Synthesis and anticancer activity of bile acid dendrimers with triazole as bridging unit through click chemistry

Article abstract of DOI:10.1016/j.steroids.2017.06.007

Triazole-based novel dendrimers with bile acid surface groups have been synthesized through click chemistry by divergent approach and characterized by spectral data. All the dendrimers exhibit excellent anticancer activity. Higher-generation dendrimers exhibit better anticancer activity than the lower-generation dendrimers.

Full text of DOI:10.1016/j.steroids.2017.06.007

Steroidsxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and anticancer activity of bile acid dendrimers with triazole as
bridging unit through click chemistry
Devaraj Anandkumar, Perumal Rajakumar⁎
Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai 600025, TamilNadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Dendrimers
Click chemistry
Bile acid
Triazole-based novel dendrimers with bile acid surface groups have been synthesized through click chemistry by
divergent approach and characterized by spectral data. All the dendrimers exhibit excellent anticancer activity.
Higher-generation dendrimers exhibit better anticancer activity than the lower-generation dendrimers.
Triazole
1. Introduction
membrane transfection [14].
Bile acids can self-assemble because of their amphiphilic properties,
Dendrimers are well-defined, hyperbranched macromolecules with
precisely defined structures and multiple controllable functionalities
[1]. Dendrimers offer a variety of applications in the fields of material
science and biological science such as drug delivery systems [2], sen-
sors [3], solar cells [4] and efficient light harvesting antenna [5], mo-
lecular encapsulation [6] and medical applications [7]. The Cu (I)-
catalyzed click reaction involving 1, 3-dipolar cycloaddition between
an azide and a terminal alkyne has been used for the synthesis of
multifunctional and star-like dendrimers. Click reaction is highly effi-
cient, regioselectivity, results in high yields [8] under mild reaction
conditions, without any protection and deprotection protocol, involving
inexpensive, economical, environmentally as well as eco-friendly re-
agents.
which make them potential building blocks for the design of polymeric
materials for biomedical applications [15] and have gained consider-
able attention in supramolecular chemistry in recent years. More re-
cently, bile acids have became much more attractive in the construction
of star-shaped derivatives called “molecular pockets” and they have
been used as a drug delivery vehicles [16], nonpolymeric hydrogelators
[17], chemo sensors for metal ions [18] and molecular containers [19].
Hence, based on such extensive applications of bile acid based den-
drimers, it is worth to synthesize the following dendrimers with bile
acid surface group using click chemistry (Fig. 1).
2. Result and discussion
Steroids are large class of natural compounds, many of which show
very important roles in plants and animals and are also the main sex
hormones in mammals which play important role in regulating meta-
bolism [9]. Bile acid science has a history of more than a century with
continuing importance in biology and medicine. In recent years, this
class of compounds has gained considerable attention in supramole-
cular chemistry [10]. Bile acids are biological compounds with inter-
esting properties due to their large, rigid, and curved steroidal skele-
tons, chemically different hydroxy groups, enantiomeric purities, and
their unique amphiphilicity. Bile acids and their derivatives are at-
tractive for synthetic chemists because these facially amphiphilic mo-
lecules are biological surfactants with multiple physiological functions.
Modified bile acids have pharmaceutical importance as novel drug
delivery systems and also have been used for regulating of cholesterol
level [11], dissolution of gallstones [12], cancer treatment [13], and
Terminal alkynes were prepared by esterification of bile acids 13
and 14 using an excess of propargyl alcohol and a catalytic amount of
para-toluenesulfonic acid to give propargyl esters 15 and 16 in 96% and
95% yields, respectively. In the 1H NMR spectrum the compounds 15
and 16 displayed the ester acetylenic proton as a triplet at δ 2.48 ppm
and –OCH2 as doublet at δ 4.68 ppm in addition to the signal for the
other aliphatic protons. The 13C NMR spectrum of compound 15 and 16
showed signals at δ 173.4 for the carbonyl carbon in addition to the
other carbon signals. The propargyl esters 15 and 16 were acetylated
using Ac2O and TMSOTf in DCM to give the acetylated terminal alkyne
compounds 17 and 18 in 92% and 89% yields, respectively (Scheme 1).
In the 1H NMR spectrum, the compound 17 displayed two singlets at
δ 2.04 and 2.11 for the two CH3COO-protons and the ester acetylenic
proton appeared as a triplet at δ 2.48 ppm and -OCH2 protons appeared
as a doublet at δ 4.68 ppm in addition to the signals for the other
⁎
Corresponding author.
E-mail address: perumalrajakumar@gmail.com (P. Rajakumar).
http://dx.doi.org/10.1016/j.steroids.2017.06.007
Received 21 April 2017; Received in revised form 15 June 2017; Accepted 17 June 2017
0039-128X/©2017ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:Anandkumar,D.,Steroids(2017),http://dx.doi.org/10.1016/j.steroids.2017.06.007

D. Anandkumar, P. Rajakumar
Steroidsxxx(xxxx)xxx–xxx
AcO
AcO
(a)
(b)
OAc
R2
AcO
R2
AcO
R2
OAc
O
O
O
N
O
O
O
N
N
N
N
N
R1
N
N
N
N
N
O
N
O
N
O
N
N
R1
O
N
N
O
N
R1
O
N
N
N
R1
O
O
N
N
N
N
AcO
R2
N
N
OAc
R2
R1
O
O
OA
c
R1
AcO
OAc
R2
N
N
N
OAc
R2
OAc
O
1 R1, R2 = H
AcO
2 R1 = CH3, R2 = H
3 R1 = H, R2 = OAc
4 R1 = CH3, R2 = OAc
N
N
N
N
N
N
O
O
O
O
OAc
OAc
R2
R
OAc
OAc
5 R1, R2 = H
6 R1 = CH3, R2 = H
7 R1 = H, R2 = OAc
8 R1 = CH3, R2 = OAc
AcO
OAc
(c)
R2
AcO
R2
OAc
O
O
O
OAc
O
R2
AcO
R2
OAc
AcO
N
N
N
N
O
O
N N
O
O
N
N
N
N
N
N
O
O
N
N
N
N
N
N
OAc
AcO
R2
O
N
R2
OAc
N
N
AcO
R1
R1
O
O
O
O
N
N
N
N
N
N
R1
O
N
N
N
N
N
N
N
O
N
O
N
N
N
N
N
N
O
N
N
N
N
N
O
N
N
O
N
N
N
O
O
O
OAc
R2
AcO
O
R2
N
N
OAc
N
N
O
AcO
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
OAc
R2
OAc
R2
OAc
AcO
AcO
R2
OAc
AcO
R2
OAc
9 R1, R2
10 R1 = CH3, R2
11 R1 = H, R2 = OAc
12 R1 = CH3, R2 OAc
= H
=
H
=
Fig. 1. The structure of bile acid dendrimers 1–12.
aliphatic protons. The 13C NMR spectrum of the compound 17 showed
signals at δ 173.2 for the propargyloxy ester carbonyl carbon and the
carbonyl carbons of the two CH3COO-group appeared at δ 170.4 and δ
170.6 respectively in addition to the signals for the other aliphatic
carbons. The ESI mass spectrum of the compound 17 showed the mo-
lecular ion peak at m/z 515 (M+1). Further the structure of the
O
O
O
O
OH
O
i
ii
OAc
OH
OH
R
R
R
OAc
OH
OH
17 R= H
15 R= H
16 R= OH
13 R= H
18 R= OAc
14 R= OH
Scheme 1. Reagents and conditions: (i) PTSA (10 mol %), propargyl alcohol (5–10 mL), 55–60 °C, 7 h, 15 (96%) and 16 (95%). (ii) Ac2O/TMSOTf/CH2Cl2, 0 °C, 10 min, 17 (92%) and 18
(89%).
2

D. Anandkumar, P. Rajakumar
Steroidsxxx(xxxx)xxx–xxx
Y
Y
O
N
N
N
N3
X
N
X
Y
O
X
ii
X
N
O
N
N
i
+
N3
Y
X
ii
N
N
X
N3
Cl
Cl
20 X = H
21 X = CH3
19
(3.1 equiv.)
O
22 X = H, Y = Cl
23 X = CH3, Y = Cl
Y
24 X = H, Y = N3
25 X = CH3, Y = N3
i
Y
Y
Y
19
(6.1 equiv.)
Y
O
N
N
N
Y
N
N
O
N
Y
O
Y
O
N
O
N
N
N
X
N
N
X
N
N
O
N
N
26 X = H, Y = Cl
N
ii
N
N
27 X = CH3, Y = Cl
X
N
N
ii
28 X = H, Y = N3
29 X = CH3, Y = N3
O
Y
N
N
Y
Y
O
N
N N
N
Y
O
Y
Y
Scheme 2. Reagents and conditions: (i) CuSO4·5H2O (5 mol %), sodium ascorbate (10 mol %), H2O–THF (1:3); rt, 12 h. 22 (86%), 23 (90%), 26 (88%) and 27 (88%) (ii) 9/15 equiv.
NaN3, DMF, 90 °C, 8 h. 24 (92%), 25 (93%), 28 (85%) and 29 (80%).
compound 17 was also confirmed from elemental analysis. Similarly,
the structure of the triacetyl terminal alkyne 18 was confirmed from
spectral and analytical data.
different –N–CH2 protons and –O–CH2 protons and a sharp singlet at δ
8.26 ppm for the triazolyl proton in addition to the signals for the other
aliphatic and aromatic protons. The 13C NMR spectrum of compound
24 displayed the –Ph–CH2 carbon at δ 52.4 ppm and signals at δ 53.3,
61.2 ppm for –N–CH2 and –O–CH2 carbons, respectively, and the tria-
zolyl carbon appeared at δ 158.3 ppm in addition to the signals for the
other aliphatic and aromatic carbons. The ESI mass spectrum of the
compound 24 showed the molecular ion peak at m/z 970 (M+1).
Further, the structure of azidodendrimer 24 was also confirmed from
analytical data. Similarly, the structure of the azidodendrimer 25 was
also confirmed from spectral and analytical data.
The divergent synthesis begins with the reaction of 1,3,5-tris(azi-
domethyl)benzene 20 [20] and 1,3,5-tris(azidomethyl)-2,4,6-tri-
methylbenzene 21 [21] with 3.1 equivalents of 1,3-bis(chloromethyl)-
5-(propargyloxy)-benzene 19 [22] under Cu(I) catalyzed click reaction
conditions to give the first generation chlorodendrimers 22 and 23 in
86% and 90% yields, respectively, which on further treatment with
NaN3 in DMF at 90 °C afforded the corresponding azidodendrimers 24
and 25 in 92% and 93% yields, respectively (Scheme 2). The 1H-NMR
spectrum of the compound 24 displayed a singlet at δ 4.40 ppm for the
–Ph–CH2 proton and two different singlets at δ 5.15, 5.58 ppm for two
Reaction of 6.1 equiv. of 1,3-bis(chloromethyl)-5-(propargyloxy)-
benzene 19 with 1.0 equiv. of each of the azidodendrimers 24 and 25
3

D. Anandkumar, P. Rajakumar
Steroidsxxx(xxxx)xxx–xxx
under Cu(I) catalyzed click reaction conditions gave the second gen-
eration chlorodendrimers 26 and 27 in 80% and 88% yields, respec-
tively, which on further treatment with NaN3 in DMF at 90 °C afforded
the corresponding azidodendrimers 28 and 29 in 85% and 80% yields,
respectively (Scheme 2). The 1H-NMR spectrum of the compound 29
displayed a singlet at δ 4.40 ppm for the –Ph–CH2 proton and four
different singlets at δ 5.06, 5.15, 5.53 and 5.68 ppm for the two dif-
ferent –N–CH2 protons and –O–CH2 protons and a sharp singlet at δ
8.09 and 8.26 ppm for the two different triazolyl protons in addition to
the signals for the other aliphatic and aromatic protons. The 13C NMR
spectrum of compound 29 displayed the –Ph–CH2 carbon at δ 53.3 ppm
and signals at δ 52.6, 61.2 ppm for –N–CH2 and –O–CH2 carbons, re-
spectively, and the triazolyl carbon appeared at δ 158.3 and 158.4 ppm
in addition to the signals for the other aliphatic and aromatic carbons
(Scheme 2).
The zeroth generation bile acid dendrimers (G0) 1, 2, 3 and 4 could
be achieved by click chemistry approach using a suitable triazide as
core unit. Reaction of 1.0 equiv. of 1,3,5-tris(azidomethyl)benzene 20
and 1,3,5-tris(azidomethyl)-2,4,6-trimethylbenzene 21 with 3.0 equiv.
of each of the acetylated alkyne dendrons 17 and 18 in the presence of
CuSO4·5H2O (5 mol %), with sodium ascorbate (10 mol %) in a mixture
of water and THF (1:3) at room temperature for 10 h afforded the
zeroth generation dendrimers 1, 2, 3 and 4 in 85%, 82%, 81% and 71%
yields, respectively (Scheme 3). The 1H NMR spectrum of compound 1
displayed singlets at δ 0.70, 5.20 and 5.47 ppm for C18-Me, O-CH2- and
N-CH2-protons and at δ 7.57 ppm for the triazolyl proton in addition to
the signals for the other aliphatic and aromatic protons. The 13C NMR
spectrum of compound 1 showed the carbonyl carbons at δ 170.5, 170.6
and δ 174.0, in addition to the signals for the other aliphatic and aro-
matic carbons. The mass spectrum (MALDI-TOF) of the dendrimer 1
showed the molecular ion peak at m/z 1810 [M+Na]+. Further the
structure of the dendrimer 1 was also confirmed from elemental ana-
lysis. Similarly, the structure of the dendrimer 2, 3 and 4 was confirmed
from spectral and analytical data.
addition to the signals for the other aliphatic and aromatic protons. The
13C NMR spectrum of compound 6 showed the carbonyl carbons at δ
170.4, 170.5 and δ 173.9, in addition to the signals for the other ali-
phatic and aromatic carbons. The mass spectrum (MALDI-TOF) of the
dendrimer 6 showed the molecular ion peak at m/z 4081 [M+Na]+
.
Further, the structure of the dendrimer 6 was also confirmed from
elemental analysis. Similarly, the structure of the dendrimer 5, 7 and 8
was confirmed from spectral and analytical data.
The synthetic pathway leading to the bile acid dendrimers 9, 10, 11
and 12 is shown in Scheme 3. Reaction of 1.0 equiv. of azidodendrimer
28 and 29 with 12.1 equiv. of each of the acetylated alkyne dendrons
17 and 18 in the presence of Cu (I) catalyzed click reaction conditions
afforded the second generation dendrimers 9, 10, 11 and 12 in 56%,
57%, 57% and 60% yields, respectively (Scheme 3). The 1H NMR
spectrum of the bile acid dendrimer 12 showed singlet at δ 0.70 ppm for
18-Me and three sharp singlets at δ 2.04, 2.08 and 2.12 for the three
CH3COO-protons and four sharp singlets at δ 5.06, 5.15, 5.42 and
5.65 ppm for different N–CH2 and O–CH2 protons and δ 7.51, 7.62 and
7.67 ppm for the three different triazolyl protons, respectively in ad-
dition to the signals for the other aliphatic and aromatic protons. The
13C NMR spectrum of compound 12 showed the carbonyl carbons at δ
170.3, 170.4, 170.5 and δ 173.8, in addition to the signals for the other
aliphatic and aromatic carbons. The mass spectrum (MALDI-TOF) of the
dendrimer 12 showed the molecular ion peak at m/z 9361 [M+Na]+
.
Further the structure of the dendrimer 12 was also confirmed from
elemental analysis. Similarly, the structure of the dendrimer 9, 10 and
11 was confirmed from spectral and analytical data.
3. Anti-cancer activity of the bile acid dendrimers
The cytotoxicity of bile acid dendrimers 1–12 was evaluated based
on their effect on cell growth with C6 glioma cell lines by MTT assay
method [23]. The cytotoxicity of bile acid dendrimers 1–12 was studied
and shows that proliferation of cancer cells was inhibited significantly
with increase in the concentration and generation of dendrimers. The
results are shown in Tables 1 and 2 and Figs. 2 and 3. In-vitro anti-
proliferation activity of dendrimers 1, 3, 5, 7, 9 and 11 were carried
against C6 glioma cell lines and the result reveals that the second
generation dendrimer 11 with IC50 value of 12.13 µM concentration
shows the high efficacy towards the anti-proliferation of the cancer cells
(Table 1 and Fig.2). In general, the efficacy increases with increase in
the generation of the dendrimers. It was also observed in another set of
dendrimers the efficacy against C6 glioma cell cancer cytotoxicity in-
creases by increasing the generation of the dendrimers in the order 2, 4,
6, 8, 10 and 12. The second generation dendrimer 12 has the highest
anti cancer efficacy with IC50 value of 10.48 µM (Table 2 and Fig.3)
The first generation bile acid dendrimers (G1) 5, 6, 7 and 8 could be
achieved by click chemistry approach. Reaction of 1.0 equiv. of the
azidodendrimer 24 and 25 with 6.1 equiv. of each of the acetylated
alkyne dendrons 17 and 18 in the presence of CuSO4·5H2O (5 mol %)
and sodium ascorbate (10 mol %) in a mixture of water and THF (1:3)
at room temperature for 10 h afforded the first generation dendrimers
5, 6, 7 and 8 in 74%, 73%, 71% and 68% yields, respectively (Scheme
3). The 1H NMR spectrum of the bile acid dendrimer 6 showed singlet at
δ 0.70 ppm for 18-Me and two sharp singlets at δ 2.03 and 2.09 for the
two CH3COO-protons and four sharp singlets at δ 5.06, 5.14, 5.45 and
5.67 ppm for N–CH2 and O–CH2 protons and singlets at δ 7.46 and
7.60 ppm for the two different triazolyl protons, respectively in
O
O
20 and 21
28 and 29
1, 2, 3
4
and
OAc
9, 10, 11
12
and
i
R
i
OAc
17 R= H
18 R= OAc
(3.1 equiv./6.1 equiv./12.1 equiv.)
i
24 and 25
5, 6, 7 and 8
Scheme 3. Reagents and conditions: (i) CuSO4·5H2O (5 mol %), sodium ascorbate (10 mol %), THF: H2O (3:1), rt, 10 h, 1 (85%), 2 (82%), 3 (81%), 4 (71%), 5 (74%), 6 (73%), 7 (71%), 8
(68%), 9 (56%), 10 (57%), 11 (57%) and 12 (60%).
4

D. Anandkumar, P. Rajakumar
Steroidsxxx(xxxx)xxx–xxx
Table 1
Anti-cancer activity of dendrimers 1, 2, 5, 6, 9 and 10 against C6 glioma cell lines concentration are in µM and represent the % of inhibition. Inhibition concentration 50 (IC50) calculated
and shown in µM.
Dendrimers
Activity (% of inhibition)
10 µM
IC50 (µM)
20 µM
30 µM
40 µM
50 µM
1
2
5
6
9
10
27.02
30
35.91
36.12
41.82
44.5
1.04
0.68
0.9
1.01
0.75
1.20
40.13
42.1
54.52
57.89
62.01
69.2
1.02
1.02
1.10
0.92
1.43
50.83
51.4
63.12
66.42
74.68
76.82
1.92
1.20
2.01
1.76
1.87
1.80
64.78
67.7
73.46
77.41
82.91
85.38
1.54
1.82
1.82
1.58
2.03
1.92
80.31
84.09
90.02
92.15
97.31
98.48
1.20
1.73
2.17
2.10
2.89
2.11
29.20
28.28
17.44
16.54
14.04
12.13
1.32
among the all synthesized dendrimers.
at room temperature for 18 h in a 3:1 mixture of THF/water. After
confirming the completion of reaction on TLC, chloroform was added to
the reaction mixture and washed with water (10 mL), and brine
(10 mL). The organic layer was separated, dried over Na2SO4 and
evaporated to obtain the crude product, which was purified by column
chromatography (SiO2), using CHCl3-MeOH as eluent to give the cor-
responding dendrimers.
The morphological study by light microscope images of second
generation dendrimers 9 and 11 has shown almost shrink and less po-
pulation of cells in C6 glioma cell line (Fig. 1 in SI). It is evident from
the images that 50 µM of dendrimers 9 and 11 treated for 24 h almost
inhibited cell population.
In conclusion the synthesis of bile acid dendrimers with triazole as
branching unit and benzene as core unit is described. Zero, first and
second generation bile acid dendrimers were synthesized successfully
using click chemistry in good yields through divergent strategy.
Further, the bile acid dendrimers showed cytotoxicity against C6
glioma cell lines by in vitro approach. Anti-cancer activity was carried
out by analyzing MTT assay. From the above study it has been con-
firmed that the second generation dendrimers have better anti-cancer
activity than the lower generation dendrimers.
4.3. General procedure for Synthesis of terminal acetylenes (B)
To a solution of bile acids (1 equiv., 1 mmol) in propargyl alcohol
(10 ml), catalytic amount (10 mol%) of para-toluene sulfonic acid
(PTSA) was added. The reaction mixture was then heated at 55–60 °C
for 7 h. It was then poured on to the crushed ice and extracted with
EtOAc (3 × 25 ml). The combined organic layer was washed with
water (3 × 25 ml), brine (25 ml), and dried over Na2SO4. Solvent was
evaporated under reduced pressure to afford the crude product. The
crude product was then purified by column chromatography (SiO2),
using the eluent as mentioned under each compound.
4. Experimental section
4.1. General information
All chemicals and solvents were purchased commercially and used
as such without further purification. All melting points were de-
termined using a Toshniwal melting point apparatus by open capillary
tube method and are uncorrected. Column chromatography was per-
formed on silica gel (ACME, 100–200 mesh). TLC was performed either
on glass plates coated with silica gel-G (ACME) of about 0.25 mm
thickness and visualized with iodine. 1H NMR and 13C NMR spectra
were recorded on Bruker 300 MHz instruments. Chemical shifts are
given in parts per million, and J values are in hertz. Elemental analyses
were carried out using a Perkin-Elmer CHNS 2400 instrument. Matrix-
assisted laser desorption ionization time-of-flight mass spectrometry
(MALDI-TOF MS) was performed with a 2,5-dihydroxy benzoic acid
(DHB) matrix.
4.4. General procedure for Synthesis of acetylated terminal acetylenes (C)
A solution of propargyl ester compound (1 equiv., 1 mmol) in
CH2Cl2 (30 mL) was treated with acetic anhydride (1.00 mL) at 0 °C,
followed by a solution of trimethylsilyl trifluoromethanesulfonate
(26 µL, 0.14 mmol) in CH2Cl2 (5 mL). The reaction upon completion
(TLC) is treated with saturated aq. NaHCO3 (5 mL), and the aqueous
phase was extracted with CH2Cl2 (2 × 50 mL). The combined organic
extract was washed with aq. NaHCO3 (50 mL), and water (50 mL), dried
(Na2SO4) and the solvent evaporated. The crude product was then
purified by column chromatography (SiO2), using the eluent as men-
tioned under each compound.
4.5. General procedure for the first and second generation of
azidodendrimers (D)
4.2. General procedure for Cu (I)-catalyzed azide-alkyne cycloaddition (A)
A mixture of polypropargylated derivatives (1.0 equiv) and azide
functionalized compound (1.2 equiv per azide functional unit),
CuSO4·5H2O (0.3 equiv), and sodium l-ascorbate (0.3 equiv) was stirred
The reaction flask was charged with chloro compound (1 equiv.,
1 mmol), DMSO (10 mL) and NaN3 (9.0 equiv./18.0 equiv. (1.5 equiv,
per chloride) and the reaction mixture was kept at 90 °C for 3 h. Then
Table 2
Anti-cancer activity of dendrimers 3, 4, 7, 8, 11 and 12 against C6 glioma cell lines concentration are in µM and represent the % of inhibition. Inhibition concentration 50 (IC50
)
calculated and shown in µM.
Dendrimers
Activity (% of inhibition)
10 µM
IC50 (µM)
20 µM
30 µM
40 µM
50 µM
3
4
7
8
11
12
30.47
0.92
0.58
1.01
1.01
1.26
1.10
55.1
0.96
0.85
1.25
1.25
1.92
62.56
63.56
70.5
74.52
83.67
76.59
1.07
1.09
1.92
1.92
2.11
1.87
67.9
68.9
84.53
86.43
89.9
90
1.43
1.23
2.13
1.87
2.32
1.92
82.91
84.3
92.73
94.1
99.31
98.4
1.87
1.57
2.17
2.2
2.89
2.30
17.77
25.18
14.49
14.86
10.68
10.48
31.92
38.12
39.72
48.03
49.01
57.34
62.42
63.26
74.62
76
1.45
5

D. Anandkumar, P. Rajakumar
Steroidsxxx(xxxx)xxx–xxx
Fig. 2. C6 glioma cell are treated for 24 hours in compound 1, 2, 5, 6, 9 and 10 were showing Inhibition under different concentration of 10 µM, 20 µM, 30 µM, 40 µM and 50 µM.
the reaction mixture was cooled to room temperature and then diluted
with water (100 mL) and extracted with CH2Cl2 (250 mL). The organic
layer was washed with saturated NaCl (aq.) (100 mL), dried over
Na2SO4 and evaporated to give the corresponding azido compound as a
solid. MeOH: CHCl3 (1:19) was used as eluent for TLC.
as colorless gel from the propargyl ester 15 (2 g, 4.69 mmol, 1.0 equiv.)
and acetic anhydride (1.1 ml, 11.74 mmol, 2.5 equiv.) after purification
by eluting from the column with 10%CHCl3/Hexane. Yield: 2.2 g, 92%;
1H NMR: (300 MHz, CDCl3): δH 0.73 (s, 3H, 18-Me); 0.81 (d, 3H,
J = 5.7 Hz); 0.91 (s, 3H); 2.04 (s, 3H); 2.10 (s, 3H); 2.47 (t, 1H,
J = 2.4 Hz); 4.71 (m, 3H); 5.08 (s, 1H). 13C NMR: (75 MHz, CDCl3): δC
12.4, 17.5, 21.36, 21.44, 23.1, 23.4, 25.6, 25.9, 26.6, 27.3, 28.9, 30.7,
30.9, 32.2, 34.1, 34.4, 34.6, 34.7, 35.7, 41.9, 45.1, 47.6, 49.5, 51.8,
74.2, 74.7, 75.9, 77.8, 170.4, 170.6, 173.2. MS (ESI): m/z = 515 [M
+1]. Anal. Calcd. For C31 H46 O6: C, 72.34; H, 9.01. Found: C, 72.45; H,
8.93.
4.6. Synthesis of terminal acetylenes 15
Following the general procedure B, the terminal acetylene 15 was
obtained as white solid from deoxycholic acid 13 (1 g, 2.5 mmol) and
propargyl alcohol (7 ml) and by eluting from the column with CHCl3:
MeOH (99:1). Yield: 1.045 g, 96%, M. P.: 159 °C; 1H NMR: (300 MHz,
CDCl3): δH 0.68 (s, 3H, 18-Me); 0.91 (s, 3H, 19-Me); 0.97 (d, 3H,
J = 6 Hz); 2.48 (t, 1H, J = 2.4 Hz); 3.62 (m, 1H); 3.98 (s, 1H); 4.68 (d,
2H, J = 2.4 Hz). 13C NMR: (75 MHz, CDCl3): δC 12.7, 17.3, 23.1, 23.7,
26.1, 27.1, 27.5, 28.7, 30.4, 30.7, 31.0, 33.6, 34.1, 35.1, 35.2, 36.0,
36.4, 42.1, 46.5, 47.3, 48.2, 51.8, 71.7, 73.1, 74.7, 77.8, 173.4. MS
(ESI): m/z = 431 [M+1]. Anal. Calcd. For C27 H42 O4: C, 75.31; H,
9.83. Found: C, 75.45; H, 9.93.
4.9. Synthesis of compound 18
Following the general procedure C, the compound 18 was obtained
as white solid from the propargyl ester 16 (2 g, 4.52 mmol, 1.0 equiv.)
and acetic anhydride (1.5 ml, 15.84 mmol, 3.5 equiv.) after purification
by eluting from the column with 10% CHCl3/Hexane. Yield: 2.3 g, 89%;
M. P.: 144 °C; 1H NMR: (300 MHz, CDCl3): δH 0.73 (s, 3H, 18-Me); 0.82
(d, 3H, J = 5.7 Hz); 0.92 (s, 3H); 1.07–2.40 (m, 24H); 2.05 (s, 3H); 2.09
(s, 3H); 2.14 (s, 3H); 2.48 (t, 1H, J = 2.4 Hz); 4.58 (m,1H); 4.67 (d, 2H,
J = 2.1 Hz); 4.91 (s, 1H); 5.09 (s, 1H). 13C NMR: (75 MHz, CDCl3): δC
12.2, 17.5, 21.4, 21.5, 21.6, 22.6, 22.8, 25.6, 26.9, 27.2, 28.9, 30.6,
30.8, 31.3, 34.3, 34.5, 34.6, 34.7, 37.8, 40.9, 43.4, 45.1, 47.3, 51.8,
70.7, 74.1, 74.8, 75.4, 77.7, 170.3, 170.47,170.50, 173.5. MS (ESI): m/
z = 572 [M+1]. Anal. Calcd. For C33 H48 O8: C, 69.20; H, 8.45. Found:
C, 69.35; H, 8.63.
4.7. Synthesis of terminal acetylenes 16
Following the general procedure B, the terminal acetylene 16 was
obtained as white solid from cholic acid 14 (1 g, 2.4 mmol) and pro-
pargyl alcohol (7 ml) and by eluting from the column with CHCl3:
MeOH (99:1). Yield: 1.04 g, 95%, M. P.: 115 °C; 1H NMR: (300 MHz,
CDCl3): δH 0.67 (s, 3H, 18-Me); 0.89 (s, 3H, 19-Me); 0.99 (d, 3H,
J = 5.7 Hz); 2.48 (t, 1H, J = 2.1 Hz); 3.07 (s, 3H); 3.43 (m, 1H); 3.84
(s, 1H); 3.96 (s, 1H); 4.68 (d, 2H, J = 2.4 Hz). 13C NMR: (75 MHz,
CDCl3): δC 12.5, 17.3, 22.5, 23.2, 26.4, 27.5, 28.2, 30.4, 30.8, 31.0,
34.7, 34.8, 35.2, 35.3, 39.5, 41.5, 41.7, 46.5, 47.0, 51.8, 68.5, 71.9,
73.1, 74.7, 77.9, 173.4. MS (ESI): m/z = 447 [M+1]. Anal. Calcd. For
C27 H42 O5: C, 72.61; H, 9.48. Found: C, 72.55; H, 9.33.
4.10. 1,3-bis(chloromethyl)-5-(prop-2-ynyloxy)benzene 19
1,3-bis(chloromethyl)-5-(prop-2-ynyloxy)benzene 19 was obtained
as white solid as per the reported procedure [22] from 1,3-bis(hydro-
xymethyl)-5-(prop-2-ynyloxy)benzene (5.6 g, 29.2 mmol, 1.0 equiv.)
and thionyl chloride (8.52 mL, 116.84 mmol, 4.0 equiv.). Yield: 6.0 g,
90%, M. P.: 89 °C; 1H NMR: (300 MHz, CDCl3): δH 2.54 (t, 1H,
J = 2.1 Hz); 4.55 (s, 4H); 4.71 (d, 2H, J = 2.4 Hz); 6.90 (s, 2H); 7.04
(s, 1H). 13C NMR: (75 MHz, CDCl3): δC 45.7, 56.0, 76.0, 78.1, 115.1,
4.8. Synthesis of compound 17
Following the general procedure C, the compound 17 was obtained
Fig. 3. C6 glioma cell are treated for 24 h in compound 3, 4, 7, 8, 11 and 12 were showing Inhibition under different concentration of 10 µM, 20 µM, 30 µM, 40 µM and 50 µM.
6

D. Anandkumar, P. Rajakumar
Steroidsxxx(xxxx)xxx–xxx
121.8, 139.4, 158.0. MS (ESI): m/z = 229 [M+1]. Anal. Calcd. For C11
H10 Cl2 O: C, 57.67; H, 4.40. Found: C, 57.45; H, 4.23.
d6): δH 2.42 (s, 9H); 4.42 (s, 12H); 5.13 (s, 6H); 5.71 (s, 6H); 6.96 (s,
3H); 7.02 (s, 6H); 8.12 (s, 3H). 13C NMR: (75 MHz, DMSO-d6): δC 16.3,
48.5, 53.3, 61.2, 114.3, 120.6, 124.4, 130.8, 137.6, 139.4, 142.3,
158.4. MS (ESI): m/z = 1012 [M+1]. Anal. Calcd. For C45 H45 N27 O3:
C, 53.41; H, 4.48; N, 37.37. Found: C, 53.25; H, 4.58; N, 37.57.
4.11. Synthesis of 1,3,5-tris(azidomethyl)benzene 20
The 1,3,5-tris(azidomethyl)benzene 20 was obtained as light yellow
oil from 1,3,5-tris(bromomethyl)benzene (2.0 g, 5.60 mmol) and NaN3
(0.97 g, 18.43 mmol) as reported in the literature [20]. Yield: 88%.
4.17. Second generation chloro dendrimer 26
From the reaction of 1,3-bis(chloromethyl)-5-propargyloxybenzene
19 (0.14 g, 0.64 mmol, 6.2 equiv.) with the azido dendrimer 24 (0.1 g,
0.10 mmol, 1.0 equiv.) using the general procedure A for click chem-
istry, the chloro dendrimer 26 was obtained as light brown amorphous
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.2 g, 80%; M. P.: 185 °C; 1H NMR: (300 MHz, DMSO-d6):
4.12. Synthesis of 1,3,5-tris(azidomethyl)-2,4,6-trimethylbenzene 21
The 1,3,5-tris(azidomethyl)-2,4,6-trimethylbenzene 21 was ob-
tained as white solid from 1,3,5-tris(bromomethyl)-2,4,6-tri-
methylbenzene (1.0 g, 1.0 mmol) and NaN3 (0.5 g, 3.50 mmol) as re-
ported in the literature [21]. Yield: 86%.
δ
H 4.68 (s, 24H); 5.04 (s, 6H); 5.13 (s, 12H); 5.54 (s, 12H); 5.57 (s, 6H);
6.93–7.01 (s, 27H); 7.25 (s, 3H); 8.23 (s, 3H); 8.28 (s, 6H). 13C NMR:
(75 MHz, DMSO-d6): δC 46.7, 52.6, 61.1, 61.2, 114.2, 115.0, 121.6,
130.7, 137.8, 139.4, 158.1, 158.4. MS (MALDI-TOF): m/z = 2467 [M
+Na]+. Anal. Calcd. For C108 H99 Cl12 N27 O9: C, 55.33; H, 4.26; N,
16.13. Found: C, 55.55; H, 4.38; N, 16.27.
4.13. First generation chloro dendrimer 22
From the reaction of 1,3-bis(chloromethyl)-5-propargyloxybenzene
19 (0.6 g, 2.63 mmol, 3.2 equiv.) with 1,3,5-tris(azidomethyl)benzene
20 (0.2 g, 0.82 mmol, 1.0 equiv.) using the general procedure A for
click chemistry, the chloro dendrimer 22 was obtained as light brown
amorphous solid after purification by eluting from the column with
CHCl3: MeOH (19:1). Yield: 0.65 g, 88%; M. P.: 201 °C; 1H NMR:
(300 MHz, DMSO-d6): δH 4.70 (s, 12H); 5.14 (s, 6H); 5.59 (s, 6H); 7.07
(s, 6H); 7.09 (s, 3H); 7.28 (s, 3H); 8.26 (s, 3H). 13C NMR: (75 MHz
DMSO-d6): δC 45.6, 53.2, 61.9, 114.9, 121.4, 123.3, 127.7, 136.7,
139.4, 142.9, 158.5. MS (ESI): m/z = 928 [M+1]. Anal. Calcd. For C42
H39 Cl6 N9 O3: C, 54.21; H, 4.22; N, 13.55. Found: C, 54.15; H, 4.33; N,
13.67.
4.18. Second generation chloro dendrimer 27
From the reaction of 1,3-bis(chloromethyl)-5-propargyloxybenzene
19 (0.21 g, 0.92 mmol, 6.2 equiv.) with the azido dendrimer 25 (0.15 g,
0.15 mmol, 1.0 equiv.) using the general procedure A for click chem-
istry, the chloro dendrimer 27 was obtained as light brown amorphous
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.3 g, 88%; M. P.: 201 °C; 1H NMR: (300 MHz, DMSO-d6):
δ
H 2.40 (s, 9H); 4.66 (s, 18H); 5.05 (s, 6H); 5.13 (s, 12H); 5.53 (s, 12H);
4.14. First generation chloro dendrimer 23
5.67 (s, 6H); 6.93 (s, 9H); 7.05 (s, 18H); 8.09 (s, 3H); 8.27 (s, 6H). 13C
NMR: (75 MHz, DMSO-d6): δC 16.3, 45.6, 52.6, 61.1, 61.2, 114.2,
115.0, 121.6, 130.7, 137.8, 139.4, 158.1, 158.4. MS (MALDI-TOF): m/
z = 2367 [M+Na]+. Anal. Calcd. For C108 H99 Cl12 N27 O9: C, 55.33;
H, 4.26; N, 16.13. Found: C, 55.55; H, 4.38; N, 16.27.
From the reaction of 1,3-bis(chloromethyl)-5-propargyloxybenzene
19 (0.50 g, 0.70 mmol, 3.1 equiv.) with 1,3,5-tris(azidomethyl)benzene
21 (0.2 g, 2.17 mmol, 3.1 equiv.) using the general procedure A for
click chemistry, the chloro dendrimer 23 was obtained as light brown
amorphous solid after purification by eluting from the column with
CHCl3: MeOH (19:1). Yield: 0.6 g, 90%; M. P.: 210 °C; 1H NMR:
(300 MHz, CDCl3 + DMSO): δH 2.41 (s, 9H); 4.53 (s, 12H); 5.16 (s, 6H);
5.67 (s, 6H); 6.96 (s, 6H); 7.01 (s, 3H); 7.54 (s, 3H). 13C NMR: (75 MHz,
CDCl3 + DMSO): δC 21.4, 50.4, 53.8, 66.7, 119.8, 126.2, 127.5, 135.3,
144.2, 144.6, 163.3. MS (ESI): m/z = 970 [M+1]. Anal. Calcd. For C45
H45 Cl6 N9 O3: C, 55.57; H, 4.66; N, 12.96. Found: C, 55.75; H, 4.48; N,
13.07.
4.19. Second generation azido dendrimer 28
Following the general procedure D, reaction of the second genera-
tion chloro dendrimer 26 (0.25 g, 0.10 mmol, 1.0 equiv.) with NaN3
(0.10 g, 1.53 mmol, 15.0 equiv.) gave the azido dendrimer 28 as a light
brown solid after purification by eluting from the column with CHCl3:
MeOH (19:1). Yield: 0.21 g, 85%; M. P.: 175 °C; 1H NMR: (300 MHz,
DMSO-d6): δH 4.41 (s, 24H); 5.10 (s, 6H); 5.15 (s, 12H); 5.55 (s, 12H);
5.58 (s, 6H); 6.9–7.02 (m, 27H); 7.29 (s, 3H); 8.25 (s, 3H); 8.29 (s, 6H).
13C NMR: (75 MHz, DMSO-d6): δC 52.3, 52.6, 53.3, 61.2, 114.2, 114.3,
120.0, 120.6, 124.8, 127.6, 137.1, 137.6, 137.9, 142.5, 142.8, 158.3.
MS (MALDI-TOF): m/z = 2446 [M+Na]+. Anal. Calcd. For C108 H99
N63 O9: C, 53.53; H, 4.12; N, 36.41. Found: C, 53.45; H, 4.28; N, 36.60.
4.15. First generation azido dendrimer 24
Following the general procedure D, reaction of the first generation
chloro dendrimer 22 (0.68 g, 0.73 mmol, 1.0 equiv.) with NaN3 (0.38 g,
5.87 mmol, 8.0 equiv.) gave the azido dendrimer 24 as light brown
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.65 g, 94%; M. P.: 180 °C; 1H NMR: (300 MHz, DMSO-
d6): δH 4.40 (s, 12H); 5.15 (s, 6H); 5.58 (s, 6H); 6.96 (s, 3H); 7.01 (s,
6H); 7.29 (s, 3H); 8.26 (s, 3H). 13C NMR: (75 MHz, DMSO-d6): δC 52.4,
53.3, 61.1, 114.3, 120.7, 124.8, 127.6, 137.0, 137.7, 158.3. MS (ESI):
m/z = 970 [M+1]. Anal. Calcd. For C42 H39 N27 O3: C, 52.01; H, 4.05;
N, 38.99. Found: C, 52.25; H, 4.18; N, 38.77.
4.20. Second generation azido dendrimer 29
Following the general procedure D, reaction of the second genera-
tion chloro dendrimer 27 (0.2 g, 0.083 mmol, 1.0 equiv.) with NaN3
(0.08 g, 1.25 mmol, 15.0 equiv.) gave the azido dendrimer 29 as light
brown solid after purification by eluting from the column with CHCl3:
MeOH (19:1). Yield: 0.17 g, 80%; M. P.: 160 °C; 1H NMR: (300 MHz,
DMSO-d6): δH 2.41 (s, 9H); 4.40 (s, 24H); 5.06 (s, 6H); 5.15 (s, 12H);
5.54 (s, 12H); 5.68 (s, 6H); 6.94 (s, 9H); 7.01 (s, 18H); 8.09 (s, 3H);
8.27 (s, 6H). 13C NMR: (75 MHz, DMSO-d6): δC 16.3, 48.4, 52.6, 53.3,
61.2, 114.3, 120.6, 124.8, 130.7, 137.6, 137.9, 139.3, 158.3, 158.4. MS
(MALDI-TOF): m/z = 2487 [M+Na]+. Anal. Calcd. For C111 H105 N63
O9: C, 54.08; H, 4.29; N, 35.79. Found: C, 54.25; H, 4.38; N, 35.67.
4.16. First generation azido dendrimer 25
Following the general procedure D, reaction of the first generation
chloro dendrimer 23 (0.67 g, 0.69 mmol, 1.0 equiv.) with NaN3 (0.36 g,
5.53 mmol, 8.0 equiv.) gave the azido dendrimer 25 as light brown
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.95 g, 93%; M. P.: 150 °C; 1H NMR: (300 MHz, DMSO-
7

D. Anandkumar, P. Rajakumar
Steroidsxxx(xxxx)xxx–xxx
4.21. Synthesis of zeroth generation dendrimers 1
(0.1 g, 0.10 mmol, 1.0 equiv.) and the alkyne dendron 17 (0.32 g,
0.64 mmol, 6.2 equiv.) using the click chemistry procedure A, as white
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.31 g, 74%; M. P.: 160 °C; 1H NMR: (300 MHz, CDCl3):
δH 0.70 (s, 18H, 18-Me); 0.78–2.28 (m, 228H); 4.64 (s, 6H); 5.00 (s,
6H); 5.04 (s, 6H); 5.10 (s, 12H); 5.38 (s, 12H); 5.41 (s, 6H); 6.73 (s,
3H); 6.77 (s, 6H); 7.11 (s, 3H); 7.55 (s, 9H). 13C NMR: (75 MHz, CDCl3):
Following the general procedure A, the dendrimer 1 was obtained as
white solid from the triazide 20 (0.05 g, 0.20 mmol, 1.0 equiv.) and the
alkyne dendron 17 (0.32 g, 0.64 mmol, 3.1 equiv.) after purification by
eluting from the column with CHCl3: MeOH (19:1). Yield:0.32 g, 85%;
M. P.: 100 °C; 1H NMR: (300 MHz, CDCl3): δH 0.70 (s, 9H, 18-Me); 0.77
(d, 9H, 19-Me, J = 6 Hz); 0.91 (s, 9H); 0.98–2.42 (m, 96H); 4.72 (m,
3H); 5.06 (s, 3H); 5.20 (s, 6H); 5.47 (s, 6H); 7.16 (s, 3H); 7.57 (s, 3H).
13C NMR: (75 MHz, CDCl3): δC 12.4, 17.5, 21.4, 21.5, 23.1, 23.4, 25.7,
25.9, 26.6, 26.9, 27.3, 30.7, 31.1, 32.3, 34.0, 34.4, 34.6, 34.7, 35.7,
41.8, 45.0, 47.6, 49.4, 53.3, 57.4, 74.2, 75.9, 127.7, 136.8, 170.5,
170.6, 174.0. MS (MALDI-TOF): m/z = 1810 [M+Na]+. Anal. Calcd.
For C102 H147 N9 O18: C, 68.54; H, 8.29; N, 7.05. Found: C, 68.35; H,
8.38; N, 7.17.
δ
C 12.4, 14.1, 17.5, 21.4, 21.4, 22.6, 23.1, 23.4, 25.3, 25.6, 25.9, 26.6,
26.9, 27.3, 28.9, 30.7, 31.1, 31.5, 32.3, 34.0, 34.4, 34.6, 34.7, 35.7,
41.8, 45.0, 47.6, 49.4, 53.3, 53.6, 57.4, 61.9, 74.2, 75.8, 114.7, 120.0,
124.0, 127.8, 136.8, 137.2, 159.1, 170.4, 170.5, 173.9. MS (MALDI-
TOF): m/z = 4081 [M+Na]+. Anal. Calcd. For C228 H315 N27 O39: C,
67.48; H, 7.82; N, 9.32. Found: C, 67.65; H, 7.68; N, 9.21.
4.26. Synthesis of first generation dendrimer 6
4.22. Synthesis of zeroth generation dendrimers 2
The dendrimer 6 was synthesized from the azido dendrimer 25
(0.1 g, 0.099 mmol, 1.0 equiv.) and the alkyne dendron 17 (0.31 g,
0.61 mmol, 6.2 equiv.) using the click chemistry procedure A, as white
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.29 g, 73%; M. P.: 157 °C; 1H NMR: (300 MHz, CDCl3):
δH 0.70 (s, 18H, 18-Me); 0.76 (d, 18H, J = 5.7 Hz); 0.85–2.37 (m,
210H); 2.42 (s, 9H); 4.64–4.70 (m, 6H); 5.05 (s, 6H); 5.08 (s, 6H); 5.17
(s, 12H); 5.44 (s, 12H); 5.67 (s, 6H); 6.78 (s, 3H); 6.84 (s, 6H); 7.45 (s,
3H); 7.60 (s, 6H). 13C NMR: (75 MHz, CDCl3): δC 12.4, 14.1, 16.8, 17.5,
21.4, 21.4, 22.6, 23.1, 23.4, 25.3, 25.7, 25.9, 26.6, 26.9, 27.3, 29.7,
30.7, 31.1, 31.6, 32.3, 34.0, 34.4, 34.6, 34.7, 35.7, 41.8, 45.0, 47.6,
49.4, 53.6, 57.4, 62.0, 74.2, 75.8, 114.8, 120.0, 122.7, 124.0, 130.6,
137.2, 139.8, 143.5, 159.2, 170.4, 170.5, 173.9. MS (MALDI-TOF): m/
z = 4123 [M+Na]+. Anal. Calcd. For C231 H321 N27 O39: C, 67.67; H,
7.89; N, 9.22. Found: C, 67.75; H, 7.78; N, 9.41.
Following the general procedure A, the dendrimer 2 was obtained as
white solid from the triazide 18 (0.07 g, 0.25 mmol, 1.0 equiv.) and the
alkyne dendron 17 (0.0.39 g, 0.76 mmol, 3.1 equiv.) after purification
by eluting from the column with CHCl3: MeOH (19:1). Yield: 0.37 g,
82%; M. P.: 130 °C 1H NMR: (300 MHz, CDCl3): δH 0.71(s, 9H, 18-Me);
0.76(d, 9H, 19-Me, J = 6 Hz); 0.83–2.43 (m, 114H); 4.70 (m, 3H); 5.06
(s, 3H); 5.16 (s, 6H); 5.65 (s, 6H); 7.39 (s, 3H). 13C NMR: (75 MHz,
CDCl3): δC 12.4, 16.7, 17.5, 21.4, 23.1, 23.4, 25.7, 25.9, 26.6, 26.9,
27.3, 30.6, 31.1, 32.3, 34.0, 34.5, 34.7, 34.7, 35.7, 41.8, 45.0, 47.6,
49.0, 49.4, 57.3, 74.2, 75.8, 130.5, 143.0, 170.5, 170.6, 174.0. MS
(MALDI-TOF): m/z = 1852 [M+Na]+. Anal. Calcd. For C105 H153 N9
O18: C, 68.94; H, 8.43; N, 6.89. Found: C, 68.85; H, 8.58; N, 6.77.
4.23. Synthesis of zeroth generation dendrimers 3
4.27. Synthesis of first generation dendrimer 7
Following the general procedure A, the dendrimer 3 was obtained as
white solid from the triazide 20 (0.05 g, 0.20 mmol, 1.0 equiv.) and the
alkyne dendron 18 (0.36 g, 0.64 mmol, 3.1 equiv.) after purification by
eluting from the column with CHCl3: MeOH (19:1). Yield: 0.32 g, 81%;
M. P.: 110 °C; 1H NMR: (300 MHz, CDCl3): δH 0.71 (s, 9H, 18-Me); 0.78
(d, 9H, 19-Me, J = 6 Hz); 0.83–2.36 (m, 108H); 4.57 (m, 3H); 4.91 (s,
3H); 5.07 (s, 3H); 5.19 (s, 6H); 5.47 (s, 6H); 7.16 (s, 3H); 7.58 (s, 3H).
13C NMR: (75 MHz, CDCl3): δC 12.2, 14.1, 17.5, 21.4, 21.5, 21.6, 22.6,
22.8, 25.2, 25.6, 26.9, 27.1, 28.9, 30.6, 30.9, 31.3, 31.5, 34.3, 34.5,
34.6, 34.7, 37.7, 40.9, 43.4, 45.1, 47.3, 53.3, 57.3, 70.7, 74.1, 75.3,
127.7, 136.8, 170.4, 170.5, 170.5, 173.9. MS (MALDI-TOF): m/
z = 1984 [M+Na]+. Anal. Calcd. For C108 H153 N9 O24: C, 66.13; H,
7.86; N, 6.43. Found: C, 66.25; H, 7.78; N, 6.57.
The dendrimer 7 was synthesized from the azido dendrimer 24
(0.1 g, 0.10 mmol, 1.0 equiv.) and the alkyne dendron 18 (0.36 g,
0.64 mmol, 6.2 equiv.) using the click chemistry procedure A, as white
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.32 g, 71%; M. P.: 180 °C; 1H NMR: (300 MHz, CDCl3):
δH 0.70 (s, 18H, 18-Me); 0.77 (d, 18H, J = 6 Hz); 0.92 (s, 18H);
1.02–2.35 (m, 198H); 4.57 (m, 6H); 4.91 (s, 6H); 5.06 (s, 6H); 5.12 (s,
6H); 5.17 (s, 12H); 5.45 (s, 12H); 5.49 (s, 6H); 6.80 (s, 3H); 6.84 (s,
6H); 7.18 (s, 3H); 7.61 (s, 9H). 13C NMR: (75 MHz, CDCl3): δC 12.2,
17.5, 21.4, 21.6, 22.6, 22.8, 25.6, 26.9, 27.1, 28.9, 30.6, 30.9, 31.3,
34.3, 34.5, 34.6, 34.7, 37.8, 41.0, 43.4, 45.1, 47.3, 53.4, 53.6, 57.4,
61.9, 70.7, 74.1, 75.3, 114.8, 120.0, 123.4, 124.0, 127.8, 136.8, 137.2,
143.4, 143.6, 159.1, 170.3, 170.4, 170.5, 173.8. MS (MALDI-TOF): m/
z = 4429 [M+Na]+. Anal. Calcd. For C240 H327 N27 O51: C, 65.42; H,
7.48; N, 8.58. Found: C, 65.55; H, 7.28; N, 8.71.
4.24. Synthesis of zeroth generation dendrimers 4
Following the general procedure A, the dendrimer 4 was obtained as
white solid from the triazide 21 (0.07 g, 0.25 mmol, 1.0 equiv.) and the
alkyne dendron 18 (0.43 g, 0.76 mmol, 3.1 equiv.) after purification by
eluting from the column with CHCl3: MeOH (19:1). Yield: 0.35 g, 71%;
M. P.: 150 °C; 1H NMR: (300 MHz, CDCl3): δH 0.71(s, 9H, 18-Me); 0.77
(d, 9H, 19-Me, J = 6 Hz); 0.83–2.13 (m, 108H); 2.41 (s, 9H); 4.58 (m,
3H); 4.90 (s, 3H); 5.07 (s, 3H); 5.15 (s, 6H); 5.64 (s, 6H); 7.39 (s,
3H).13C NMR: (75 MHz, CDCl3): δC 12.2, 16.7, 17.5, 21.4, 21.5, 21.6,
22.6, 22.8, 25.3, 25.6, 26.9, 27.2, 28.9, 30.6, 30.9, 31.3, 34.3, 34.5,
34.6, 34.7, 37.7, 40.9, 43.4, 45.0, 47.3, 49.0, 57.3, 70.7, 74.1, 75.3,
127.7, 130.5, 139.8, 170.4, 170.5, 170.5, 173.9. MS (MALDI-TOF): m/
z = 2026 [M+Na]+. Anal. Calcd. For C111 H159 N9 O24: C, 66.54; H,
8.00; N, 6.29. Found: C, 66.65; H, 8.18; N, 6.17.
4.28. Synthesis of first generation dendrimer 8
The dendrimer 8 was synthesized from the azido dendrimer 25
(0.1 g, 0.099 mmol, 1.0 equiv.) and the alkyne dendron 18 (0.35 g,
0.61 mmol, 6.2 equiv.) using the click chemistry procedure A, as white
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.3 g, 68%; M. P.: 160 °C; 1H NMR: (300 MHz, CDCl3): δH
0.71 (s, 18H, 18-Me); 0.77 (d, 18H, J = 5.7 Hz); 0.83–2.38 (m, 216H);
2.42 (s, 9H); 4.53–4.65 (m, 6H); 4.90 (s, 6H); 5.12 (s, 12H); 5.17 (s,
12H); 5.44 (s, 12H); 5.67 (s, 6H); 6.79 (s, 3H); 6.84 (s, 6H); 7.46 (s,
3H); 7.59 (s, 6H). 13C NMR: (75 MHz, CDCl3): δC 12.2, 14.1, 17.5, 21.4,
21.4, 21.6, 22.5, 22.6, 22.8, 25.3, 25.6, 26.9, 27.1, 28.9, 29.0, 29.7,
30.6, 30.9, 31.3, 31.6, 34.3, 34.5, 34.7, 34.7, 37.8, 41.0, 43.4, 45.1,
47.3, 53.6, 57.4, 62.0, 70.7, 74.1, 75.3, 77.5, 114.8, 120.0, 122.7,
123.4, 123.9, 130.6, 137.2, 139.8, 143.1, 143.4, 159.2, 170.3, 170.4,
170.5, 173.8. MS (MALDI-TOF): m/z = 4471 [M+Na]+. Anal. Calcd.
4.25. Synthesis of first generation dendrimer 5
The dendrimer 5 was synthesized from the azido dendrimer 24
8

D. Anandkumar, P. Rajakumar
Steroidsxxx(xxxx)xxx–xxx
For C243 H333 N27 O51: C, 65.61; H, 7.55; N, 8.50. Found: C, 65.53; H,
7.68; N, 8.33.
5.42 (s, 36H); 5.65 (s, 6H); 6.76 (s, 6H); 6.83 (s, 18H); 7.51 (s, 3H);
7.62 (s, 12H); 7.68 (s, 6H). 13C NMR: (75 MHz, CDCl3): δC 11.4, 12.2,
14.1, 14.3, 17.5, 18.7, 19.4, 20.4, 20.7, 21.4, 21.4, 21.6, 22.5, 22.6,
22.6, 22.8, 25.3, 25.6, 26.9, 27.1, 27.6, 28.9, 29.0, 30.6, 30.9, 31.3,
31.6, 34.3, 34.7, 34.7, 36.1, 37.8, 41.0, 41.3, 43.4, 45.1, 47.4, 53.6,
57.4, 61.8, 70.7, 74.1, 75.3, 76.6, 114.8, 120.1, 124.0, 137.2, 143.4,
159.1, 170.3, 170.4, 170.5, 173.8. MS (MALDI-TOF): m/z = 9361 [M
+Na]+. Anal. Calcd. For C507 H681 N63 O105: C, 65.21; H, 7.35; N, 9.45.
Found: C, 65.15; H, 7.47; N, 9.32.
4.29. Synthesis of second generation dendrimer 9
The dendrimer 9 was synthesized from the azido dendrimer 28
(0.05 g, 0.021 mmol, 1.0 equiv.) and the alkyne dendron 17 (0.13 g,
0.25 mmol, 12.2 equiv.) using the click chemistry procedure A, as white
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.1 g, 56%; M. P.: 158 °C; 1H NMR: (300 MHz, CDCl3): δH
0.70 (s, 36H, 18-Me); 0.76 (d, 36H, J = 5.7 Hz); 0.86–2.34 (m, 432H);
4.66–4.73 (m, 12H); 5.05 (s, 24H); 5.15 (s, 24H); 5.41 (s, 36H); 6.75 (s,
9H); 6.83 (s, 18H); 7.16 (s, 3H); 7.62 (s, 18H); 7.68 (s, 3H). 13C NMR:
(75 MHz, CDCl3): δC 12.4, 14.1, 17.5, 21.4, 21.4, 22.7, 23.1, 23.4, 25.7,
25.9, 26.6, 26.9, 27.3, 29.7, 30.7, 31.1, 31.9, 32.3, 34.0, 34.4, 34.6,
34.7, 35.7, 41.8, 45.0, 47.6, 49.4, 53.6, 57.4, 74.2, 75.8, 114.8, 120.1,
123.7, 124.1, 136.8, 137.2, 143.4, 159.1, 170.4, 170.5, 173.9. MS
(MALDI-TOF): m/z = 8622 [M+Na]+. Anal. Calcd. For C480 H651 N63
O81: C, 67.01; H, 7.63; N, 10.26. Found: C, 67.21; H, 7.43; N, 10.38.
4.33. Inhibition of cancer cell growth
The C6 glioma cells were cultured in DMEM media containing 10%
fetal bovine serum at 37 °C and 5% CO2. 5000 cells were seeded in each
well containing 200 µl of DMEM medium in 96 well plates. After 24 h
dendrimers 1–12 were added in triplicate. Five different test con-
centrations 10 µM, 20 µM, 30 µM, 40 µM and 50 µM were added and
cell viability was assessed; after 24 h of treatment, 20 μl per well of
MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
5 mg/ml; stock solution, Sigma) was added. The plates were incubated
at 37 °C for additional four hours. The medium was discarded and the
formazan blue, which formed in the cells, was dissolved with 200 μl of
DMSO. Cell viability and IC50 value was calculated for compound ef-
ficacy on cancer cell.
4.30. Synthesis of second generation dendrimer 10
The dendrimer 10 was synthesized from the azido dendrimer 29
(0.1 g, 0.040 mmol, 1.0 equiv.) and the alkyne dendron 17 (0.25 g,
0.49 mmol, 12.2 equiv.) using the click chemistry procedure A, as white
solid after purification by eluting from the column with CHCl3: MeOH
(19:1). Yield: 0.2 g, 57%; M. P.: 168 °C; 1H NMR: (300 MHz, CDCl3): δH
0.70 (s, 36H, 18-Me); 0.76 (d, 36H, J = 4.8 Hz); 0.83–2.39 (m, 441H);
4.69–4.73 (m, 12H); 5.05 (s,18H); 5.15 (s, 24H); 5.42 (s, 36H); 5.65 (s,
6H); 6.76 (s, 9H); 6.83 (s, 18H); 7.50 (s, 3H); 7.62 (s, 6H); 7.67 (s,
12H). 13C NMR: (75 MHz, CDCl3): δC 11.4, 12.4, 14.1, 14.3, 17.5, 18.7,
19.4, 20.4, 20.7, 21.4, 21.4, 22.6, 22.6, 22.8, 23.1, 23.4, 25.3, 25.7,
25.9, 26.6, 26.9, 27.3, 27.7, 29.0, 29.4, 29.7, 30.7, 31.1, 31.6, 32.3,
34.0, 34.4, 34.5, 34.7, 34.7, 35.7, 36.1, 41.3, 41.8, 45.0, 47.6, 49.4,
53.6, 57.4, 61.8, 74.2, 75.9, 114.8, 120.1, 124.1, 137.2, 143.4, 159.1,
170.4, 170.5, 173.9. MS (MALDI-TOF): m/z = 8664 [M+Na]+. Anal.
Calcd. For C483 H657 N63 O81: C, 67.13; H, 7.66; N, 10.21. Found: C,
67.25; H, 7.48; N, 10.35.
Acknowledgements
The authors thank UGC, New Delhi, for financial assistance under
UPE Phase II programme and DST-FIST for providing NMR facility to
the department.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.steroids.2017.06.007.
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10