Synthesis, photophysical and anticancer study of D-ring extended estrone analogues

Abstract

A concise route for the highly substituted ring extended estrone derivatives has been established. This protocol involves very simple, facile and one step ring transformation and cyclization process. The preliminary absorption, emission spectroscopic and biological studies of these compounds revealed the possible use of such prototypes in cancer chemotherapy as fluorescence probes.

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Steroidal modifications are an important area of research¹ due to their established utility in the pharmaceutical industry for development of therapeutics to combat various diseases such as inflammation (I, II),^2a asthma (I),^2b rheumatoid arthritis (I, II)^2c crohn's disease (I),^2d ulcerative colitis (II),^2e eczema, leukemia, swelling of brain, contraception, ovulation inhibitor^2f (III) and breast cancer (I–V),^2g–h Fig. 1.

Fig. 1 Steroids modified clinically useful drugs.

These modifications are achieved either through substitution of different groups at defined positions or through manipulations in steroidal core itself in a receptor friendly manner. In case of estrane nucleus, modification and/or ring extension approach of ring D in steroidal core leads to mainly enzyme inhibitors or mixed enzyme inhibitor-antiestrogens.^4,5 Following this approach, Fischer et al. reported pyrazole fused estrone derivatives as 17β-hydroxysteroid dehydrogenase type 1 inhibitors (VI, IC₅₀: 300 nM),³ while Mohareb et al. reported pyran (VII), pyrimidine and thiazole extended derivatives of estrone possessing anticancer activity in various carcinoma cell lines (upto 22 nM) (Fig. 2).⁶ Numerous protocols for the ring extension of estrone such as Pd(OAc)₂ catalyzed synthesis of benzo[b][1,4]thiazepines and 2-arylsubstitutedbenzo[b][1,4]thiazepines⁷ fused steroids and other heterocyclic rings extended estrone have been delineated in the literature.^1,3–5 Silyl enol ether formation or ring closing metathesis of steroids,^6,8 intermolecular Pauson–Khand cycloaddition for the synthesis of estrone-derived cyclopentenone⁹ were also reported recently.

Fig. 2 Ring extended biologically active estrone analogs.

Our research interest in the development of novel D ring extended estrone derivatives as possible fluorescence probe, fascinated us to explore a convenient and effective method for the synthesis of target molecules through reaction of estrone with substituted 2H-pyran-2-ones. 2H-Pyran-2-ones were chosen owing to their widespread use as a synthon for the development of diversified heterocycles.¹⁰ Structurally, 2H-pyran-2-ones (5) seize three electron deficient centers C-2, C-4 and C-6 (Fig. 3) which make these molecules proficient to yield different reaction products on reaction with nucleophiles under diverse reaction conditions. Position C-6 is the most electron deficient and therefore, preferentially attacked by nucleophiles yielding cyclic arenes and heteroarenes.

Fig. 3 Synthesis of precursor pyran-2-one (5).

Thus, our synthetic strategy was based on employing 6-aryl-4-sec amino-2H-pyran-2-one-3-carbonitriles (5) as key precursor, which were synthesized from the reaction of aryl methyl ketone (2) and methyl 2-cyano-3,3-dimethylthioacrylate (1) and further amination of 3 with sec amines (4).¹² Conventionally, potassium hydroxide or sodium hydroxide in DMF or DMSO at room temperature are often required for the reaction of pyran-2-one with α-hydrogen containing ketones.¹¹

Our initial attempts for reaction of estrone (6a) with 5a under these reaction conditions were unsuccessful probably due to partial solubility of estrone (6) in DMF or DMSO. Therefore, we preferred to use tetrahydrofuran (THF) as a solvent of choice in which 6 was completely soluble and accordingly sodium hydride (NaH) was chosen as base for this reaction. Employing NaH-THF at room temperature, reaction of 6a with 5a did not yield any product rather starting materials were recovered, however, NaH-THF at refluxing condition gave trace amount of products along with significant quantities of 6a recovered. In order to optimize reaction conditions, we further explored the reaction of 6a with 5a using a strong base potassium hydride (KH) in THF at 100 °C for 6 h and succeeded to synthesize ring extended estrone derivative 7a along with an unexpected reaction product 8a in 36% and 38% yields respectively, Table 1. Interestingly, we could not observe the formation of other expected product 9. Under palladium acetate catalyzed reaction conditions, 7a was produced in low yield (19%).

Table 1 Optimization of conditions for the ring extension reaction of estrone (6a)

S. no.	Base	Solvent	T (°C)	t (h)	Yield 7a (%)	Yield 8a (%)
a No reaction.b With recovery of 6a.c 10 mol%.d Not formed.
1	KOH	DMF	rt	48	^a	^a
2	KOH	DMF	80	48	^a	^a
3	NaOH	DMF	100	48	^a	^a
4	NaOH	DMSO	100	48	^a	^a
5	^tBuOK	nBuOH	110	06	^a	^a
6	NaH	THF	rt	48	^a	^a
7	NaH	THF	100	48	Trace^b	^d
8	KH	THF	100	03	26	37
9	KH	THF	100	06	36	38
10	KH/Pd(OAc)2^c	THF	100	06	19	^d
11	KH	Pyridine	100	06	03	^d

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To expand the scope of this synthetic process, we explored the reaction of methylsulfonyl containing lactone (3a) with estrone (6a) since methylsulfonyl group is a convenient handle for further functionalization. The reaction of 6a with 3a under these reaction conditions did not yield desired product instead an inseparable complex mixture was obtained. In our observations, we found that sec amine containing lactones (5) were showing selectivity towards the ring annulations reactions, Scheme 1.

Scheme 1 Ring extension reaction of estrones (6) and plausible mechanism for the formation of the products 7, 8 and 9.

Plausible mechanistic propositions for the formation of products 7, 8 and other expected product 9 are outlined in Scheme 1. The reaction involving the formation of 7 (route a) was proposed to be proceeded through a Michael addition (i), followed by protonation of carbonyl group and cyclization (ii) subsequent decarboxylation (iii) and dehydration while the formation of 8 (route b) proceeded through almost the same mechanism with a hydroxyl shift followed by dehydration and elimination of sec amine. The reaction involving the formation of 9 (route c) was expected through Michael addition (v), followed by enolization (vi), subsequent decarboxylation (vii), cyclization (viii) and elimination of sec amine.

On the basis of above results (Table 1), we explored various reactions of different 6-aryl-4-sec amino-2H-pyran-2-one-3-carbonitriles analogues (5a–e) with estrone derivatives (6a–d) under optimized reaction conditions and results have been shown in Table 2. Results showed that in these reactions, the yield of product 8 was not uniform. In some cases products 8e–i could be realized only with the help of mass spectroscopy.

Table 2 Yields of expended estrone products 7 and 8, Scheme 1

S. no.	Com.	R	R1	X	Con.^b (6, %)	Yields^c (7, %)	Yields^c (8, %)
a Trace.b Conversion.c Yields are based on % of conversion.
1	a	Me	Br	CH2	100	36	38
2	b	Me	Me	O	96	20	38
3	c	Me	Cl	CH2	95	39	11
4	d	Me	OMe	O	90	31	05
5	e	Me	H	CH2	67	56	36
6	f	Et	Cl	CH2	94	36	^a
7	g	THP	Cl	CH2	87	32	^a
8	h	OH	Cl	CH2	84	30	^a
9	i	Et	Me	O	82	29	^a

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The unique photophysical, chemical and electronic properties of helical molecules¹³ inspired us to examine the synthetic utility of this estrone ring transformation protocol for the synthesis of helical steroids, which are hither to unreported. We further explored the reaction of estrone (6) with tricyclic lactones (10) under the above reaction conditions which yielded tricyclic helical ring extended estrone analogues (12) instead of anticipated product (11) as shown in Scheme 2.

Scheme 2 Synthesis of tricyclic helical ring extended estrones (12).

The structures of the synthesized products were assigned by different spectroscopic techniques such as IR, 1D and 2D NMR, mass and X-ray diffraction analyses (see ESI†). Single crystals of 8a for X-ray diffraction study were obtained by slow evaporation of ethyl acetate solution. The compound was crystallized with two independent molecules in the asymmetric unit. A co-crystallized water molecule was present in the asymmetric unit, which was hydrogen bonded to the hydroxyl group of one of the steroid molecules (Fig. 4). In addition, as many as three disordered water molecules were identified in the asymmetric unit which appears to be trapped in the crystal structure, forming a continuous channel (Fig. S1†).^13,14

Fig. 4 ORTEP drawing (ellipsoids at 30% probability) of the two independent molecules of 8a (CCDC no. 1046576) in the asymmetric unit (left) molecule (i) hydrogen bonded to an ordered water molecule. Methyl group attached to the OCH₃ group is disordered over two positions with an occupancy ratio of 0.66 : 0.34 (right) molecule (ii). H atoms of the disordered methyl group and the disordered oxygen atoms of water molecules in the asymmetric unit are not shown (C-dark grey, O-red, N-pink, H-light grey).

Interestingly, as anticipated all synthesized compounds were found to have significant florescence under ultraviolet light. To the best of our knowledge, estrone based fluorescent probes have not been reported so far. Therefore, it was imperative to study their photophysical properties.

The UV-Vis absorption spectra of 7a–7i, 8a, 8b, 8e, 12a and 12b in acetonitrile are shown in Fig. 5 and the characteristic data are summarized in Table 3. The UV-Vis spectra revealed that all new steroid based molecules had longest absorption maxima between 310–355 nm, which could be tentatively ascribed to the π–π* transition. In this region the molar extinction coefficient for steroidal derivatives was 2300–6200 M⁻¹ cm⁻¹.

Fig. 5 UV-Vis Spectra of 7a–7i, 8a, 8b, 8e, 12a and 12b in acetonitrile.

Table 3 Photophysical properties of compounds 7a–7i, 8a, 8b, 8e, 12a and 12b in acetonitrile medium

Com.	λabs^a (nm)	λem^b (nm)	ε^c (M^−1 cm^−1)	Δν^d (nm)	Φ^e
a λabs (nm): absorption wavelength.b λem (nm): emission wavelength.c ε: molar extinction coefficient.d Δν: stokes shifts.e Φ: fluorescence quantum yield.
7a	342	450	5233.4	108	0.12
7b	324	431	4786.5	107	0.22
7c	341	450	4585.5	109	0.20
7d	326	427	6174.1	101	0.15
7e	339	441	4126.5	102	0.25
7f	342	450	4796.6	108	0.16
7g	341	450	5096.2	109	0.19
7h	342	450	3510.3	108	0.19
7i	332	433	4269.7	101	0.16
8a	316	370	9342.7	54	0.12
8b	336	433	1524.9	97	0.16
8e	313	364	5340.7	51	0.29
12a	353	444	3515.8	91	0.08
12b	353	444	2371.1	91	0.07

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There was not much change in the absorption maxima (339–342 nm) and ε (4100–5300 M⁻¹ cm⁻¹) for 7a, 7c and 7e irrespective of the location of the substituents like -bromophenyl, -chlorophenyl and -phenyl at the 12^th position of the molecule. Interestingly, compound 7d had an increment in the ε observed which may be attributed to intra molecular charge transfer due to presence of methoxy group on phenyl ring pendent at 12^th position of the molecule. Likewise, the similar λ_max were found for the 7c, 7f, 7g and 7h in the range 339–341 nm, irrespective of the substituent at 4^th position of the molecule.

In contrast, compounds 8a, 8b and 8e exhibited low λ_max compared to the other molecules, whereas 12a and 12b had an increment in the λ_max (353 nm) which was observed due to the presence of phenanthrene ring system.

The florescence spectra of 7a–7i, 8a, 8b, 8e, 12a and 12b were measured in acetonitrile and the corresponding emission wavelengths were in the region 360–450 nm, upon excitation of at their corresponding λ_max value (Fig. 6). Among all, compounds 7a, 7c, 7f–7h showed higher emission at 450 nm. In general, compounds exhibited relatively large stokes shifts (51–109 nm) possibly due to high intramolecular charge-transfer excitation of an electron from HOMO state to the LUMO of the chromophores. Compounds 7a–7i, 8a, 8b and 8e showed higher quantum yields compared to 12a and 12b which are presented in Table 3.

Fig. 6 Electronic absorption & emission spectra of 7a–i, 8a–b, 8e, 12a–b in acetonitrile.

Furthermore, the florescence properties of compounds 7a–7i, 8a, 8b, 8e, 12a and 12b were studied at different P^H in acetonitrile–phosphate buffer (AcN : PBS) medium in order to elucidate the effect of pH on fluorescence of compounds. However, it was observed that pH of the medium has no impact on florescence properties of compounds 7a–7i, 8a, 8b, 8e, 12a and 12b, Table 4.

Table 4 Fluorescence properties of compounds 7a–7i, 8a, 8b, 8e, 12a and 12b in AcN : PBS buffer medium at different pH^a

Compound	λem (nm) (AcN : 1 mM PBS at pH = 5)	λem (AcN : 1 mM PBS at pH = 7.4)	λem (AcN : 1 mM PBS at pH = 8)
a λem (nm): emission wavelength.
7a	460	460	459
7b	439	439	439
7c	460	460	460
7d	437	437	437
7e	453	453	453
7f	460	459	460
7g	459	457	459
7h	460	460	460
7i	440	441	440
8a	377	381	377
8b	445	445	445
8e	364	364	364
12a	458	459	458
12b	458	456	458

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Steroids modified clinically useful drugs (I–V, Fig. 1) for the treatment of breast cancer prompted us to investigate in vitro antiproliferative activity of ring extended estrone analogues (7–8). The compounds 7a–i, 8a–b were explored for in vitro anticancer activity in various human carcinoma cell lines such as breast (MCF-7), colon (DLD1), lung (A549), prostate (Du145) using SRB assay as shown in Table 5. The results exhibited that compound 8a and 8b had significant anticancer activity (IC₅₀ value 10.48–20.92 μM) against MCF-7, DLD1, A549, and (Du145) cell lines (Table 5) while 7a–i were devoid of any activity at 30 μM concentration. SAR showed that possibly, the free hydroxyl group present in 8a–b could be responsible for anticancer activity. Further, compounds 8a–b was evaluated in normal healthy monkeykidneyepithelial (Vero) cells and results showed some cytotoxicity of the compounds (Table 5).

Table 5 Growth inhibition (IC₅₀ values) of 8a and 8b in carcinoma cell lines MCF-7, DLD1, A549, Du145

Comp.	IC50 (μM, 48 h) (Mean ± SE)^a
	MCF-7^b	DLD^b	A549^b	DU145^b	Vero^c
a SRB assay repeated three time.b Breast (MCF-7), colon (DLD1), lung (A549), prostate (Du145) carcinoma cell lines.c Healthy monkey kidney epithelial cells.d Values from single assay performed in 6 replicates, ND = not done.
8a	17.13 ± 1.78	14.17 ± 2.51	17.84 ± 0.93	20.92 ± 2.94	5.39 ± 0.06^d
8b	14.50 ± 0.78	10.48 ± 1.61	12.97 ± 0.77	14.81 ± 2.75	5.54 ± 0.07^d
Tamoxifen	8.74 ± 1.17	14.56	10.37 ± 0.84	12.14 ± 0.62	ND

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Conclusion

In summary, an efficient method for synthesis of highly substituted ring extended estrone analogues has been delineated via ring transformation. This methodology is applicable to generate variety of D-ring extended estrone derivatives. The preliminary results of photophysical and biological studies of these compounds showed that such prototypes may have capability to function as possible fluorescence probes in bio-imaging. However, further structural refinement and biological evaluation of compounds is required to establish their true use as fluorescent probe in cell bio-imaging. Our efforts in this direction are underway and results will be reported in due course.

Experimental section

General

The reagents and solvents used in this study were of analytical or laboratory grade and used without further purification unless stated otherwise. All the reactions were monitored on Merck aluminium silica gel thin layer chromatography (TLC, UV_{254 nm}) plates. Column chromatography was carried out on silica gel (100–200 mesh). The melting points were determined on Buchi melting point M560 apparatus in open capillaries and are uncorrected. Commercial reagents were used without purification. ¹H (300 MHz) and ¹³C NMR (75 MHz) spectra were recorded on a Bruker WM-300 using CDCl₃ as the solvent. Chemical shift are reported in parts per million (ppm) shift (δ-value) based on the middle peak of the solvent (CDCl₃). Signal patterns are indicated as s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet. Coupling constants (J) are given in Hertz. Infrared (IR) spectra were recorded on a Perkin-Elmer AX-1 spectrophotometer in KBr disc and reported in wave number (cm⁻¹). ESI-MS mass spectra were recorded on Shimadzu LC-MS and/or LC-MS-MS APC3000 (Applied Biosystem).

General procedure for the synthesis of extended estrons (7, 8)

Potassium hydride (150 mg) was taken in a dry round bottom (RB) flask (obtained after rinsed with freshly dried tetrahydrofuran three times) and taken 30 mL freshly dried THF in RB. Further, estrone (6, 629 mg, 2.0 mmol) was added in RB and reflux the reaction mixture for 10 min at 100 °C. Consequently lactone (5, 2.05 mmol) added in the reaction mixture at 100 °C. Reflux the reaction mixture for 6 h. Cool the reaction mixture and the crude residue poured onto crushed ice with vigorous stirring. The aqueous suspension was neutralized with dil. HCl and the precipitate obtained was filtered, washed with water and dried. Residue was purified by silica gel column chromatography using ethyl acetate (0–20%)/hexane.

(+)(6bS,8aS,13aS,13bS)-12-(4-Bromophenyl)-4-methoxy-8a-methyl-10-(piperidin-1-yl)-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7a). White solid with white florescence in long UV (365 nm); R_f 0.38 (3% EtOAc/hexane); yield 36%; mp 214–215 °C; IR (KBr): 2216 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.16 (s, 3H, CH₃), 1.25 (m, 1H, CH), 1.44 (bs, 3H, CH₂), 1.78 (m, 8H, 4 × CH₂), 2.31 (m, 1H, CH), 2.45 (m, 1H, CH), 2.61 (m, 2H, CH₂), 2.86–2.96 (m, 3H, CH₂), 3.07–3.14 (m, 4H, 2 × NCH₂), 3.77 (s, 3H, OMe), 6.63–6.78 (m, 3H, Ar–H), 7.21–7.30 (m, 3H, Ar–H), 7.58 (m, 2H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 17.9 (CH₃), 24.5 (CH₂), 26.6 (2 × CH₂), 26.9 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.3 (CH₂), 34.9 (CH₂), 37.9 (CH), 44.3 (CH), 47.8, 54.4 (2 × NCH₂), 55.6, 56.9 (CH₂), 101.3, 111.9 (Ar–H), 114.3 (Ar–H), 117.0 (Ar–H), 117.3 (CN), 122.5, 126.5 (Ar–H), 130.4 (2 × Ar–H), 132.0 (2 × Ar–H), 132.7, 134.8, 138.0, 139.5, 142.3, 157.1, 158.0, 159.6; MS: m/z = 581.3 (M⁺); _D[α]^24.7 = +68 (0.001 g mL⁻¹, EtOAc); anal. calcd (C₃₅H₃₇BrN₂O): C, 72.28; H, 6.41; N, 4.82. Found: C, 72.18; H, 6.38; N, 4.80.; HRMS (ESI): calc. for C₃₅H₃₈BrN₂O: 581.2168 (M⁺ + H); found: 581.2167.

(+)(6bS,8aS,13aS,13bS)-12-(4-Bromophenyl)-10-hydroxy-4-methoxy-8a-methyl-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (8a). White solid; R_f 0.14 (3% EtOAc/hexane); yield 38%; mp 170–171 °C; IR (KBr): 2227 (CN), 3411 (OH) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.18 (s, 3H, CH₃), 1.41–1.50 (m, 1H, CH), 1.68–1.99 (m, 6H, 2 × CH₂ & CH), 2.32–2.39 (m, 1H, CH), 2.47–2.51 (m, 1H, CH), 2.64–2.67 (m, 2H, CH₂), 2.83–2.96 (m, 3H, CH₂ & CH), 3.81 (s, 3H, OCH₃), 6.67 (s, 1H, Ar–H), 6.75–6.82 (m, 2H, Ar–H), 7.23–7.26 (m, 1H, Ar–H), 7.30 (d, J = 8.4 Hz, 2H, Ar–H), 7.60 (d, J = 8.4 Hz, 2H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 18.2 (CH₃), 26.8 (CH₂), 28.0 (CH₂), 29.9 (CH₂), 31.3 (CH₂), 34.8 (CH₂), 37.9 (CH), 44.3 (CH), 47.8, 55.6 (OCH₃), 57.0 (CH), 94.2, 111.9 (Ar–H), 113.8 (Ar–H), 114.3 (Ar–H), 115.6, 122.8, 126.5 (Ar–H), 130.3 (2 × Ar–H), 132.1 (2 × Ar–H), 132.6, 133.8, 138.0, 138.7, 143.6, 157.9 (2C), 159.0; MS: m/z = 512 (M⁺ − H); _D[α]^24.7 = +80 (0.0008 g mL⁻¹, EtOAc); anal. calcd (C₃₀H₂₈BrNO₂): C, 70.04; H, 5.49; N, 2.72. Found: C, 69.96; H, 5.50; N, 2.70; HRMS (ESI): calc. for C₃₀H₂₉BrNO₂: 514.1382 (M⁺ + H); found: 514.1381.

(+)(6bS,8aS,13aS,13bS)-4-Methoxy-8a-methyl-10-morpholino-12-p-tolyl-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7b). White solid with white florescence in long UV (365 nm); R_f 0.50 (10% EtOAc/hexane); yield 20%; mp 150–151 °C; IR (KBr): 2218 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 0.85 (m, 1H, CH), 1.16 (s, 3H, CH₃), 1.42 (m, 1H, CH), 1.69–1.93 (m, 5H, 2 × NCH₂ & CH), 2.32–2.42 (m, 5H, CH & CH₂), 2.64–2.70 (m, 2H, CH₂), 2.86–2.95 (m, 3H, CH & CH₂), 3.10–3.25 (m, 3H, CH & CH₂), 3.77 (s, 3H, OCH₃), 3.90–3.91 (m, 4H, OCH₂), 6.63 (d, 1H, J = 2.1 Hz, Ar–H), 6.73 (dd, 1H, J = 2.4 Hz & 8.4 Hz, Ar–H), 6.83 (s, 1H, Ar–H), 7.20–7.33 (m, 5H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 18.0 (CH₃), 21.6 (CH₃), 26.9 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.5 (CH₂), 34.9 (CH₂), 38.0 (CH), 44.3 (CH), 47.9, 53.0 (2 × NCH₂), 55.6 (OCH₃), 56.9 (CH), 67.5 (2 × OCH₂), 100.6, 111.9 (Ar–H), 114.3 (Ar–H), 117.2 (Ar–H), 117.3 (CN), 126.5 (Ar–H), 128.6 (2 × Ar–H), 129.6 (2 × Ar–H), 132.7, 136.1, 137.5, 138.0, 138.3, 143.9, 155.4, 158.0, 159.8; MS: m/z = 519.31 (M⁺ + H); _D[α]^25.2 = +98 (0.00057 g mL⁻¹, MeCN); anal. calcd (C₃₅H₃₈N₂O₂): C, 81.05; H, 7.38; N, 5.40. Found: C, 80.92; H, 7.35; N, 5.38; HRMS (ESI): calc. for C₃₅H₃₉N₂O₂: 519.30060 (M⁺ + H); found: 519.30075.

(+)(6bS,8aS,13aS,13bS)-10-Hydroxy-4-methoxy-8a-methyl-12-p-tolyl-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (8b). White solid; R_f 0.20 (10% EtOAc/hexane); yield 38%; mp 164–165 °C; IR (KBr): 2227 (CN), 3413 (OH) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.13 (s, 3H, CH₃), 1.40–1.42 (m, 1H, CH), 1.67–1.84 (m, 5H, 2 × CH₂ & CH), 1.87–1.95 (m, 1H, CH), 2.30 (m, 1H, CH), 2.39 (s, 3H, CH₃), 2.46 (m, 1H, CH), 2.64–2.66 (m, 2H, CH₂), 2.79–2.91 (m, 3H, CH₂ & CH), 3.77 (s, 3H, OCH₃), 6.63 (d, J = 2.4 Hz, 1H, Ar–H), 6.73 (dd, J = 2.7 & 8.7 Hz, 1H, Ar–H), 6.80 (s, 1H, Ar–H), 7.18–7.31 (m, 5H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 18.2 (CH₃), 21.6 (CH₃), 26.8 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.4 (CH₂), 34.9 (CH₂), 37.9 (CH), 44.3 (CH), 47.8, 55.6 (OCH₃), 57.0 (CH), 93.6, 111.9 (Ar–H), 113.9 (Ar–H), 114.3 (Ar–H), 115.8 (CN), 126.5 (Ar–H), 128.6 (2 × Ar–H), 129.6 (2 × Ar–H), 132.8, 133.8, 137.0, 138.1, 138.4, 144.8, 157.9 (2 × Ar), 158.7; MS: m/z = 448 (M⁺ − H); _D[α]^24.7 = +71 (0.0009 g mL⁻¹, MeCN); anal. calcd (C₃₁H₃₁NO₂): C, 82.82; H, 6.95; N, 3.12. Found: C, 82.91; H, 6.97; N, 3.14; HRMS (ESI): calc. for C₃₁H₃₂NO₂: 450.36047 (M⁺ + H); found: 450.36334.

(+)(6bS,8aS,13aS,13bS)-12-(4-Chlorophenyl)-4-methoxy-8a-methyl-10-(piperidin-1-yl)-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7c). White solid with white florescence in long UV (365 nm); R_f 0.45 (5% EtOAc/hexane); yield 39%; mp 206–207 °C; IR (KBr): 2216 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.16 (s, 3H, CH₃), 1.42 (m, 1H, CH), 1.59 (bs, 2H, CH₂), 1.65–1.80 (m, 8H, 4 × CH₂), 1.90–1.95 (m, 1H, CH), 2.32 (m, 1H, CH), 2.45 (m, 1H, CH), 2.61 (m, 2H, CH₂), 2.86–2.96 (m, 3H, CH₂ & CH), 3.03–3.18 (m, 4H, 2 × NCH₂), 3.77 (s, 3H, OMe), 6.63 (s, 1H, Ar–H), 6.71–6.78 (m, 2H, Ar–H), 7.21–7.25 (m, 1H, Ar–H), 7.33–7.43 (m, 4H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 17.9 (CH₃), 24.5 (CH₂), 26.6 (2 × CH₂), 26.9 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.3 (CH₂), 34.9 (CH₂), 37.9 (CH), 44.3 (CH), 47.8, 54.4 (2 × NCH₂), 55.6 (OCH₃), 56.9 (CH), 101.2, 111.9 (Ar–H), 114.3 (Ar–H), 117.1 (Ar–H), 117.3 (CN), 126.5 (Ar–H), 129.0 (2 × Ar–H), 130.1 (2 × Ar–H), 132.7, 134.3, 134.9, 138.0, 139.1, 142.3, 157.1, 158.0, 159.6; MS: m/z = 537.35 (M⁺ + H); _D[α]^24.2 = +80 (0.0007 g mL⁻¹, EtOAc); anal. calcd (C₃₅H₃₇ClN₂O): C, 78.26; H, 6.94; N, 5.22. Found: C, 78.14; H, 6.90; N, 5.20; HRMS (ESI): calc. for C₃₅H₃₈ClN₂O: 537.26672 (M⁺ + H); found: 537.26677.

(+)(6bS,8aS,13aS,13bS)-12-(4-Chlorophenyl)-10-hydroxy-4-methoxy-8a-methyl-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (8c). White solid; R_f 0.41 (17% EtOAc/hexane); yield 11%; mp 167–168 °C; IR (KBr): 2222 (CN), 3326 (OH) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.18 (s, 3H, CH₃), 1.27–1.31 (m, 2H, CH₂), 1.43–1.49 (m, 1H, CH), 1.69–2.02 (m, 3H, CH & CH₂), 2.08 (m, 1H, CH), 2.33–2.36 (m, 1H, CH), 2.48–2.49 (m, 1H, CH), 2.53–2.67 (m, 2H, CH₂) 2.84–2.96 (m, 2H, CH₂), 3.81 (s, 3H, OCH₃), 6.15 (h, 1H, OH), 6.67 (s, 1H, Ar–H), 6.75–6.82 (m, 2H, Ar–H), 7.28 (d, J = 6.3 Hz, 1H, Ar–H), 7.36 (d, J = 8.4 Hz, 2H, Ar–H), 7.45 (d, J = 8.4 Hz, 2H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 18.2 (CH₃), 26.8 (CH₂), 28.0 (CH₂), 29.9 (CH₂), 31.3 (CH₂), 34.8 (CH₂), 37.9 (CH), 44.3 (CH), 47.8, 55.6 (OCH₃), 57.0 (CH), 94.2, 111.9 (Ar–H), 113.8 (Ar–H), 114.3 (Ar–H), 115.5 (CN), 126.5 (Ar–H), 129.1 (2 × Ar–H), 130.0 (2 × Ar–H), 132.6, 133.9, 134.6, 138.0, 138.2, 143.6, 157.8 (2 × C = O), 158.9; MS: m/z = 468 (M⁺ − H); _D[α]^24.9 = +80 (0.001g mL⁻¹, MeCN); anal. Calcd (C₃₀H₂₈ClNO₂): C, 76.66; H, 6.00; N, 2.98. Found: C, 76.59; H, 5.98; N, 2.96;

(+)(6bS,8aS,13aS,13bS)-4-Methoxy-12-(4-methoxyphenyl)-8a-methyl-10-morpholino-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7d). White solid with white florescence in long UV (365 nm); R_f 0.30 (20% EtOAc/hexane); yield 31%; mp 183–184 °C; IR (KBr): 2215 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.20 (s, 3H, CH₃), 1.47 (m, 1H, CH), 1.67–1.89 (m, 5H, 2 × CH₂ & CH), 2.36 (m, 1H, CH), 2.49 (m, 1H, CH), 2.68–2.74 (m, 2H, CH₂), 2.90–2.99 (m, 3H, CH₂ & CH), 3.14–3.30 (m, 4H, 2 × NCH₂), 3.81 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 3.95 (t, J = 3.9 Hz, 4H, 2 × OCH₂), 6.67 (d, J = 2.4 Hz, 1H, Ar–H), 6.77 (dd, J = 2.7 & 8.7 Hz, 1H, Ar–H), 6.85 (s, 1H, Ar–H), 7.03 (d, J = 8.4 Hz, 2H, Ar–H), 7.28 (d, J = 2.7 Hz, 1H, Ar–H), 7.40 (d, J = 9.0 Hz, 2H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 18.0 (CH₃), 26.9 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.5 (CH₂), 34.9 (CH₂), 38.0 (CH), 44.3 (CH), 47.9, 53.0 (2 × NCH₂), 55.6 (OCH₃), 55.7 (OCH₃), 56.9 (CH), 67.5 (2 × OCH₂), 100.4, 111.9 (Ar–H), 114.3 (CN), 114.4 (2 × Ar–H), 117.0 (Ar–H), 117.3, 126.5 (Ar–H), 130.0 (2 × Ar–H), 132.7 (2 × Ar), 135.9, 138.0, 143.6, 155.4, 158.0, 159.8, 159.9; MS: m/z = 535.32 (M⁺ + H); _D[α]^24.9 = +96 (0.0008 g mL⁻¹, MeCN); anal. calcd (C₃₅H₃₈N₂O₃): C, 78.62; H, 7.16; N, 5.24. Found: C, 78.50; H, 7.13; N, 5.21; HRMS (ESI): calc. for C₃₅H₃₈N₂O₃: 535.29552 (M⁺ + H); found: 535.29528.

(6bS,8aS,13aS,13bS)-10-Hydroxy-4-methoxy-12-(4-methoxyphenyl)-8a-methyl-2,6b,7,8,8a,9,10,13,13a,13b-decahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (8d). White solid with white florescence in long UV (365 nm); R_f 0.35 (30% EtOAc/hexane); yield 05%; mp 170–171 °C; IR (KBr): 2224 (CN), 3367 (OH) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.18 (s, 3H, CH₃), 1.45–1.48 (m, 1H, CH), 1.73–1.89 (m, 4H, 2 × CH₂), 1.96–1.97 (m, 1H, CH), 2.21 (m, 1H, CH), 2.08 (m, 1H, CH), 2.35 (m, 1H, CH₂), 2.66–2.71 (m, 2H, CH₂), 2.84–2.94 (m, 3H, CH & CH₂), 3.81 (s, 3H, OMe), 3.89 (s, 3H, OMe), 6.67 (s, 1H, Ar–H), 6.76 (dd, J = 2.4 & 8.4 Hz, 1H, Ar–H), 6.82 (s, 1H, Ar–H), 7.00 (d, J = 8.7 Hz, 2H, Ar–H), 7.28 (d, J = 5.1 Hz, 1H, Ar–H), 7.37 (d, J = 8.7 Hz, 2H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 18.2 (CH₃), 26.8 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.5 (CH₂), 34.8 (CH₂), 37.9 (CH), 44.3 (CH), 47.7, 55.6 (OCH₃), 55.7 (OCH₃), 57.0 (CH), 66.2, 93.3, 111.9 (Ar–H), 113.7 (Ar–H), 114.3 (2 × Ar–H), 115.8 (CN), 126.5 (Ar–H), 129.9 (2 × Ar–H), 132.2, 132.7, 133.7, 138.0, 144.5, 157.7 (C=O), 157.9, 158.7, 159.9; MS: m/z = 464 (M⁺ − H); _D[α]^24.9 = +93 (0.0008 g mL⁻¹, MeCN); anal. calcd (C₃₁H₃₁NO₃): C, 79.97; H, 6.71; N, 3.01. Found: C, 79.89; H, 6.69; N, 2.99.

(+)(6bS,8aS,13aS,13bS)-4-Methoxy-8a-methyl-12-phenyl-10-(piperidin-1-yl)-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7e). White solid with white florescence in long UV (365 nm); R_f: 0.31 (5% EtOAc/hexane); yield 56%; mp 175–176 °C; IR (KBr): 2218 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 0.88–0.91 (m, 2H, 2 × CH), 1.20 (s, 3H, CH₃), 1.46 (m, 1H, CH), 1.56–1.64 (m, 4H, 2 × CH₂), 1.73–1.94 (m, 6H, 3 × CH₂), 2.35 (m, 1H, CH), 2.55 (m, 1H, CH), 2.66–2.71 (m, 2H, CH₂), 2.89–3.01 (m, 2H, CH₂), 3.09–3.22 (m, 4H, 2 × NCH₂), 3.81 (s, 3H, OMe), 6.66 (s, 1H, Ar–H), 6.76 (dd, J = 2.4 & 8.4 Hz, 1H, Ar–H), 6.87 (s, 1H, Ar–H), 7.28 (s, 2H, Ar–H), 7.41–7.48 (m, 4H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 17.9 (CH₃), 24.5 (CH₂), 26.7 (2 × CH₂), 26.9 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.3 (CH₂), 34.9 (CH₂), 38.0 (CH), 44.4 (CH), 47.8, 54.4 (2 × NCH₂), 55.6 (CH), 56.9 (OCH₃), 60.7, 100.9, 111.9 (Ar–H), 114.3 (Ar–H), 117.4 (Ar–H), 117.5 (CN), 126.5 (Ar–H), 128.1 (Ar–H), 128.7 (Ar–H), 128.8 (2 × Ar–H), 132.8, 135.0, 138.1, 140.7, 143.6, 157.0, 157.9, 159.4; MS: m/z = 503.37 (M⁺ + H); _D[α]^24.9 = +70 (0.0008 g mL⁻¹, MeCN); anal. calcd (C₃₅H₃₈N₂O): C, 83.63; H, 7.62; N, 5.57. Found: C, 83.51; H, 7.58; N, 5.56; HRMS (ESI): calc. for C₃₅H₃₉N₂O: 503.30569 (M⁺ + H); found: 503.30561.

(6bS,8aS,13aS,13bS)-10-Hydroxy-4-methoxy-8a-methyl-12-phenyl-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (8e). White solid; R_f 0.27 (20% EtOAc/hexane); yield 36%; mp 174–175 °C IR (KBr): 2222 (CN), 3421 (OH) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.18 (s, 3H, CH₃), 1.29–1.31 (m, 1H, CH), 1.45–1.47 (m, 2H, CH₂), 1.73–1.99 (m, 5H, CH₂ & CH), 2.08 (m, 1H, CH), 2.36 (m, 1H, CH₂), 2.54 (m, 1H, CH), 2.66–2.70 (m, 1H, CH), 2.85–2.90 (m, 2H, CH₂), 3.81 (s, 3H, OMe), 6.67–6.78 (m, 2H, Ar–H), 6.85 (s, 1H, Ar–H), 7.24–7.28 (m, 1H, Ar–H), 7.39–7.53 (m, 5H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 18.2 (CH₃), 26.8 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.3 (CH₂), 34.9 (CH₂), 37.9 (CH), 44.3 (CH), 47.8, 55.6 (OCH₃), 57.0 (CH), 93.9, 111.9 (Ar–H), 114.0 (Ar–H), 114.3 (Ar–H), 115.8 (CN), 126.5 (Ar–H), 128.4 (2 × Ar–H), 128.7 (2 × Ar–H), 128.8 (Ar–H), 132.7, 133.8, 138.1, 139.9, 144.8, 157.9, 158.0, 158.7; MS: m/z = 435.25 (M⁺); _D[α]^24.9 = +78 (0.001g mL⁻¹, MeCN); anal. calcd (C₃₀H₂₉NO₂): C, 82.73; H, 6.71; N, 3.22. Found: C, 82.64; H, 6.68; N, 3.19; HRMS (ESI): calc. for C₃₀H₃₀NO₂: 436.22711 (M⁺ + H); found: 436.22701.

(+)(6bS,8aS,13aS,13bS)-12-(4-Chlorophenyl)-4-ethoxy-8a-methyl-10-(piperidin-1-yl)-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7f). White solid with white florescence in long UV (365 nm); R_f 0.38 (3% EtOAc/hexane); yield 36%; mp 226–227 °C; IR (KBr): 2221 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.15 (s, 3H, CH₃), 1.17–1.27 (m, 1H, CH), 1.36–1.40 (m, 4H, 2 × CH₂), 1.40–1.84 (m, 9H, CH & CH₂), 2.03 (s, 1H, CH), 2.32–2.35 (m, 1H, CH), 2.44–2.49 (m, 1H, CH), 2.59–2.63 (m, 2H, CH₂), 2.85–2.96 (m, 3H, CH₂ & CH), 3.06–3.18 (m, 4H, 2 × NCH₂), 4.00 (q, J = 7.2 Hz, 2H, OCH₂), 6.62 (s, 1H, Ar–H), 6.71 (dd, J = 2.4 & 8.4 Hz, 1H, Ar–H), 6.77 (s, 1H, Ar–H), 7.20 (d, J = 8.7 Hz, 1H, Ar–H), 7.33 (d, J = 8.4 Hz, 2H, Ar–H), 7.41 (d, J = 8.4 Hz, 2H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 15.3 (CH₃), 17.9 (CH₃), 24.5 (CH₂), 26.6 (2 × CH₂), 26.9 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.3 (CH₂), 34.9 (CH₂), 38.0 (CH), 44.3 (CH), 47.9, 54.3 (2 × NCH₂), 56.9 (CH), 63.7 (OCH₂), 101.2, 112.4 (Ar–H), 115.0 (Ar–H), 117.1 (Ar–H), 117.3 (CN), 126.4 (Ar–H), 129.0 (2 × Ar–H), 130.0 (2 × Ar–H), 132.6, 134.3, 134.9, 138.0, 139.1, 142.3, 157.1, 157.3, 159.6; MS: m/z = 551.33 (M⁺ + H); _D[α]^24.3 = +45 (0.0008 g mL⁻¹, EtOAc); anal. calcd (C₃₆H₃₉ClN₂O): C, 78.45; H, 7.13; N, 5.08. Found: C, 78.35; H, 7.11; N, 5.05; HRMS (ESI): calc. for C₃₆H₄₀ClN₂O: 551.28237 (M⁺ + H); found: 551.28272.

(+)(6bS,8aS,13aS,13bS)-12-(4-Chlorophenyl)-8a-methyl-10-(piperidin-1-yl)-4-(tetrahydro-2H-pyran-2-yloxy)-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7g). White solid with white florescence in long UV (365 nm); R_f 0.40 (6% EtOAc/hexane); yield 32%; mp 229–230 °C; IR (KBr): 2217 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.18 (s, 3H, CH₃), 1.26–1.31 (m, 2H, CH₂), 1.45 (m, 1H, CH), 1.64–2.07 (m, 17H, CH & CH₂), 2.19 (s, 1H, CH), 2.35 (m, 1H, CH), 2.53 (m, 1H, CH), 2.62–2.67 (m, 1H, CH), 2.99 (m, 2H, CH & CH₂), 3.08–3.21 (m, 2H, CH & CH₂), 3.64 (m, 1H, CH), 3.95 (m, 1H, OCH), 4.14 (m, 1H, CH), 5.41 (s, 1H), 6.81–6.91 (m, 3H, Ar–H), 7.23–7.28 (m, 1H, Ar–H), 7.36–7.46 (m, 4H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 17.9 (CH₃) 19.2 (CH₂), 24.5 (CH₂), 25.6 (CH₂), 26.6 (CH₂), 26.8 (2 × CH₂), 28.1 (CH₂), 29.9 (CH₂), 30.8 (CH₂), 31.3 (CH₂), 34.9 (CH₂), 37.9 (CH), 44.4 (CH), 47.8, 54.3 (2 × NCH₂), 56.9 (CH), 62.3 (OCH₂), 96.8 (OCH), 101.2, 114.5 (Ar–H), 117.0 (Ar–H), 117.1 (Ar–H), 117.3, 126.4 (Ar–H), 129.0 (2 × Ar–H), 130.0 (2 × Ar–H), 133.7, 134.3, 134.9, 137.9, 139.1, 142.3, 155.4, 157.1, 159.6; MS: m/z = 607.36 (M⁺ + H); _D[α]^24.4 = +40 (0.001 g mL⁻¹, EtOAc); anal. calcd (C₃₉H₄₃ClN₂O₂): C, 77.14; H, 7.14; N, 4.61. Found: C, 76.99; H, 7.10; N, 4.58; HRMS (ESI): calc. for C₃₉H₄₄ClN₂O₂: 607.30858 (M⁺ + H); found: 607.30860.

(+)(6bS,8aS,13aS,13bS)-12-(4-Chlorophenyl)-4-hydroxy-8a-methyl-10-(piperidin-1-yl)-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7h). White solid with white florescence in long UV (365 nm); R_f 0.29 (10% EtOAc/hexane); yield 30%; mp 265–266 °C; IR (KBr): 2226 (CN), 3370 (OH) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.19 (s, 3H, CH₃), 1.29 (m, 1H, CH), 1.44–1.61 (m, 1H, CH), 1.63–1.94 (m, 10H, 5 × CH₂), 2.34 (m, 1H, CH), 2.47 (m, 1H, CH), 2.62–2.67 (m, 1H, CH), 2.86–2.99 (m, 3H, CH & CH₂), 3.08–3.21 (m, 4H, NCH₂), 4.85 (bs, 1H, OH), 6.61 (d, J = 2.4 Hz, 1H, Ar–H), 6.69 (dd, J = 2.4 & 8.4 Hz, 1H, Ar–H), 6.81 (s, 1H, Ar–H), 7.20 (d, J = 8.4 Hz, 1H, Ar–H), 7.38 (d, J = 8.4 Hz, 2H, Ar–H), 7.45 (d, J = 8.4 Hz, 2H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 17.9 (CH₃), 24.5 (CH₂), 26.6 (2 × CH₂), 26.9 (CH₂), 28.0 (CH₂), 29.8 (CH₂), 31.3 (CH₂), 34.9 (CH₂), 37.9 (CH), 44.3 (CH), 47.8, 54.4 (2 × NCH₂), 56.8 (CH), 101.2, 113.2 (Ar–H), 115.6 (Ar–H), 117.1 (Ar–H), 117.3 (CN), 126.7 (Ar–H), 129.0 (2 × Ar–H), 130.0 (2 × Ar–H), 132.8, 134.3, 134.9, 138.3, 139.1, 142.4, 153.9, 157.1, 159.6; MS: m/z = 523.32 (M⁺ + H); _D[α]^24.8 = +81 (0.0008 g mL⁻¹, MeCN); anal. calcd (C₃₄H₃₅ClN₂O): C, 78.06; H, 6.74; N, 5.36. Found: C, 77.95; H, 6.70; N, 5.33; HRMS (ESI): calc. for C₃₄H₃₆ClN₂O: 523.25107 (M⁺ + H); found: 523.25110.

(+)(6bS,8aS,13aS,13bS)-4-Ethoxy-8a-methyl-10-morpholino-12-p-tolyl-2,6b,7,8,8a,13,13a,13b-octahydro-1H-indeno[2,1-a]phenanthrene-9-carbonitrile (7i). White solid with white florescence in long UV (365 nm); R_f 0.60 (10% EtOAc/hexane); yield 29%; mp 226–227 °C; _D[α]^24.5 = +66 (0.0007 g mL⁻¹, EtOAc); IR (KBr): 2219 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.21 (s, 3H, CH₃), 1.43 (t, J = 6.9 Hz, 3H, CH₃), 1.63 (s, 1H, CH), 1.73–1.94 (m, 5H, CH & 2 × CH₂), 2.35–2.49 (m, 5H, CH₂ & CH₃), 2.68–2.74 (m, 2H, CH₂), 2.89–2.99 (m, 3H, CH & CH₂), 3.14–3.29 (m, 4H, 2 × NCH₂), 3.94–3.95 (m, 4H, 2 × OCH₂), 4.03 (q, J = 6.9 Hz, 2H, OCH₂), 6.66 (s, 1H, Ar–H), 6.75 (dd, J = 2.4 & 8.4 Hz, 1H, Ar–H), 6.87 (s, 1H, Ar–H), 7.23–7.37 (m, 5H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 15.3 (CH₃), 18.0 (CH₃), 21.6 (CH₃), 26.9 (CH₂), 28.1 (CH₂), 30.0 (CH₂), 31.5 (CH₂), 34.9 (CH₂), 38.0 (CH), 44.3 (CH), 47.9, 53.0 (2 × NCH₂), 56.9 (CH), 63.7 (OCH₂), 67.5 (2 × OCH₂), 100.6, 112.4 (Ar–H), 115.0 (Ar–H), 117.1 (Ar–H), 117.3 (CN), 126.4 (Ar–H), 128.6 (2 × Ar–H), 129.6 (2 × Ar–H), 132.6, 136.0, 137.5, 138.0, 138.3, 143.9, 155.4, 157.3, 159.8; MS: m/z = 533.34 (M⁺ + H); _D[α]^24.5 = +66 (0.0007 g mL⁻¹, EtOAc); anal. calcd (C₃₆H₄₀N₂O₂): C, 81.17; H, 7.57; N, 5.26. Found: C, 81.11; H, 7.55; N, 5.23; HRMS (ESI): calc. for C₃₆H₄₁N₂O₂: 533.31626 (M⁺ + H); found: 533.31630.

(+)(6bS,8aS,17aS,17bS)-4-Ethoxy-8a-methyl-10-(piperidin-1-yl)-1,2,6b,7,8,8b,11,12,17a,17-decahydro-17H-cyclopenta[a:c]bisphenanthrene-9-carbonitrile (12a). White solid with white florescence in long UV (365 nm); R_f 0.35 (5% EtOAc/hexane); yield 26%; mp 210–211 °C; IR (KBr): 2213 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.26 (s, 3H, CH₃), 1.43 (t, J = 6.9 Hz, 3H, CH₃), 1.46 (m, 1H, CH); 1.61–1.88 (m, 10H, 5 × CH₂), 2.05–2.15 (m, 1H, CH), 2.35–2.52 (m, 3H, CH₂ & CH), 2.68 (m, 1H, CH), 2.81–2.98 (m, 7H, 3 × CH₂ & CH), 3.15 (m, 2H, CH₂), 3.30–3.35 (m, 1H, CH), 3.68 (m, 1H, CH), 4.08 (q, J = 6.9 Hz, 2H, OCH₂), 6.68 (d, J = 2.1 Hz, 1H, Ar–H), 6.77 (dd, J = 2.4 & 8.4 Hz, 1H, Ar–H), 7.24–7.37 (m, 4H, Ar–H), 7.65 (d, J = 6.3 Hz, 1H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 15.3 (CH₃), 17.5 (CH₃), 24.6 (CH₂), 24.7 (CH₂), 26.9 (CH₂), 28.1 (CH₂), 29.7 (2 × CH₂), 30.0 (CH₂), 33.9 (2 × CH₂), 34.9 (CH₂), 38.0 (CH), 44.4 (CH), 47.2, 52.6 (2 × NCH₂), 57.1 (CH), 63.7 (OCH₂), 102.9, 112.4 (Ar–H), 115.0 (Ar–H), 118.3 (Ar–H), 126.5 (Ar–H), 126.6 (Ar–H), 127.6 (Ar–H), 127.9 (Ar–H), 128.4, 132.7, 134.7 (2 × Ar), 136.1, 137.8, 138.0, 139.9, 152.4, 157.3, 157.7; MS: m/z = 543.40 (M⁺ + H); _D[α]^24.4 = +20 (0.0007 g mL⁻¹, EtOAc); anal. calcd (C₃₈H₄₂N₂O): C, 84.09; H, 7.80; N, 5.16. Found: C, 83.97; H, 7.78; N, 5.14; HRMS (ESI): calc. for C₃₈H₄₃N₂O: 543.33699 (M⁺ + H); found: 543.33716.

(+)(6bS,8aS,17aS,17bS)-4-(Tetrahydro-2H-pyranoxy)-8a-methyl-10-(piperidin-1-yl)-1,2,6b,7,8,8b,11,12,17a,17-decahydro-17H-cyclopenta[a:c]bisphenanthrene-9-carbonitrile (12b). White solid with white florescence in long UV (365 nm); R_f 0.40 (6% EtOAc/hexane); yield 25%; mp 229–230 °C; IR (KBr): 2222 (CN) cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.25 (s, 3H, CH₃), 1.29–1.88 (m, 17H, CH & 8 × CH₂), 2.04–2.07 (m, 2H, CH₂), 2.36–2.52 (m, 3H, CH & CH₂), 2.69 (m, 1H, CH), 2.81–2.98 (m, 6H, 3 × CH₂), 3.19 (m, 2H, CH₂), 3.30–3.35 (m, 1H, CH), 3.61–3.64 (m, 2H, OCH₂), 3.96 (m, 1H, CH), 5.42 (s, 1H, CH–O), 6.84 (s, 1H, Ar–H), 6.90 (dd, J = 1.8 & 8.4 Hz, 1H, Ar–H), 7.24–7.35 (m, 4H, Ar–H), 7.64 (d, J = 6.6 Hz, 1H, Ar–H); ¹³C NMR (75 MHz, CDCl₃): δ 17.5 (CH₃), 19.2 (CH₂), 24.6 (CH₂), 24.7 (CH₂), 25.6 (2 × CH₂), 26.8 (CH₂), 28.1 (CH₂), 29.6 (CH₂), 30.0 (CH₂), 30.8 (2 × CH₂), 33.9 (CH₂), 34.9 (CH₂), 37.9 (CH), 44.4 (CH), 47.2, 52.6 (2 × NCH₂), 57.1 (CH), 62.3 (OCH₂), 96.8 (CH–O), 102.9, 114.5 (Ar–H), 116.9 (Ar–H), 118.3 (CN), 126.4 (Ar–H), 126.5 (Ar–H), 127.6 (Ar–H), 127.9 (Ar–H), 128.4 (Ar–H), 133.8, 134.7, 136.1, 137.8, 138.0, 139.9, 152.4, 155.4, 157.7; MS: m/z = 599.42 (M⁺ + H); _D[α]^24.4 = +40 (0.001 g mL⁻¹, EtOAc); anal. calcd (C₄₁H₄₆N₂O₂): C, 82.24; H, 7.74; N, 4.68. Found: C, 82.18; H, 7.71; N, 4.65; HRMS (ESI): calc. for C₄₁H₄₇N₂O₂: 599.36321 (M⁺ + H); found: 599.36363.

X-ray crystallography

Single crystals of 8a (C₃₀H₂₈NBr·4H₂O, M = 546.44) were obtained by slow evaporation from ethyl acetate. The compound crystallized in orthorhombic space group P2₁2₁2₁ with two independent molecules in the asymmetric unit along with co-crystallized solvent, water. The unit cell dimensions were determined to be a = 6.8589 (5) Å, b = 26.1040 (18) Å, c = 30.252 (2) Å, Z = 8, V = 5416.5 (7) ų, D_calc = 1.340 g cm⁻³, F(000) = 2256. X-ray diffraction data were collected on a Bruker AXS SMART APEX CCD diffractometer using MoK_α radiation (λ = 0.71073 Å). Data were acquired using ω scan mode at room temperature (293 K). The structure was solved by direct methods using SHELXS¹⁴ and was refined against F² with full-matrix least squares method by using SHELXL.¹⁵ Positional disorder was assigned for the methyl group of the OMe moiety of molecule-1 and for three of the four co-crystallized water molecules. The two positions of the disordered methyl group have an occupancy ratio of 0.66 : 0.34. The best refined model was obtained with three disordered solvent oxygen positions refined with major occupancy factors of 0.50, 0.54 and 0.63. Anisotropic refinement was performed on all the non-hydrogen atoms, except the disordered oxygen atoms of co-crystallized water. Hydrogen atoms were geometrically fixed and were refined as riding over the atoms to which they are bonded. Bond length constraint was applied to the O–C bond of the disordered methyl group. 40 547 reflections were measured (13 487 independent reflections) with R_int = 0.076. The final R-value was 0.0626 (wR = 0.1214) for 5461 observed reflections with [I > 2sigI] and for 659 parameters. The goodness-of-fit is 0.931. Flack parameter is refined as 0.003 (9). The largest difference peak and largest difference hole were found to be 0.57e Å⁻³ and −0.46e Å⁻³, respectively, both near Br atom positions (ESI†).

In vitro anticancer activity

Cell growth inhibition assay. In vitro anticancer activity of synthesized compounds was studied by Sulphorhodamine B (SRB) dye based plate assay. The stock solution of all the compounds was made at 20 mM concentration in DMSO. While incubating with cells in cell growth inhibition assay, working dilutions of 60 μM onwards (2 fold dilutions) were made in culture media (aqueous solution) and equal amount (100 μL) was added to existing culture media in each well. The plates were then checked microscopically with no apparent crystals in any well. Thus 8a and 8b were aqueous soluble at ≥60 μM which might go further high. In brief, 10⁴ cells per well were added in 96-well culture plates and incubated at 37 °C in 5% carbondioxide concentration. After overnight incubation of cells, serial dilutions of synthesized compound was added to the wells. Untreated cells served as control. After 48 h, cells were fixed with ice-cold tri-chloroacetic acid (50% w/v, 100 mL per well), stained with SRB (0.4% w/v in 1% acetic acid, 50 mL per well), washed and air-dried. Bound dye was solubilize with 10 mM Tris base (150 mL per well) and absorbance was read at 540 nm on a plate reader. The cytotoxic effect of compound was calculated as % inhibition in cell growth as per formula: [1 − (absorbance of drug treated cells/absorbance of untreated cells) × 100]. Determination of IC₅₀ (50% inhibitory concentration) was based on dose–response curves.

Acknowledgements

HKM acknowledged SERB, DST New Delhi, India for financial support as DST Fast Track Young Scientist Fellowship. Technical Support received from Mr Jaipal Kandhadi is duly acknowledged. Authors are thankful to Director CSIR-CIMAP, CSIR-CDRI, Lucknow and CSIR-IICT, Hyderabad.

References

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1046576. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra11112a

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