Synthesis and biological activity of new steroidal heterocyclic compounds

Introduction

It is known from the literature that both steroids and heterocyclic compounds have medicinal and biological activity. They are also used as drugs, antibacterial and antiviral agents.The presence of heterocycles in all kinds of organic compounds of interest in biology, pharmacology, optics, electronics and material sciences and so on is very well known². Among them sulfur and nitrogen-containing heterocyclic compounds have maintained the interest of researchers through decades of historical development of organic synthesis. The ground of this interest was their biological activity and unique structure that led to several applications in different areas of pharmaceutical and agrochemical research, ormore recently in material sciences³.

On the other hand steroids have great effects as anti-inflammatory, antibacterial and antiviral drugs⁴.

Nonsteroidal anti-inflammatory drugs (NSIDs) are an inhomogeneous family of pharmacologically active compounds which are widely used in treatment of acute and chronic inflammation, pain and fever, However, long-term clinical employment of NSIDs is associated with significant side effects. Therefore, the discovery of new safer drugs is a challenging goal for research⁵.

Attention has been devoted in the literature to synthesis of several steroidal heterocyclic derivatives that exhibit marked medicinal activity^6,7,8. In recent years, heterocyclic pregnane derivatives have been found to possess a variety of interesting pharmacological and biological activities⁹. Recently, reactivity of some steroidal hormones towards lawessons’s reagent as sulfur moiety produced acyclic and cyclic sulfur compounds containing a steroidal unit which have an antibacterial and antifungal activities¹⁰.

These observations led us to attempt to make new heterocyclic systems bearing steroidal moieties.

Results and Discussion

17a-Hydroxyandrost-4-en-3-one (I) was refluxed with hydrazinotriazine to afford 17a-hydroxyandrost-4-en-3-(5,6-diphenyl-1,2,4-triazin-3-hydrazone (II). The IR spectrum showed a signal at 3301 cm^-1 (NH) and the absence of the carbonyl group. The ¹H NMR spectrum of the product contained new signals at 7.4-7.8 (10H, m, phenyl protons), 10.75, (1H, brs, NH). The ¹³C NMR revealed a signal at d 157.65 which was confirmed the presence of (C=N) group. These results confirmed that the product was 17a-hydroxyandrost-4-en-3-(5,6-diphenyl-1,2,4-triazin-3-hydrazone(II). (Scheme 1)

Experimental

Melting points were determined by Thomas-Hoover capillary melting point apparatus and are uncorrected. IR spectra were recorded using KBr disks on a Nicolet Magna 520 Fourier transform spectrometer. ¹H NMR spectra were determined in deuteriochloroform with TMS as an internal standard reference at 600 MHz on a Brucker Avance DPX 600 spectrometer while ¹³C NMR spectra were recorded in deuteriochloroform at 150 MHz with a Brucker Avance DPX 400 spectrometer.

3b-Hydroxypregn-5-en-20-one (III) was refluxed with hydrazinotriazine to give 3b-hydroxypregn-5-en-20-(5,6-diphenyl-1,2,4-triazin-3-hydrazone (IV). The IR spectrum showed a signal at 3311 cm^-1 (NH) and the absence of the signal of carbonyl group. The 1H NMR spectrum of the product contained new signals at 7.4-7.8 (10H, m, phenyl protons) and 10.61,(1H, brs, NH). The ¹³C NMR revealed signal at d 165.6 which confirmed the presence of (C=N) group. These results confirmed that the product was 3b-hydroxypregn-5-en-20-(5,6-diphenyl-1,2,4-triazin-3-hydrazone (XII). (Scheme 2)

Mass spectra were recorded on a VG Autospec.

Micro-analysis were carried out using Perkin Elmar.

1.  17b-Hydroxyandrost-4-en-3-(5,6-diphenyl-1,2,4-triazin-3-hydrazone (II)

A mixture of 17b-hydroxyandrost-4-en-3-one (I) (0.01 mol),hydrazinotriazine (0.01mol) in absolute ethanol (50ml) with few drops oftriethyl amine was refluxed for 4 hrs, cooled and poured onto ice. The solid product was filtered off to give 17β-hydroxyandrost-4-en-3-(5,6-diphenyl-1,2,4-triazin-3-hydrazone (II) (1.2 g, 65% ) which was  recrystallized from ethyl acetate as yellow plates. m.p. 203-205°C HRMS found 533.3143 C34H39N5O, Calculated 533.3155 FTIR νmax/cm-1 3382 (OH), 3401 (NH) and 1616 (C=N) ¹H NMR (CDCl3, 600 MHz) δ 0.85 (3H, s, 18-H), 1.85 (3H, s, 19-H), 3.69 (1H, t, J= 8.4 Hz, 17a-H), 5.85 (1H, s, 4-H), 7.4-7.8 (10H, m, phenyl protons), 10.75,(1H, brs, NH).

2. 3b-Hydroxypregn-5-en-20-(5,6-diphenyl-1,2,4-triazin-3-hydrazone (IV)

A mixture of 3b-hydroxypregn-5-en-20-one (III) (0.01 mol), hydrazinotriazine (0.01mol) in absolute ethanol (50ml) with few drops of triethyl amine was refluxed for 4 hrs, cooled and poured onto ice. The solid product was filtered off to give 3β-hydroxypregn-5-en-20-(5,6-diphenyl-1,2,4- triazin-3-hydrazone (IV) (1.3 g, 73% ) which was recrystallized from ethyl acetate as yellow cubes.

m.p. 189-192°C HRMS found 561. 3460 C36H43N5O , Calculated 561.3468 FTIR νmax/cm-1 3402 (OH), 3311 (NH) and 1621 (C=N) ¹H NMR (CDCl3, 400 MHz) δ 1.06 (3H, s, 18-H), 1.10 (3H, s, 19-H), 3.64 (1H, tt, J= 8.4, 11.0 Hz, 3b-H), 5.77 (1H, s, 6-H) , 7.4-7.8 (10H, m, phenyl protons), 10.61 (1H, brs, NH).

13C NMR data determined in CDCl3 at 150 MHz of new compounds II and IV

Biological effects

Upon the irradiation of visible light withappropriate wavelength, the photosensitizer can derive molecular oxygen into excited triplet state, transferring energy into ground state molecular oxygen to produce singlet molecular oxygen. Activated singlet oxygen, or reactive oxygen species (ROS) in general, plays an important role in cytotoxic effects on affected tissues. A variety of attractive pharmacological effects were attributed to sulfur mono- and polycyclic heterocyclic systems. Thus we aimed to investigate the biological effects of the new prepared compounds towards some microorganisms and use as photochemical probe agents, as well as strong bacterial and fugicidal agents.

Table 1: The preliminary screening antimicrobial activity of some synthetic compounds

Compound	Gram+ve Bacteria			Gram-ve Bacteria			Fungi
No	B	S	E	P	K	C	A
XI	16	20	14	22	24	12	6
XII	15	20	14	20	24	11	6
Na.	32	30	30	12	22	6	6
Ny.	6	6	6	6	10	10	32

+ve Bacteria : B : Bacillus subtilis; S: Staphylococcus aureus

-ve Bacteria : E : Escherichia coli; P: Pesudomonas aeuruginosa. K: Klebsiella Pneumonia.

Fungi: C: Candida albicans (Aucc 1720), A : Aspergillus fumigatus (Aucc 1924).

Na: Nalidixic acid, 30 mg/disk, Bioanalize, Egypt.

Ny: Nystatin: Manufactured by Pasteur Lab. Egypt, NS 100 Units.

Table 2: MIC of the biological active compounds towards Gram -ve bacteria

Compound						Inhibition zone (nm)
No.			E. coli			P. aeuruginosa						K. pneumonia
	50	40	30	20	10	50	40	30	20	10	50	40	30	20	10
XI	14	10	10	8	6	22	18	14	8	6	24	18	14	8	6
XII	14	14	11	8	6	20	13	11	8	6	24	18	12	8	6

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