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

Steroids xxx (xxxx) xxx–xxx

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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

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.

1. Introduction 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], sensors [3], solar cells [4] and efficient light harvesting antenna [5], molecular 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 efficient, 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 reagents. 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 metabolism [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 supramolecular chemistry [10]. Bile acids are biological compounds with interesting properties due to their large, rigid, and curved steroidal skeletons, chemically different hydroxy groups, enantiomeric purities, and their unique amphiphilicity. Bile acids and their derivatives are attractive for synthetic chemists because these facially amphiphilic molecules 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

⁎

membrane transfection [14]. Bile acids can self-assemble because of their amphiphilic properties, which make them potential building blocks for the design of polymeric materials for biomedical applications [15] and have gained considerable attention in supramolecular chemistry in recent years. More recently, 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 dendrimers, it is worth to synthesize the following dendrimers with bile acid surface group using click chemistry (Fig. 1). 2. Result and discussion 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: [email protected] (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/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Anandkumar, D., Steroids (2017), http://dx.doi.org/10.1016/j.steroids.2017.06.007

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(a)

AcO

(b)

AcO OAc

R2

R2

AcO

AcO R2 OAc

O

O

O

O

O

N N

N

N

R1

N N N

O

N

R1 N

O

N R1 N N

O N N N N

N N N

N

O

N R1 N

O

N

AcO

N N N R1

R2

O

OAc

O

O O OAc

R2 OA c

R1 N N N

AcO

OAc R2

O R2

N

O

OAc O

1 R1, R2 = H 2 R1 = CH3, R2 = H 3 R1 = H, R2 = OAc 4 R1 = CH3, R2 = OAc

AcO

N

N N N

N N

O

O O

O

OAc OAc

R2

R

OAc

OAc

(c)

AcO

OAc

R2

R2

AcO

AcO

OAc

O

O

N NN

N NN

O

R2 AcO O

O

N

5 R 1, R 2 = H 6 R1 = CH3, R 2 = H 7 R1 = H, R2 = OAc 8 R1 = CH3, R 2 = OAc

OAc R2

O

N N O

O

N N N

OAc

O

O N N

N N N

N

AcO

OAc

O

R2 AcO O O

O

N NN O

NN N

O

N NN

R1 NN N

N NN R1

R2 OAc O

N R1 N N

O

N N N

O

N NN

O

NN

NN N

N NN

O

AcO R2

N

O

O O

OAc R2

O N N N

AcO O

O

NN N

NN N

O O

OAc

N N N O

O

O O

R2

AcO

OAc

OAc R2

OAc

AcO R2

OAc AcO

R2 OAc 9 R1, R2 = H 10 R1 = CH3, R2 = H 11 R1 = H, R2 = OAc 12 R1 = CH3, R2 = OAc

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 molecular ion peak at m/z 515 (M+1). Further the structure of the

O OH

OH

i

O

OH

R OH

ii

OAc

O

R

R OAc

OH 13 R= H 14 R= OH

O

O

15 R= H 16 R= OH

17 R= H 18 R= OAc

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

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Y Y

O N3

O

X

+

X

O

i

N3 Cl

X

Cl

19 (3.1 equiv.)

N NN X

Y N N X N

Y

N3

20 X = H 21 X = CH3

X

N N N O

22 X = H, Y = Cl 23 X = CH3, Y = Cl

Y

Y ii

24 X = H, Y = N3

i Y

ii

Y

25 X = CH3, Y = N3

19 (6.1 equiv.)

Y

O N N N Y

N N N

Y

O

O

Y

O N NN N NN O

O

N NN X

N N X N X

26 X = H, Y = Cl 27 X = CH3, Y = Cl

N

N N

28 X = H, Y = N3

ii

29 X = CH3, Y = N3

O

Y

ii

Y N N N

N N N

Y

O

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%).

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 triazolyl 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. 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

compound 17 was also confirmed from elemental analysis. Similarly, the structure of the triacetyl terminal alkyne 18 was confirmed from spectral and analytical data. The divergent synthesis begins with the reaction of 1,3,5-tris(azidomethyl)benzene 20 [20] and 1,3,5-tris(azidomethyl)-2,4,6-trimethylbenzene 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 3

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D. Anandkumar, P. Rajakumar

addition to the signals for the other aliphatic and aromatic protons. The C 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 aliphatic 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 addition to the signals for the other aliphatic and aromatic protons. The 13 C 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.

under Cu(I) catalyzed click reaction conditions gave the second generation chlorodendrimers 26 and 27 in 80% and 88% yields, respectively, 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 different –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, respectively, 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 aromatic 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 analysis. Similarly, the structure of the dendrimer 2, 3 and 4 was confirmed from spectral and analytical data. 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

13

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 antiproliferation 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 increases 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) O

28 and 29

O

OAc

9, 10, 11 and 12 i

20 and 21 1, 2, 3 and 4 i

R 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

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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

1 2 5 6 9 10

IC50 (µM)

Activity (% of inhibition) 10 µM

20 µM

30 µM

40 µM

50 µM

27.02 ± 1.04 30 ± 0.68 35.91 ± 0.9 36.12 ± 1.01 41.82 ± 0.75 44.5 ± 1.20

40.13 ± 1.02 42.1 ± 1.02 54.52 ± 1.10 57.89 ± 0.92 62.01 ± 1.43 69.2 ± 1.32

50.83 ± 1.92 51.4 ± 1.20 63.12 ± 2.01 66.42 ± 1.76 74.68 ± 1.87 76.82 ± 1.80

64.78 ± 1.54 67.7 ± 1.82 73.46 ± 1.82 77.41 ± 1.58 82.91 ± 2.03 85.38 ± 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

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 corresponding dendrimers.

among the all synthesized dendrimers. The morphological study by light microscope images of second generation dendrimers 9 and 11 has shown almost shrink and less population 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 confirmed 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 determined using a Toshniwal melting point apparatus by open capillary tube method and are uncorrected. Column chromatography was performed 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. Matrixassisted 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 mentioned under each compound.

4.2. General procedure for Cu (I)-catalyzed azide-alkyne cycloaddition (A)

4.5. General procedure for the first and second generation of azidodendrimers (D)

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

3 4 7 8 11 12

30.47 31.92 38.12 39.72 48.03 49.01

± ± ± ± ± ±

0.92 0.58 1.01 1.01 1.26 1.10

IC50 (µM) 20 µM

30 µM

40 µM

50 µM

55.1 ± 0.96 57.34 ± 0.85 62.42 ± 1.25 63.26 ± 1.25 74.62 ± 1.92 76 ± 1.45

62.56 ± 1.07 63.56 ± 1.09 70.5 ± 1.92 74.52 ± 1.92 83.67 ± 2.11 76.59 ± 1.87

67.9 ± 1.43 68.9 ± 1.23 84.53 ± 2.13 86.43 ± 1.87 89.9 ± 2.32 90 ± 1.92

82.91 ± 1.87 84.3 ± 1.57 92.73 ± 2.17 94.1 ± 2.2 99.31 ± 2.89 98.4 ± 2.30

5

17.77 25.18 14.49 14.86 10.68 10.48

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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.

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%; 1 H 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.

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. 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 propargyl 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(hydroxymethyl)-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

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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.

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. 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 chemistry, 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): δH 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.

4.12. Synthesis of 1,3,5-tris(azidomethyl)-2,4,6-trimethylbenzene 21 The 1,3,5-tris(azidomethyl)-2,4,6-trimethylbenzene 21 was obtained as white solid from 1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene (1.0 g, 1.0 mmol) and NaN3 (0.5 g, 3.50 mmol) as reported in the literature [21]. Yield: 86%. 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 chemistry, 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): δH 2.40 (s, 9H); 4.66 (s, 18H); 5.05 (s, 6H); 5.13 (s, 12H); 5.53 (s, 12H); 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.

4.14. First generation chloro dendrimer 23 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 generation 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). 13 C 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, DMSOd6): 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 generation 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, DMSO7

Steroids xxx (xxxx) xxx–xxx

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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): δC 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 (MALDITOF): 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.

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). 13 C 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.

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). 13 C 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

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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.

For C243 H333 N27 O51: C, 65.61; H, 7.55; N, 8.50. Found: C, 65.53; H, 7.68; N, 8.33. 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 concentrations 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 efficacy 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. References [1] (a) D.A. Tomalia, A.M. Naylor, W.A. Goddard, Angew. Chem. Int. Ed. Engl. 29 (1990) 138; (b) G.R. Newkome, C.N. Moorefield, F. Vögtle, Dendritic Molecules: Concepts, Syntheses, , PerspectiVes, VCH, Weinheim, 1996; (c) O.A. Matthews, A.N. Shipway, J.F. Stoddart, Prog. Polym. Sci. 23 (1998) 1. [2] (a) S.H. Medina, M.E.H. El-Sayed, Chem. Rev. 109 (2009) 3141; (b) N.A. Peppas, T. Nagai, M. Miyajima, Pharm. Tech. Jpn. 10 (1994) 611; (c) S. Svenson, D.A. Tomalia, Adv. Drug Delivery Rev. 57 (2005) 2106; (d) I.J. Majoros, A. Myc, T. Thomas, C.B. Mehta, J.R. Baker Jr., Biomacromolecules 7 (2006) 572. [3] (a) V. Balzani, P. Ceroni, S. Gestermann, C. Kauffmann, M. Gorka, F. Vogtle, Chem. Commun. (2000) 853; (b) T.D. Jamnes, H. Shinmori, M. Takeuchi, S. Shinkai, Chem. Commun. (1996) 705. [4] D. Astruc, E. Boisselier, C.T. Orneals, Chem. Rev. 110 (2010) 1857. [5] (a) G.M. Stewart, M.A. Fox, J. Am. Chem. Soc. 118 (1996) 4354; (b) T.H. Ghaddar, J.F. Wishart, D.W. Thompson, J.K. Whitesell, M.A. Fox, J. Am. Chem. Soc. 124 (2002) 8285; (c) A. Adronov, J.M.J. Frechet, Chem. Commun. (2000) 1701; (d) S.L. Gilat, A. Adronov, J.M.J. Frechet, Angew. Chem. Int. Ed. 38 (1999) 1422. [6] A.W. Kleiji, R.A. Gossage, R.J.M.K. Gebbink, N. Brinkmann, E. Reijerse, F. Vogtle, V. Vicinelli, V. Ceroni, M. Maestri, V. Balzani, Angew. Chem. Int. Ed. 41 (2002) 3595. [7] (a) T. Zhou, H. Neubert, D.Y. Liu, Z.D. Liu, Y.M. Ma, X.L. Kong, W. Luo, S. Mark, R.C. Hider, J. Med. Chem. 49 (2006) 4171. [8] (a) R. Huisgen, Angew. Chem. 80 (1968) 329; (b) R. Huisgen, Angew. Chem. Int. Ed. Engl. 7 (1968) 321; (c) H.C. Kolb, M.G. Finn, K.B. Sharpless, Angew. Chem. 113 (2001) 2056; (d) H.C. Kolb, M.G. Finn, K.B. Sharpless, Angew. Chem. Int. Ed. 40 (2001) 2004; (e) V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless, Angew. Chem. 114 (2002) 2708. [9] (a) I. Kirson, E.J. Glotter, Nat. Prod. 44 (1981) 633–647; (b) H. Gao, J.R. Dias, Org. Prep. Proced. Int. 32 (1999) 145–166; (c) M. Fetizon, F.J. Kakis, V.J. Ignatiadou-Ragoussis, Org. Chem. 38 (1973) 4308–4311. [10] (a) H. Danielsson, P.P. Nair, D. Kritchevsky (Eds.), The Bile Acids: Chemistry,

4.31. Synthesis of second generation dendrimer 11 The dendrimer 11 was synthesized from the azido dendrimer 28 (0.05 g, 0.021 mmol, 1.0 equiv.) and the alkyne dendron 18 (0.14 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: 0.11 g, 57%; M. P.: 260 °C; 1H NMR: (300 MHz, CDCl3): δH 0.70 (s, 36H, 18-Me); 0.78 (d, 36H, J = 5.4 Hz); 0.83–2.28 (m, 430H); 4.53–4.61 (m, 12H); 4.90 (s, 6H); 5.04 (s, 12H); 5.06 (s, 24H); 5.14 (s, 24H); 5.42 (s, 44H); 6.75 (s, 9H); 6.83 (s, 18H); 7.17 (s, 3H); 7.62 (s, 18H); 7.68 (s, 3H). 13C NMR: (75 MHz, CDCl3): δC 11.4, 12.2, 14.1, 17.5, 21.4, 21.4, 21.5, 21.6, 22.6, 22.8, 25.3, 25.6, 26.9, 27.1, 28.9, 29.0, 30.6, 31.0, 31.3, 31.6, 34.3, 34.5, 34.7, 34.7, 37.8, 41.0, 43.4, 45.1, 47.4, 53.6, 57.4, 70.7, 74.1, 75.3, 76.6, 114.7, 120.1, 124.1, 137.2, 143.4, 159.1, 170.3, 170.4, 170.5, 173.8. MS (MALDI-TOF): m/ z = 9319 [M+Na]+. Anal. Calcd. For C504 H675 N63 O105: C, 65.12; H, 7.32; N, 9.49. Found: C, 65.25; H, 7.43; N, 9.28. 4.32. Synthesis of second generation dendrimer 12 The dendrimer 12 was synthesized from the azido dendrimer 29 (0.1 g, 0.40 mmol, 1.0 equiv.) and the alkyne dendron 18 (0.28 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: 0.23 g, 60%; M. P.: 180 °C; 1H NMR: (300 MHz, CDCl3): δH 0.70 (s, 36H, 18-Me); 0.78 (d, 36H, J = 5.4 Hz); 0.83–2.40 (m, 450H); 4.53–4.57 (m, 12H); 4.90 (s, 12H); 5.06 (s, 24H); 5.15 (s, 24H); 9

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[11] [12] [13] [14] [15] [16] [17]

(2001) 2281–2283. [18] J.W. Zhang, J. Luo, X.X. Zhu, M.J.N. Junk, D. Hinderberger, Langmuir 26 (2010) 2958–2962. [19] E.H. Ryu, Y. Zhao, Org. Lett. 6 (2004) 3187. [20] J.W. Lee, J.H. Kim, B. Kim, W.S. Shin, S. Jin, Tetrahedron 62 (2006) 894. [21] Y. Song, E.K. Kohlmeir, T.J. Meade, J. Am. Chem. Soc. 130 (2008) 6662. [22] C. He, L.W. Li, W.D. He, W.X. Jiang, C. Wu, Macromolecules 44 (2011) 6233. [23] (a) M. Tim, Immunol. Methods 65 (1983) 55; (b) Y.P. Wang, Z.F. Wang, Cell Res. 14 (2004) 467; (c) Z. Wang, M. Cui, L. Sun, Z. Jia, Yun Bai, K. Ma, C. Zhou, F. Chen, Biochem. Biophys. Res. Commun. 359 (2007) 685.

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