Access to steroidal pyridazines via modified thiohydrazides

Abstract

An approach to steroids annulated with pyridazines via cascade imination/electrocyclization of chlorovinyl aldehydes with oxamic acid thiohydrazides is disclosed. A mechanistic rationalization was performed using real-time ¹H NMR spectroscopy and computational studies. A series of 18-nor-5α-androsta-2,13-diene[3,2-d]pyridazines, androsta-2-ene[3,2-d]pyridazines and Δ^1,3,5(10)-estratrieno[16,17-d]pyridazines were synthesized from native hormones. These compounds were screened for cytotoxicity against the human estrogen-responsive breast cancer cell line MCF-7 and the estrogen-independent breast cancer cell line MDA-MB-231. The structure–activity relationship analysis revealed that the annulation of the pyridazine moiety to the A-ring of the 17β-hydroxy-5α-androsta-2-ene core provides high antiproliferative activity. Compounds 7a and 10b exhibited higher antiproliferative potency than the drug cisplatin. 5α-Androsta-2-ene[3,2-d]pyridazine 10c showed good selectivity against the MCF-7 breast cancer cells.

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Introduction

The development of methods for rapid access to N-heterocycles from simple and readily available starting compounds via the formation of several bonds as a one-pot reaction has become one of the main challenges in organic synthesis. In this regard, transformations of hydrazones have emerged as a powerful tool for the atom-economical chemoselective construction of (hetero)cyclic systems with complexity and diversity.¹ The synthetic utility of hydrazones extends far beyond the preparation of indoles via the classical Fischer reaction.² Hydrazones have found applications in the total synthesis of natural products as practical chiral auxiliaries³ and have been reported as useful ligands⁴ and catalysts for asymmetric reactions.⁵ The unique reactivity of N-tosylhydrazones⁶ as precursors of unstabilized diazo compounds enabled the synthesis of isoindolines,⁷ 1H-indazoles,⁸ azetidin-2-ones,⁹ pyrroles,¹⁰ pyrrolidines,¹¹ etc.;¹² N,N′-dialkylhydrazones^1b proved to be excellent Michael acceptors,¹³ good azomethine nitrogen atom nucleophiles¹⁴ and radical acceptors in cyclizations.¹⁵ Besides, α,β-unsaturated hydrazones bearing the 1-azadiene unit are employed in hetero-Diels–Alder reactions.¹⁶ Examples of the use of donor–acceptor substituted hydrazones as acyl anion equivalents that undergo heterocyclization with α,β-unsaturated aldehydes or keto esters were reported.^13c,d,17 Modified hydrazones with C=O, C=S, C=N and P=O functional groups are applied in the synthesis of diverse five- and six-membered O,N,S-heterocycles,¹⁸ including such widely used pharmacophores as 1,3,4-thiadiazoles¹⁹ and pyrazoles.²⁰ All this clearly attests to the power of hydrazone chemistry to reach into new areas of (hetero)cyclic chemical space, aromatic and saturated alike.

Recently, we have reported a new type of reactivity of α,β-unsaturated hydrazones of thiohydrazides as 2,3-diazahexatriene synthons (Scheme 1).²¹ We elaborated a two-step procedure for the synthesis of highly substituted pyridazines employing (1) the Vilsmeier–Haack reaction of enolizable ketones giving chlorovinyl aldehydes and (2) imination of the former with oxamic acid thiohydrazides followed by cascade electrocyclization/aromatization of the resulting 2,3-diazahexatrienes. The aim of this work was to develop an efficient method for transformations of complex natural products in order to modulate the biological activity of substrates.

Scheme 1 Transformation of ketones into pyridazines via α,β-unsaturated hydrazones of thiohydrazides.

In this respect, we sought to steroids comprising one of the largest and most diverse classes of natural products. Native hormones are versatile modulators that regulate most of the key functions in the mammalian body. Their A- and D-ring modified heterocyclic derivatives, over the years, have been considered to be a privileged scaffold for the drug discovery, including the design of anticancer agents.²² Synthetic azasteroids encompass a wide range of compounds with antiproliferative activity, e.g., reductase inhibitors such as finasteride,²³ high-affinity agonist ligands for the glucocorticoid receptor, e.g., cortivazol,²⁴ GnRH agonists such as danazol,²⁵ aromatase inhibitors such as formestane and exemestane²⁶ (Fig. 1). Herein, we report the synthesis of unique derivatives of the androstene and estrane series containing pyridazine motifs annulated to the A- and D-rings of the steroid core and the evaluation of their inhibitory activity against breast cancer cells.

Fig. 1 Synthetic heterosteroid drugs.

Results and discussion

Chemistry

Based on our previous work, we initially prepared 3-chloro-2-formylandrostane 2 from 3-keto-17α-methyl-17β-hydroxyandrostane (1) by the treatment with the Vilsmeier–Haack reagent under standard conditions (Scheme 2). The chlorovinylation was accompanied by the Wagner–Meerwein rearrangement resulted in the elimination of 17-OH group and migration of 18-Me group to C-17 position. Meanwhile, the subsequent reactions of 3-chloro-2-formyl-17,17-dimethyl-18-norandrostane-2,13-diene (2) with thiohydrazides 3a–c containing both electron-donating and withdrawing substituents on the benzoyl group proceeded smoothly. The reflux for 2 h in ethanol in the presence of a catalytic amount of TsOH afforded A-ring modified 18-nor-5α-androsta-2,13-diene[3,2-d]pyridazines 4a–c in 51–82% yields.

Scheme 2 Synthesis of 18-nor-5α-androsta-2,13-diene[3,2-d]pyridazines 4.

In order to avoid side rearrangements and ensure an efficient synthesis process, we decided to use 3-keto-17β-hydroxyandrostane 5. The reaction of ketone 5 with the Vilsmeier–Haack reagent during prolonged storage at rt produced 3-chloro-2-formylandrostane 6 in 61% yield (Scheme 3). In this case, the chlorovinylation was followed by esterification of 17-OH group. Fortunately, the desired heterocyclization of 6 with oxamic acid thiohydrazides 3a, 3b was accompanied by the release of 17-OH group, giving appropriate 17β-hydroxy-5α-androsta-2-ene[3,2-d]pyridazines 7a, 7b. The reactions were completed in 3 h in ethanol under reflux with TsOH (10 mol%) resulting in the formation of 7a, 7b in 73% and 67% yields.

Scheme 3 Synthesis of 5α-androsta-2-ene[3,2-d]pyridazines 7.

Then we made an attempt to generate D-ring modified androstene derivatives from 17-chloro-16-formyl steroid 9 derived from 3β-acetoxyandrostene 8 (Table 1). Having so far observed a seemingly general transformation pattern, we found that the reaction of chlorovinyl aldehyde 9 with thiohydrazides 3 affords androst-5-ene[16,17-d]pyridazines 10a–f in high yields. Acid thiohydrazides 3a–f containing electron-donating and-withdrawing groups were tested. The reaction showed high chemoselectivity and functional group tolerance. It is remarkable that the acetyl protecting group could be preserved or removed depending on pH of the reaction mixture in the heterocyclization step. Once TsOH was washed out after the intermediate formation of hydrazone, 3β-acetoxy-androst-5-ene[16,17-d]pyridazines 10a–c were isolated as the only products (Table 1, procedure A). Meanwhile, the reaction was accompanied by the deprotection of the 3-hydroxy group producing 10a′,10b′,10e′,10f′ under reflux in the presence of TsOH (Table 1, procedure B).

Table 1 Synthesis of androsteno[16,17-d]pyridazines 10^ab

a Reaction conditions. Procedure A: androsta-5,16-diene 9 (0.1 mmol), oxamic acid thiohydrazide 3 (0.1 mmol), TsOH·H₂O (10 mol%) and ethanol (30 mL) were stored at rt for 30 min. TsOH was washed out and the reaction mixture was refluxed for 1 h. Procedure B: androsta-5,16-diene 9 (0.1 mmol), oxamic acid thiohydrazide 3 (0.1 mmol), TsOH·H₂O (10 mol%) and ethanol (30 mL) were refluxed for 3–5 h.b Isolated yield.

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Since 3-oxo-Δ⁴-azasteroids are well-known as 5-α-reductase inhibitors,²⁷ the Oppenauer oxidation was used for compounds 10a′ and 10f′ producing Δ⁴-3-oxo[17,16-d]pyridazines 11a, 11f in good yields (Scheme 4).

Scheme 4 Oppenauer oxidation of compounds 10.

We next examined the scope of the method employing 17-chloro-16-formyl-Δ^1,3,5(10),16-estratetraenol 13 – chlorovinylation product of estrone (12). The reaction shown in Scheme 5 resulted in the formation of the Δ^1,3,5(10)-estratrieno[16,17-d]pyridazines 14a, 14b, 14g in 79–91% yields. The reactions were accomplished under standard conditions by heating under reflux for 2 h in ethanol in the presence of 10 mol% of TsOH.

Scheme 5 Synthesis of pyridazines 14.

It should be noted that the structural assignments for all compounds 4, 7, 10 and 14 were confirmed by 2D NMR (¹H–¹H COSY, ¹H–¹H TOCSY, ¹H–¹H ROESY, ¹³C–¹H HMBC, and ¹³C–¹H HSQC) techniques and HRMS.

The possible mechanism for the formation of steroidal pyridazines by the reaction of chlorovinyl aldehydes with oxamic acid thiohydrazides is shown in Scheme 6 illustrating an example of product 10a. Relying on the literature,²⁸ the mechanism involves disrotatory 6π-electrocyclization of highly reactive Z-15-thiol isomer into dihydropyridine 16 followed by aromatization with elimination of hydrochloric acid and elemental sulfur.²⁹ The evaluated activation barriers for electrocyclization step are in the range of 2.5–5.7 kcal mol⁻¹ for the products obtained (ΔH_r = 3.3–7.3 kcal mol⁻¹, PM6, see ESI†).

Scheme 6 Proposed mechanistic pathway.

In order to elucidate the pattern of the critical build up of Z-15-thiol, we isolated intermediate hydrazone 15 in 91% yield after short-time storage of a mixture of aldehyde 9 and thiohydrazide 3a at rt in the presence of TsOH (10 mol%). In relatively non-polar CDCl₃, as well in the solid state,³⁰ hydrazone 15 was found to exist as the most stable E-15-thione, resulting in that no isomerization or formation of product 10a occur even after 100 h of storage at rt (for detailed spectra see ESI†). However, the solution-state structure of compound 15 in polar DMSO-d₆ immediately after dissolution corresponded to a mixture of E-15-thione and 15-thiadiazoline isomers in a 1.3 : 1.0 ratio. The minor isomer Z-15-thione was additionally detected (in DMSO-d₆) using real-time monitoring of the heterocyclization step at rt by ¹H NMR spectroscopy in an NMR tube. The observed solvent effect was attributed to the nucleophilic attack of the S-nucleophile at the C=N bond of E-15-thione giving 15-thiadiazoline, which is enhanced in polar solvents. In that context, the pathway of the formation of Z-15-thione isomer through 15-thiadiazoline via the C–S bond cleavage, at least at rt in the absence of an acidic catalyst, seems to play a significant role. The subsequent thione–thiol tautomerism results in the conversion of Z-15-thione into Z-15-thiol,³¹ which was not detected by NMR spectroscopy as a short-lived isomer involved in the fast isomerization and cyclization. This suggestion is supported by the simultaneous occurrence of both Z-15-thione and product 10a in the reaction mixture.

Antitumor evaluation

Cytotoxic effects against breast cancer cells. The antiproliferative activity of some synthesized compounds was evaluated against the human estrogen-responsive breast cancer cell line MCF-7, as well as against the estrogen-independent breast cancer cell line MDA-MB-231, using the MTT assay. Cisplatin, a chemotherapy drug, was used as the reference compound. The corresponding inhibitory concentrations IC₅₀ (IC₅₀ is the half maximal inhibitory concentration) are enlisted in Table 2.

Table 2 Antiproliferative data for the synthesized steroidal pyridazines and the reference drug (cisplatin) against the human breast cancer cells (MCF-7 and MDA-MB-231)

Entry	Comp.		R^1	R^2	IC50, μM
					MCF-7	MDA-MB-231
1	4a		p-OMe	—	12.4 ± 1.1	NA
2	4b		p-Cl	—	15.8 ± 1.2	20.3 ± 1.3
3	4c		p-CF3	—	9.5 ± 0.7	20.6 ± 1.0
4	7a		p-OMe	—	3.9 ± 0.7	7.7 ± 0.9
5	7b		p-Cl	—	7.6 ± 0.9	12.8 ± 0.7
6	10a		p-OMe	Ac	NA	12.5 ± 0.7
7	10b		p-Cl	Ac	5.1 ± 0.7	6.9 ± 0.8
8	10c		p-CF3	Ac	4.9 ± 0.8	NA
9	10d		H	Ac	6.5 ± 0.9	7.9 ± 0.7
10	10a′		p-OMe	H	NA	NA
11	10e′		m-OMe	H	NA	NA
12	10f′		p-F	H	NA	NA
13	11a		p-OMe	—	NA	NA
14	11f		p-F	—	NA	NA
15	14e		m-OMe	—	16.5 ± 0.9	NA
16		Cisplatin			6.2 ± 0.9	13.1 ± 1.1

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For a SAR evaluation, the activity of the series of the newly synthesized compounds 4, 7, 10, 11 and 14 were evaluated against MCF-7 and MDA-MB-231 in order to investigate the effects of the pyridazine annulation to A/D-rings of the steroidal core and the halogen and methoxy substituents on the benzoyl moiety. The IC₅₀ values of these compounds were determined as a measure of their relative cytotoxicity. Initially, A-ring modified androstane[3,2-d]pyridazines 4, 7 showed higher cytotoxic activity against both the MCF-7 and MDA-MB-231 cell lines as compared to D-ring annulated androstene[16,17-d]pyridazines and Δ^1,3,5(10)-estratrieno[16,17-d]pyridazines 10, 11 and 14.

The estrogen-responsive breast cancer cell line MCF-7 was more sensitive to series of compounds 4 and 7. Among these series, 19-norandrostene 4b with R¹ = Cl showed weak activity compared to the reference drug (Table 2, entry 2). However, after the replacement of chlorine with the methoxy substituent on the benzoyl moiety and of the 17,17′-dimethyl substituent with the hydroxyl group on the 18-methyl-17β-hydroxy moiety resulting in the formation of compound 7a, the latter showed the highest antiproliferative potency with IC₅₀ of 3.9 ± 0.7 μM against the MCF-7 cell line, the IC₅₀ value of compound 7a being 1.6 times smaller compared to cisplatin. Compounds 10b and 10c of the androstene[16,17-d]pyridazine series bearing a Cl and CF₃ substituent on the benzoyl group exhibited higher antiproliferative activity (IC₅₀ are 5.1 ± 0.7 μM and 4.9 ± 0.8 μM, respectively; see Table 2, entries 7 and 8) compared to the reference compound. Compound 10d containing the unsubstituted benzoyl moiety showed antiproliferative potency comparable to the drug cisplatin with the IC₅₀ value of 6.5 ± 0.9 μM (Table 2, entry 8). It is remarkable that the structurally similar compounds 10a′, 10e′, 10f′ possessing a free hydroxyl group (Table 2, entries 10–12), as well as Δ⁴-3-oxo[17,16-d]pyridazines 11a, 11f (Table 2, entries 13, 14), were completely ineffective. Compound 14e from the Δ^1,3,5(10)-estratrieno[16,17-d]pyridazine series was found to be weakly active towards MCF-7 cells with IC₅₀ 16.5 ± 0.9 μM (Table 2, entry 15).

The hormone-independent MDA-MB-231 cells were less sensitive to series of the newly synthesized compounds. Among the synthesized compounds, only seven chemicals showed toxicity against the MDA-MB-231 cells, whereas the other eight were not active. Chlorine-substituted androstene[16,17-d]pyridazine 10b proved to be the most active compound with IC₅₀ (6.9 ± 0.8 μM, Table 2, entry 7) lower than that of cisplatin. However, A-ring modified androstan[3,2-d]pyridazine 7a with the methoxy substituent also exhibited higher antiproliferative activity compared to cisplatin (IC₅₀ is 7.7 ± 0.9 μM, Table 2, entry 4).

It is noteworthy that compound 10a inhibited MDA-MB-231 cell growth, but was not active in MCF-7. On the contrary, compound 10c exhibited high activity in hormone-responsive MCF-7 and showed no effects in MDA-MB-231. Δ^1,3,5(10)-Estratrieno[16,17-d]pyridazine 14e and 19-norandrostene 4a showing activities against MCF-7 did not exhibit any cytotoxic activity against the MDA-MB-231 cells.

Conclusions

To summarize, new types of heterosteroids, steroidal pyridazines, were prepared by the 6π-electrocyclization of highly reactive thiohydrazones of oxamic acid thiohydrazides. Native hormones 1, 5, 8 and 11 were transformed into the corresponding A- and D-annulated products in good/high yields (51–92%) using a two-step sequence involving the Vilsmeier–Haack reaction and imination with oxamic acid thiohydrazides. The mechanistic rationalization of the thiohydrazone isomerization equilibrium revealed the thiadiazoline form as an important intermediate. The evaluation of cytotoxic activity of the newly synthesized compounds against the breast cancer cell lines MCF-7 and MDA-MB-231 showed that compounds 7a and 10b, 10c, 10d displayed higher cytotoxicity than the reference drug. The IC₅₀ values of compound 7a being 3.9 and 7.7 μM, which is about 1.6 times lower than that of the control cisplatin. Furthermore, the analysis of the structure–activity relationship revealed that the A-ring annulation is favorable for high cytotoxicity. Finally, this work provides evidence that steroidal pyridazines are promising lead compounds for developing novel and highly effective anticancer drugs.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and characterization data for new compounds. For ESI see DOI: 10.1039/c6ra06881b

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