Eco-friendly synthesis and study of new plant growth promoters: 3,3′-Diindolylmethane and its derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.06.025

3,3′-Diindolylmethane (DIM) derivatives 3a-k, prepared in one-pot from indoles 1a-k and hexamethylenetetramine (2) using ionic liquid [Bmim]BF₄ as eco-friendly recyclable solvent as well as catalyst, showed good plant growth promoting activity on Oryza sativa. Among the DIM derivatives synthesized 3c shows potent auxin like growth promoting activity.

Full text of DOI:10.1016/j.bmcl.2007.06.025

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4924–4928
Eco-friendly synthesis and study of new plant growth
promoters: 3,30-Diindolylmethane and its derivatives
Churala Pal,a Sumit Dey,a Sanjit Kumar Mahato,a Jayaraman Vinayagam,a
Prasun K. Pradhan,a Venkatachalam Sesha Giri,a Parasuraman Jaisankar,a,*
Tanvir Hossain,a, Shikhi Baruri,a, Debjit Raya, and Suparna Mandal Biswasb
aDepartment of Medicinal Chemistry, Indian Institute of Chemical Biology (Unit of C.S.I.R), 4,
Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India
bAgricultural and Ecology Research Unit, Indian Statistical Institute 203, B.T. Road, Calcutta 700 108, India
Received 4 May 2007; revised 4 June 2007; accepted 7 June 2007
Available online 10 June 2007
Abstract—3,30-Diindolylmethane (DIM) derivatives 3a–k, prepared in one-pot from indoles 1a–k and hexamethylenetetramine (2)
using ionic liquid [Bmim]BF4 as eco-friendly recyclable solvent as well as catalyst, showed good plant growth promoting activity on
Oryza sativa. Among the DIM derivatives synthesized 3c shows potent auxin like growth promoting activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Natural auxins constitute a group of phytohormones
which play an important role in plant growth activ-
ity.1–4 Their best applications are in their ability to pro-
mote cell division and root formation.4,5 The most
widely occurring natural auxin, indole-3-acetic acid
(IAA), is found in both free and conjugated state in dif-
ferent plants and in their seeds. The labile nature in
aqueous solution of this auxin prevents its widespread
use. This has resulted in the search for alternative auxin
analogues.4,6,7
patented formulation of diindolylmethane is also under
study for cervical dysplasia, breast pain, and human
papilloma virus (HPV) related conditions.9,12
Room temperature ionic liquids (RTILs) are recognized
as green recyclable alternatives to the traditional volatile
organic solvents because of their unique chemical and
physical properties.13 As a result of their green creden-
tials and potential to enhance rates and selectivities, io-
nic liquids find increasing applications in organic
synthesis.14 Recent reports have shown that they can
also promote and catalyze many transformations of
commercial importance under mild conditions without
the need for any additional acid catalyst.15,16 Earlier,
we have reported17 the synthesis of DIMs using InCl3-
HMTA. We now report herein the use of ionic liquid
[Bmim]BF4 for the synthesis of plant growth promoter
diindolylmethane (DIM) and its derivatives in mild con-
dition without the requirement for additional catalyst
and solvent. Their plant growth promoting activity
was tested on Oryza sativa over a wide concentration
range. The results revealed that DIMs significantly in-
crease germination and plant growth in comparison
with IAA.
3,30-Diindolylmethane (DIM) and its derivatives are
used as dietary supplements,8,9 which promote healthy
estrogen metabolism in humans by converting both es-
trone and estradiol to their respective 2-hydroxy deriva-
tives (non-carcinogenic metabolites). There are
numerous reports, which show that DIM is an effective
inhibitor of human prostate cancer cells.10 Recent
study11 shows that DIM and 5-methoxy-diindolylme-
thane possess potential radical scavenging activities
associated with cancer cells. Besides, a highly absorbable
Keywords: Ionic liquid; Diindolylmethane; Plant growth promoter;
Auxin; Oryza sativa.
*
Corresponding author. Tel.: +91 33 24733491/0492; fax: +91 33
24735197; e-mail: jaisankar@iicb.res.in
In the course of developing simple and efficient synthetic
methodology for the preparation of bioactive DIM
derivatives, it was observed that treatment of 12 equiv
Summer trainee from Bengal College of Engineering and Techno-
logy, Durgapur 713212, India.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.025

C. Pal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4924–4928
4925
of 2-phenyl-indole (1h) with 1 equiv of hexamethylene-
tetramine (HMTA) (2) in ionic liquid [Bmim]BF4 at
60 ꢁC for 7 h. resulted18 in 2,20-Diphenyl-3,30-dii-
ndolylmethane (3h) in 82% yield (Scheme 1). This effi-
cient method was applied to prepare a number of
DIMs 3a–k in very good yield (Table 1). This is the first
report of the synthesis of DIMs using HMTA as meth-
ylene donor in presence of ionic liquid [Bmim]BF4. The
reaction condition requires a catalytic amount of ionic
liquid (20 mol%) in i-PrOH at 28 ꢁC for 7–27 h. leading
to the corresponding DIMs in very good yield (Table 1).
The ionic liquid [Bmim]BF4 could be recycled three
times without affecting the yield of DIMs.
The formation of DIMs from HMTA (2) and indoles
1a–k could be explained on the basis of the earlier pro-
posed mechanism17 except for the fact that in this case
ionic liquid [Bmim]BF4 facilitates the formation of the
ammonium complex with indole and HMTA. All the
compounds 3a–k have been characterized from their
spectral data mainly NMR and mass spectra and further
compared with the authentic samples available17 with
us.
The study of growth promoting activity19 of DIM and
its derivatives 3a–k as shown in Table 2 on O. sativa
indicates that they have good root and shoot growth
3
R
3
3
R
R
a or b
(CH ) N
2 6
+
4
2
N
R
2
2
R
N
R
R
R
N
R
1
1
1
1a-k
2
3a-k
Scheme 1. Reagents and conditions: (a) i—[Bmim]BF4, 60 ꢁC, 5–20 h; ii—H2O, 80 ꢁC, 30 min.; (b) i—[Bmim]BF4 (20 mol%), i-PrOH, 28 ꢁC, 7–27 h;
ii—H2O, 80 ꢁC, 30 min.
Table 1. Formation of DIM derivatives 3a–k under Methods A and B
Entry
R1
R2
R3
Time (h)
Isolated yield (%)
Method A Method B
Method A
Method B
a
b
c
d
e
f
H
H
H
5
7
7
8
78
71
67
80
72
65
61
82
73
66
59
73
68
61
72
67
60
58
77
67
60
54
Me
H
H
H
Me
H
H
8
10
12
10
17
27
10
13
15
25
H
OMe
OMe
Br
9
Me
H
H
8
H
14
16
7
g
h
i
Me
H
H
Br
H
Ph
Ph
Me
H
Me
Me
Me
H
9
j
H
NO2
10
20
k
Method A: (i) [Bmim]BF4, 60 ꢁC, 5–20 h; (ii) H2O, 80 ꢁC, 30 min.
Method B: (i) [Bmim]BF4 (20 mol%), i-PrOH, 28 ꢁC, 7–27 h; (ii) H2O, 80 ꢁC, 30 min.
Table 2. Effect of DIM derivatives in the shoot and root growth of Oryza sativa
Compound
31.25 ppm
RL
62.5 ppm
RL
125 ppm
250 ppm
RL
500 ppm
1000 ppm
SL
8.72
SL
9.81
SL
RL
SL
SL
RL
SL
RL
IAA
3a
3b
3c
3d
3e
3f
15.61
24.56
20.42
40.20
28.35
25.70
18.57
16.82
33.62
29.56
34.17
16.24
18.73
26.33
23.47
42.78
30.57
28.25
21.05
19.57
35.87
31.24
36.42
18.92
10.6
20.26
29.50
25.41
46.50
33.78
30.45
23.24
22.64
38.74
34.46
39.37
21.40
9.25
17.62
25.87
22.52
40.66
29.42
26.72
19.72
18.56
34.20
30.27
35.42
18.16
7.66
10.95
8.37
14.45
22.37
19.66
38.52
26.5
5.28
11.72
19.85
16.72
35.12
23.62
20.78
13.80
12.90
29.76
25.60
30.72
12.12
11.24
9.20
13.41
10.70
19.60
15.26
11.53
10.97
10.12
17.43
15.44
16.37
9.96
15.67
12.23
21.05
17.62
13.56
12.73
11.41
19.47
17.23
18.74
10.96
13.01
10.30
18.57
15.23
11.42
10.70
9.72
8.45
6.1
16.43
13.40
9.78
16.32
13.40
9.66
13.34
11.62
7.80
23.26
16.56
15.92
32.62
28.24
33.36
15.21
9.50
9.05
8.53
8.12
6.70
5.92
3g
3h
3i
15.23
13.80
14.67
8.87
17.32
15.01
16.80
9.56
15.5
12.90
10.80
12.02
5.76
13.62
14.72
7.82
3j
3k
SL, Shoot length in mm; RL, Root Length in mm.

4926
C. Pal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4924–4928
promoting activities. It could be seen from Figure 1 that
all the eleven derivatives of DIM, 3a–k, at concentration
of 125 ppm showed better growth promoting activities
of both the roots and shoots of O. sativa compared to
that of IAA. But, maximum growth was observed in
presence of compound 3c at the same concentration.
Since compound 3c showed maximum growth at con-
centration of 125 ppm, it prompted us to study the effect
of different concentration of compound 3c on the
growth of root and shoot against IAA. This studies
(Fig. 2 and 3) clearly showed that a concentration of
ꢀ125 ppm of 3c is the desired one as any concentration
Comparison of Growth Rate
Shoot length
Root length
50
*
45
*
*
*
40
*
*
*
*
*
35
*
*
*
*
*
30
25
20
15
10
5
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0
IAA
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
Concentration at 125 ppm
Figure 1. Data were analyzed by ANOVA and Dunnett’s test. *Denotes significant promotion as compared to control (p < 0.05).
Comparative Studies of IAA and 3c on Shoot Growth
25
20
15
10
5
IAA
3c
*
*
*
*
*
*
0
31.25
62.5
125
250
500
1000
Concentration in ppm
Figure 2. Data were analyzed by ANOVA and Dunnett’s test. *Denotes significant promotion as compared to control (p < 0.05).
Comparative Studies of IAA and 3c on Root length
IAA
3c
60
50
40
30
20
10
0
*
*
*
*
*
*
31.25
62.5
125
Concentration in ppm
Figure 3. Data were analyzed by ANOVA and Dunnett’s test. *Denotes significant promotion as compared to control (p < 0.05).
250
500
1000

C. Pal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4924–4928
4927
12. Carter, T. H.; Liu, K.; Ralph, W., Jr.; Chen, D.; Qi, M.;
Fan, S.; Yuan, F.; Rosen, E. M.; Auborn, K. J. J. Nutr.
2002, 132, 3314.
above or below showed slow growth which is more alike
natural auxin IAA.
13. (a) Welton, T. Chem. Rev. 1999, 99, 2071; (b) Wassersc-
heid, P.; Keim, W. Angew. Chem. Int. Ed. 2000, 39, 3772.
14. (a) Jain, N.; Kumar, A.; Chauhan, S.; Chauhan, S. M. S.
Tetrahedron 2005, 61, 1015; (b) Gordon, C. M. Appl.
Catal., A. Gen. 2001, 222, 101.
15. (a) Meshram, H. M.; Reddy, P. N.; Vishnu, P.; Sadshiv,
K.; Yadav, J. S. Tetrahedron Lett. 2006, 47, 991; (b)
Yadav, J. S.; Reddy, B. V. S.; Baishya, G. J. Org. Chem.
2003, 68, 7098, and references cited therein.
16. (a) Jarikote, D. V.; Siddiqui, S. A.; Rajagopal, R.; Daniel,
T.; Lahoti, R. J.; Srinivasan, K. V. Tetrahedron Lett. 2003,
44, 1835; (b) Palimkar, S. S.; Siddiqui, S. A.; Daniel, T.;
Lahoti, R. J.; Srinivasan, K. V. J. Org. Chem. 2003, 68,
9371.
In summary, the present methodology developed by us
offers an efficient one-pot synthesis of biologically
important DIM and its derivatives in very good yield
under eco-friendly recyclable ionic liquid [Bmim]BF4
medium. The study of plant growth promoting activities
of DIMs on O. sativa revealed that among the ones stud-
ied compound 3c is the best choice for growth promot-
ing activity than all the other ones including natural
auxin IAA.
Acknowledgments
17. Pradhan, P. K.; Dey, S.; Giri, V. S.; Jaisankar, P.
Synthesis 2005, 1779.
Authors C.P., and S.D. thankfully acknowledge CSIR,
New Delhi, India, and UGC, New Delhi, India, respec-
tively, for the financial support in the form of Research
Fellowships.
18. General procedure for synthesis of 2,20-diphenyl-3,30-dii-
ndolylmethane (3h): Method A: A solution of 2-phenyl
indole (1h) (193 mg, 1 mmol) and hexamethylenetetramine
(2) (11.76 mg, 0.084 mmol) in [Bmim]BF4 (5 mL) was
stirred at 60 ꢁC for 7 h. After completion of the reaction,
water (7 mL) was added to the reaction mixture followed
by heating at 80 ꢁC for 30 min. The reaction mixture was
then extracted with diethyl ether (3· 25 mL). The organic
layer was dried over anhydrous Na2SO4, and the solvent
removed to give a solid mass. Column chromatography of
the residue over silica gel using increasing concentration of
chloroform in petroleum ether yielded 3h as white solid
(164 mg, 82%).
References and notes
1. Went, F. W. Proc. Kon. Ned. Akad 1934, 37, 445.
2. Thimann, K. V.; Went, F. W. Proc. Kon. Med. Akad. Wet.
1934, 37, 456.
3. Thimann, K. V.; Koepfli, J. B. Nature 1935, 135, 101.
4. Haissig, B. E.; Davis, T. D. A Historical Evaluation of
Adventitious Rooting Research to 1993. In Biology of
Adventitious Root Formation; Davis, T. D., Haissig, B. E.,
Eds.; Plenum Press: New York, NY, 1994.
5. Hartmann, H. T.; Kestar, D. E.; Davies, F. T.; Geneve, R.
L., Jr. In Plant Propagation: Principles and Practices;
Prentice Hall: Upper Saddle River, NJ, 1997.
6. Macdonald, B. Practical Woody Plant Propagation for
Nursery Growers; Timber Press: Portland, OR, 1986,
Chapter 10.
Recycling of ionic liquid [Bmim]BF4:The ionic liquid
[Bmim]BF4 was recovered by extracting the aqueous
phase of the above reaction with dichloromethane
(3 · 25 mL). The organic layer was separated, dried over
anhydrous Na2SO4, and the solvent removed to give the
ionic liquid. The ionic liquid thus obtained was further
dried at 80 ꢁC under reduced pressure for subsequent runs.
Method B: Ionic liquid (45.2 mg, 0.2 mmol, 20 mol%) was
added to a solution of 2-phenyl indole (1 h) (193 mg,
1 mmol) and hexamethylenetetramine (2) (11.76 mg,
0.084 mmol) in dry i-PrOH (7 mL). The reaction mixture
was then stirred at room temperature for 10 h. Water
(7 mL) was added to the reaction mixture followed by
heating at 80 ꢁC for 30 min. It was then extracted with
CHCl3 (3· 25 mL). The organic layer was dried over
anhydrous Na2SO4, and the solvent removed to give a
solid mass. Column chromatography of the residue over
silica gel using increasing concentration of chloroform in
petroleum ether yielded 3h as white solid (154 mg, 77%).
19. Comparative studies of plant growth activities with IAA
and DIMs on Rice seeds (Oryza sativa):Fresh solutions of
both IAA and DIMs in the concentration of 1000 ppm
were prepared in acetone. From this stock solution further
dilutions were made in the following manner:500
ppm—10 mL stock solution + 10 mL acetone; 250 ppm
—10 mL of 500 ppm + 10 mL acetone; 125 ppm—10 mL
of 250 ppm + 10 mL acetone; 62.5 ppm—10 mL of
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125 ppm + 10 mL
62.5 ppm + 10 mL
acetone;
acetone,
31.25 ppm—10 mL
15.62 ppm—10 mL
of
of
31.25 ppm + 10 mL acetone.10 mL solutions of each con-
centration of both IAA and DIM were soaked in Whatman I
filter papers, which were placed in the lower lid of a 15 mL
Petri dish (Borosil glass). Petri dishes were wrapped with
black electrician’s tape. Both distilled water and acetone
were soaked separately with same type of filter papers as
control. After drying of filter papers, 15 mL of distilled water
added to all Petri dishes including the control one. Seeds
11. Benabadji, S. H.; Wen, R.; Zheng, J.; Dong, X.; Yuan, S.
Acta Pharmacol. Sin. 2004, 25, 666.

4928
C. Pal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4924–4928
were surface sterilized by soaking with 0.1% HgCl2 solution
temperature varied from ꢀ15 ꢁC (night) to ꢀ30 ꢁC (day).
Level of water was maintained at the 15 mL mark of each
Petri dish before start of the days experiment to compensate
the water loss. Readings of the shoot and root growths were
measured in millimeter scale on day 5.
followed by washing with distilled water. Seeds (42) of the
same type were placed on each of the above Petri dishes in
horizontal manner; otherwise their growth could be affected.
The Petri dishes were placed in greenhouse in which the