramide
(10b):Yellow powder; 86 % yield; m.p. 135-136 °C; 1H NMR
(400 MHz CDCl3): δ: 8.15 (1H, s, NH), 7.60 (1H, d, J = 7.6
Hz), 7.35 (2H, d, J = 7.2 Hz ), 7.31 (2H, d, J = 8.4 Hz), 7.29
(2H, t, J1 = 7.6 Hz, J2 = 7.2 Hz), 7.19 (1H, t, J1 = 6.8 Hz, J2 =
7.6 Hz), 7.13 ( 2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz), 2.93 (3H, s,
CH3), 2.84 (2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz, CH2), 2.37 (2H, q,
J1 = 7.2 Hz, J2 = 7.6 Hz, J3 = 6.8 Hz, CH2), 2.13 (2H, t, J1 = 7.2
Hz, J2 = 7.6 Hz, CH2 ); 13C NMR (100 MHz, CDCl3) δ: 171.5,
139.6, 136.3, 128.6, 128.6, 127.2, 127.1, 122.8,122.3, 119.1,
118.3, 118.1, 114.0, 111.3, 111.3, 36.1, 34.0, 25.9, 24.3; H
RMS(ESI): (M + K) 331.1316 (calculated 331.4732), error =
4.0 ppm.
4-(1H-Indol-3-yl)-N-methyl-N-phenethyl butyramide
1
(12b): White solid; 83 % yield; m.p.141-142 °C; H NMR
(400 MHz CDCl3): δ: 10.5 (1H, s, NH), 7.61 (1H, d, J = 7.6
Hz), 7.49 (1H, d, J = 7.6 Hz), 7.39 (2H, d, J = 8.0 Hz), 7.16
(1H, d, J = 7.6 Hz), 7.12 (3H, t, J1 = 7.2 Hz, J2 = 7.6 Hz), 6.97
(2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz), 3.21 (2H, dd, J1 = 6.4 Hz, J2
= 6.4 Hz, CH2), 2.90 (3H, s, CH3), 2.77 (2H, t, J1 = 7.6 Hz, J2
= 7.2 Hz, CH2), 2.67 (2H, t, J1 = 7.2 Hz, J2 = 7.6 Hz, CH2),
2.13 (2H, q, J1 = 7.2 Hz, J2 = 7.2 Hz, J3 = 7.6 Hz, CH2), 2.01
(2H, t, J1 = 7.2 Hz, J2 = 7.6 Hz, CH2); 13C NMR (100 MHz,
CDCl3) δ: 170.2, 136.3, 129.1, 128.5, 127.2, 122.4, 122.3,
120.9, 120.9, 118.1, 118.1, 118.0, 115.2, 111.9, 111.6, 45.1,
37.4, 32.7, 26.1, 25.5, 24.3; H RMS (ESI): (M + H) 321.4248
(calculated 321.4359 ), error = 0.6 ppm.
Biological activity cell culture and MTT assay: LS174T,
A549, Hela, BeL-7402 were maintained in DMEM, supple-
mented with 10 % FBS, at 37 °C under 5 % CO2 in the air.
Cell proliferation was determined by standard MTT assay in
LS174T, A549, Hela, BeL-7402. Cells were inoculated in 96-
well plates at the density of 2 × 105 cells per well. Attached
cells were incubated for 24 h prior to compounds addition at
different concentrations and then the incubation period was
extended for another 48 h. The cell survival fraction was deter-
mined with 10 ul MTT (5 mg mL-1 in PBS) per well. Cells
were incubated further for 4 h at 37 °C. Finally the crystals
formed were dissolved by the addition of DMSO and measured
by the absorbance at 490 nm.
Kinase assays and IC50 determination: The kinase assays
measure the IC50s of the compounds through the depletion in
ATP concentrations occurring as a result of phosphorylation
of retinoblastoma and histone H1 by CDK4 and CDK2,
respectively10,15. Protein complexes of CDK4-cyclin D1 and
CDK2-cyclin A were extracted and purified according to
description of the CDK4-cyclin D1 or CDK2-cyclin A kinase
activity spectrum quantitative detection kit (GENMED
SCIENTIFICS INC. U.S.A). Protein content was measured in
Bradford assay. The compounds were dissolved in DMSO as
10 mM stock solutions. Compounds were further diluted in
kinase buffer in order to obtain the desired concentrations.
Purified enzyme complexes were pretreated first according to
the requirements of the kit. The assay was run in a 96 well
4-(1H-Indol-3-yl)-N-benzyl butyramide (11a): White
1
solid; 67 % yield; m.p.122-123°C; H NMR (400 MHz
CDCl3): δ: 8.14 (1H, s, NH), 7.55 (1H, d, J = 7.6 Hz), 7.31
(2H, d, J = 7.6 Hz ), 7.28 (1H, d, J = 7.6 Hz), 7.24 (2H, t, J1
= 5.6 Hz, J2 = 6.8 Hz), 7.16 (1H, t, J1 = 7.6 Hz, J2 = 7.6 Hz),
7.08 (1H, t, J1 = 7.6 Hz, J2 = 7.2 Hz), 6.90 (1H, s, CONH),
5.75 (1H, d, J = 7.2 Hz), 4.46 (2H, d, J = 5.6 Hz, CH2), 2.79
(2H, t, J1 = 7.2 Hz, J2 = 7.2 Hz, CH2), 2.24 (2H, t, J1 = 7.6
Hz, J2 = 7.6 Hz, CH2), 2.07 (2H, q, J1 = 7.2 Hz, J2 = 7.2 Hz,
J3 = 7.2 Hz, CH2 ); 13C NMR (100 MHz, CDCl3) δ: 172.1,
139.8, 136.3, 131.7, 128.3, 128.3, 127.2, 126.7, 122.4, 120.8,
120.8, 118.3, 118.1, 114.1, 111.3, 42.0, 35.2, 26.2, 24.3; H
RMS (ESI): (M + H) 293.1648 (calculated 293.3828 ), error
= 0.3 ppm.
4-(1H-Indol-3-yl)-N-methyl-N-benzyl butyramide
1
(11b): White solid; 55 % yield; m.p.129-130 °C; H NMR
(400 MHz CDCl3): d: 8.27 (1H, s, NH), 7.56 (1H, d, J = 7.2
Hz), 7.52 (2H, d, J = 7.6 Hz), 7.32 (1H, d, J = 7.6 Hz), 7.24
(2H, t, J1 = 7.2 Hz, J2 = 6.8 Hz), 7.16 (1H, t, J1 = 7.6 Hz, J2 =
7.6 Hz), 7.08 (1H, t, J1 = 7.6 Hz, J2 = 7.2 Hz), 5.75 (1H, d, J =
7.2 Hz), 4.46 (2H, d, J = 5.6 Hz, CH2), 3.01 (3H, s, CH3), 2.69
(2H, t, J1 = 7.6 Hz, J2 = 7.6 Hz, CH2), 2.31 (2H, t, J1 = 7.6 Hz,
J2 = 7.6 Hz, CH2), 2.11 (2H, q, J1 = 7.2 Hz, J2 = 7.6 Hz, J3 =
7.2 Hz, CH2); 13C NMR (100 MHz, CDCl3) δ: 171.4, 139.6,

Vol. 27, No. 5 (2015)
Design, Synthesis and Activity Evaluation of Some Novel Indole Derivatives 1733
format and was performed in 25 uL of kinase buffer with the
enzymatic solution, substrate solution (in case of CDK4-cyclin
D1 or CDK2-cyclin A) added sequentially. The plate was
incubated for 3 min at 30 °C in a humidified incubator. Then
the 20 µL pretreated sample was added and determined by
microplate reader at 30 °C for 0.5 h. In the case of the CDK4-
cyclin D1 assay, the compounds fascaplysin (Merck) with known
IC50 values were used to validate the assay. For the CDK2-
cyclin A assay, flavopiridol (Sigma) were used as standards
for the assay.
also shown in Table-1. Again we see that all compounds are
CDK4 active compared to CDK2. The IC50s were less than 60
µM for three of compounds . 8b also shows the most powerful
inhibition to CDK4 with an IC50 of 17 µM. Therefore, it is
likely that 8b inhibits cell proliferation by inhibiting the activity
of CDK4.
In addition, the product of flipping the amide bond 8a
falls a little short of the activities. But the activity would increase
significantly when we methylated the amide group (8b). In
contrast, when we changed the carbon chains length at both
ends of amide bond, the opposite result was obtained. With
the extension of the carbon chains, the activity decreased badly,
such like 10a-12b.A possibility we predict is that methylation
of the amino group may lead compound itself to combine
with CDK4 more strongly. However, the extension of the
carbon chains resists its inserting into the CDK4 binding
pocket.
RESULTS AND DISCUSSION
The strategy we adopt is to maintain most of the key
interactions thought to occur between fascaplysin and CDK4
to enhance the success. We designed a series of such comp-
ounds proceeding from the intermediate before closing the
loop in the fascaplysin synthesis.All compounds in this series
could be synthesized by two different methods. Most yields
are more than 70 %. Some compounds have been previously
synthesized, while the application as CDK4 inhibitiors has
not been studied. Spectroscopy data and melting point agreed
Conclusion
In summary, our studies of structure-activity relationship
suggests that breaking the planar structure and changing the
type of hydrogen bonds from donor to acceptor of the lead
compound fascaplysin may greatly improve the activity, which
will contribute to the discovery of a series of prospective non-
toxic (non-planar) fascaplysin-based CDK4 inhibitor. In
addition, the higher activity of 8b compared other indole
derivatives also proves the success of the efforts to flip the
amide bond and extending carbon chain. The application of
these results and further mechanism studies are underway in
our laboratory.
with those of the literature16-19
.
All compounds were tested for in vitro cytotoxicity against
four human tumor cell lines such as LS174T, A549, Hela and
BeL-7402. The inhibitory effects of compounds were quan-
tified using the MTT assay and IC50s for cell growth inhibition
were determined. The results were showed in Table-1. These
results showed that all the compounds (4a-12b) have different
degrees of cytotoxicity. In particular, 8b is most potent and
shows no differential cytotoxicity against four different tumor
cells and CDK4 with the IC50 of 10-25 µM. The results is
consistent with that seen in kinase assays. The kinase assays
measure the IC50 of all compounds through the depletion in
ATP concentrations occurring as a result of phosphorylation
by CDKs of GST-pRB152 (a substrate for both CDK4-cyclin
D1 and CDK2-cyclin A). The results for the inhibition are
ACKNOWLEDGEMENTS
The authors are thankful to the Fundamental Research
Funds for the Central Universities (lzujbky-2010-137 and
lzujbky-2014-217) and Lanzhou Science and Technology
Bureau Program Funds (2012-2-90).
TABLE-1
ACTIVITY OF FASCAPLYSIN ANALOGUES IN DIFFERENT in vitro ASSAYS
IC50 (µmol L-1)
A549 Hela
0.73 0.1
Compound
n
n1
R
CDK4-cyclin D1
0.54 0.1
CDK2- cyclin A
LS174T
0.92 0.1
BeL-7402
0.61 0.1
Fascaplysin
4a
-
-
-
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
513 11
1267 37
1020 34
625 20
584 24
763 26
731 21
987 31
784 18
1125 27
832 17
584 24
913 23
950 34
1320 35
937 27
821 23
1410 31
1190 26
1.51
134
146
87
1
5
5
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
0
0
1
1
2
2
0
0
1
1
2
2
0
0
1
1
2
2
126
117
64
9
6
7
153
110
67
5
4
4
95
103
51
91
105
3
4
7
6
5
112
108
76
4
6
6
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
8
59
88
73
5
7
8
55
87
58
2
4
5
121
136
96
7
4
94
97
88
93
35
87
14
59
3
5
3
3
2
4
1
3
3
81 3.2
7
103
36
85
17
95
7
105
28
78
11
77
6
115
12
8
1
141
52
9
3
6
2
7
6
4
4
1
3
3
95
16
94
123
103
115
95
121
202
207
3
2
6
8
4
8
126
25
6
3
122
151
156
147
133
134
275
131
4
9
5
9
6
8
9
5
91
101
130
87
6
5
6
87
136
141
171
156
257
233
8
118
76
113
92
7
3
4
CH3
H
CH3
H
7
5
3
9
6
159
175
187
265
5
4
6
6
4
8
9
8
4
117
211
4
6
CH3

1734 He et al.
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