Synthesis and Evaluation of in Vitro Biological Activity of 4-Substituted Arylpiperazine Derivatives of 1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione

Kossakowski, Jerzy; Pakosinska-Parys, Magdalena; Struga, Marta; Dybala, Izabela; Koziol, Anna E.; La Colla, Paolo; Marongiu, Laura Ester; Ibba, Cristina; Collu, David; Loddo, Roberta

doi:10.3390/molecules14125189

Open AccessArticle

Synthesis and Evaluation of in Vitro Biological Activity of 4-Substituted Arylpiperazine Derivatives of 1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione

by

Jerzy Kossakowski

¹,

Magdalena Pakosinska-Parys

¹,

Marta Struga

^1,*,

Izabela Dybala

²,

Anna E. Koziol

²,

Paolo La Colla

^3,*,

Laura Ester Marongiu

³,

Cristina Ibba

³,

David Collu

³ and

Roberta Loddo

³

¹

Department of Medical Chemistry, Medical University, 3 Oczki Str., 02-007 Warsaw, Poland

²

Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland

³

Department of Biomedical Science and Technology, University of Cagliari, Cittadella Universitaria, 09042, Monserrato, Cagliari, Italy

^*

Authors to whom correspondence should be addressed.

Molecules 2009, 14(12), 5189-5202; https://doi.org/10.3390/molecules14125189

Submission received: 5 November 2009 / Revised: 26 November 2009 / Accepted: 1 December 2009 / Published: 11 December 2009

Download

Browse Figures

Versions Notes

Abstract

:

A series of twenty arylpiperazine derivatives of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione have been prepared. These derivatives were tested in vitro with the aim of identifying novel lead compounds active against emergent and re-emergent human and cattle infectious diseases (AIDS, hepatitis B and C, tuberculosis, bovine viral diarrhea). In particular, these compounds were evaluated in vitro against representatives of different virus classes, such as a HIV-1 (Retrovirus), a HBV (Hepadnavirus) and the single-stranded RNA⁺ viruses Yellow fever virus (YFV) and Bovine viral diarrhea virus (BVDV), both belonging to the Flaviridae. Compounds 2c, 2g and 3d showed a modest activity against CVB-2. The molecular structures of the starting imide 1 and one of propyl-piperazine derivatives, 3b, have been determined by an X-ray crystallography study.

Keywords:

1,7,8,9-tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione; arylpiperazine derivatives; X-ray crystal structure analysis; antiviral activity

Graphical Abstract

Introduction

It is widely recognized that the utility of most antiviral agent is limited by the inherent toxicity of the compounds and the appearance of drug resistant virus following their continued use. As such, there is great interest in the identification of novel antiviral agents and lead compounds from which new antivirals might be synthesized. Viruses belonging to the Flaviviridae family have a single-stranded, positive-sense RNA genome (ssRNA⁺) [1]. Members of the Flaviviridae family can be classified into three genera: Hepaciviruses, Flaviviruses and Pestiviruses [2,3,4].

Some members of the Flaviviridae family can infect only animals, causing severe diseases usually followed by death [eg. bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV) and border disease virus (BDV)] [5]. In humans infections with Flaviviridae viruses may cause fulminant, hemorrhagic diseases [Yellow fever virus (YFV), dengue fever virus (DENV) and Omsk hemorrhagic fever virus (OHFV)], viral encephalitis [Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), St. Luis encephatitis virus (SLEV)] or hepatitis, formerly referred to as non-A, non-B hepatitis [hepatitis C virus (HCV)] [4,5,6]. Globally HCV is a major cause of human hepatitis [7,8]. Most infections become persistent, and about 60% of cases progress toward chronic liver disease, which in turn, can lead to development of cirrhosis, hepatocellular carcinoma, and liver failure [9,10]. There is a vaccine against the various Flaviviridae members with the exception of YFV; new therapies and preventative agents are strongly needed.

Other important ssRNA⁺ viruses are those belonging to the Picornaviridae family. This family includes nine genera, some of which comprise major human pathogens, namely, Enterovirus (including Poliovirus, Coxsackievirus, Echovirus), Rhinovirus (approximately 105 serotypes), and Hepatovirus [Hepatitis A virus (HAV)]. The range of clinical syndromes associated with enteroviruses is legion and includes acute paralysis, aseptic meningitis, encephalitis, carditis, pleurodynia, conjunctivitis, hand food and mouth disease and other enanthems [11]. While many of these diseases are relatively benign, others, such as encephalitis and carditis, may be fatal. A fascinating aspect of these clinical syndromes is that different enteroviruses may be associated with the same clinical syndrome. Conversely, the same enterovirus may cause different clinical syndromes. Moreover, these associations are not always predictable: A given virus may behave differently, both clinically and epidemiologically, in different places at different times [12]. At present, no specific antiviral therapy is available for the treatment of Picornaviridae infections.

Some arylpiperazine derivatives possess antienteroviral activity [13,14]. Piperazine fragment there are in anti-HIV agents bis(heteroaryl)piperazines (BHAPs), such as delavirdine and atevirdine [15]. Another arylpiperazine, vicriviroc, is currently under II phase of clinical investigation [16]. Inspired by these reports, we have recently synthesized and tested in vitro a series of arylpiperazine derivatives of a tricyclic imide for cytotoxicity and antiviral activity against representative viruses assays. The title compounds were also tested against representatives of other virus families. AZT (3’-azidothymidine), NM 108 (2’-β-methylguanosine), NM 176 (2’-ethynyl-D-cytidine), M 5255 (mycophenolic acid) and ACG (acycloguanosine) were used as reference inhibitors of ssRNA+, ssRNA- and DNA viruses, respectively.

Results and Discussion

The preparation of the compounds was accomplished according to the reactions described in Scheme 1. The starting compound, 1,7,8,9-tetrachloro-10,10-dimethoxy-4-azatricyclo [5.2.1.0^2,6]dec-8-ene-3,5-dione (1), was synthesized in a Diels-Alder reaction of 5,5-dimetoxy-1,2,3,4-tetracyclo-pentadiene with 1H-pyrrole-2,5-dione. 1,7,8,9-Tetrachloro-4-(4-chlorobutyl)- (2) and 1,7,8,9-tetrachloro-4-(3-chloropropyl)-10,10-dimethoxy-4-aza-tricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione (3) have been prepared by the alkylation of imide 1 with 1,4-dibromobutane and 1-bromo-3-chloropropane, respectively. The alkylated derivatives were further substituted in acetonitrile with 4-aryl- and 4-heteroaryl piperazines in the presence of potassium carbonate and potassium iodide. For biological assays, the obtained arylpiperazine derivatives were converted into the corresponding hydrochloride salts in anhydrous methanol saturated with gaseous HCl to yield compounds 2a–2j and 3a–3j (Scheme 1). The new compounds were identified and their structure has been proven by ¹H-NMR and elemental analyses (Table 1).

The molecular structure of starting imide 1 and one of propylpiperazine derivatives, 3b, has also been determined by an X-ray crystallography study (Table 2). The configuration of the cycloalkene skeleton of the imide 1 is endo, cis (Figure 1). This compound crystallizes with two symmetrically independent molecules, A and B, both adopting similar orientations of the –OCH₃ groups in the solid state. The alternating molecules A and B are linked through four intermolecular N-H...O hydrogen bonds forming centrosymmetric tetramer. This type of molecular association excludes imide groups from further hydrophilic contacts.

The N-propyl-piperazine derivative 3b adopts an extended conformation. In its protonated form it exists as a cation/anion pair (Figure 1); the observed geometry of the N⁺-H...Cl^– hydrogen bond is: N⁺...Cl^– distance of 3.032(6) Å and ∠N⁺-H...Cl^– angle of 175°.

The synthesized arylpiperazine derivatives were evaluated in vitro in parallel cell-based assays for cytotoxicity and antiviral activity (Table 3) against viruses representative of two of the three genera of the Flaviviridae family, i.e., Flaviviruses (YFV) and Pestiviruses (BVDV). The title compounds were also tested against representatives of other virus families. Among ssRNA+ were a Retrovirus (Human Immunodeficiency Virus type 1, HIV-1), two Picornaviruses (Coxsackie Virus type B2, CVB-2, and Poliovirus type-1, Sabin strain, Sb-1); among ssRNA- were a Paramyxoviridae (a Rhabdoviridae, Vesicular Stomatitis Virus, VSV) representative. Among double-stranded RNA (dsRNA) viruses was a Reoviridae representative (Respiratory Enteric Orphan Virus type-1, Reo-1). Two representatives of DNA virus families were also included: Herpes Symplex type 1, HSV-1 (Herpesviridae) and Vaccinia Virus, VV (Poxviridae). Although compound 1 was characterized by high cytotoxicity (Table 3), its derivatives possessed considerably low cytotoxicity. None of the compounds showed any activities against all viruses tested, with the exception of compounds 2c, 2g and 3d, that showed a modest activity against CVB-2-mediated diseases and potentiality as therapeutic targets (EC₅₀ range = 10–17 μM) (Table 3).

Experimental

General

Melting points were determined in a Kofler’s apparatus and are uncorrected. The ¹H NMR spectra were recorded on a Bruker AVANCE DMX 400 spectrometer, operating at 400 MHz. The chemical shift values are expressed in ppm relative to TMS as an internal standard. Elemental analyses were recorded with a CHN 2400 Perkin-Elmer model. Flash chromatography was performed on Merck silica gel 60 (200–400 mesh) using chloroform/methanol (19:1 vol.) mixture as eluent. Analytical TLC was carried out on silica gel F₂₅₄ (Merck) plates (0.25 mm thickness).

1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione (1) was synthesized as previously described [17].

1,7,8,9-Tetrachloro-4-(4-bromo-butyl)-10,10-dimethoxy-4-aza-tricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione (2). A mixture of imide 1 (0.5 g, 0.00138 mol), dibromobutane (0.9 g, 0.0027 mol) and anhydrous K₂CO₃ (0.5 g, 0.0036 mol) in acetonitrile (50 mL) was refluxed for 8 h. The inorganic precipitate was filtered off and the solvent was evaporated. For characterization data see Table 1.

1,7,8,9-Tetrachloro-4-(3-chloro-propyl)-10,10-dimethoxy-4-aza-tricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione (3). A mixture of imide 1 (0.5 g, 0.00138 mol), 1-bromo-3-chloropropane (0.6 g, 0.00415 mol) and anhydrous K₂CO₃ (0.5 g, 0.0036 mol) in acetonitrile (50 mL) was refluxed for 8 h. The inorganic precipitate was filtered off, the solvent was evaporated. For characterization data see Table 1.

General procedure for the preparation of arylpiperazine derivatives 2a–2j and 3a–3j

A mixture of compound 2 (0.3 g; 0.0006 mole) or 3 (0.3 g; 0.0007 mole), an appropriate amine (0.00137 or 0.0012 mole), anhydrous K₂CO₃ (0.3 g, 0.0022 mole) and KI (0.2 g, 0.0012 mole) was dissolved in acetonitrile (50 mL) and refluxed for 30 h. The solvent was evaporated, then the residue was purified by column chromatography (eluent: CH₂Cl₂-CH₃OH, 95:5) to give compounds 2a–2j and 3a–3j, respectively.

General procedure for the preparation of hydrochloride salts of compounds 2a–2j and 3a–3j

The solid product was dissolved in methanol saturated with gaseous HCl. The hydrochloride was precipitated by addition of diethyl ether. The crude product was crystallized from methanol/ethyl ether. Characterization data is given in Table 1.

X-Ray crystal structure analysis of 1 and 3b diethyl ether hemisolvate

Measurements were carried out at 292 K with a KM4 diffractometer using graphite monochromated CuK_α radiation (λ = 1.54178 Å) and ω/2θ scan mode. Intensities were corrected for absorption. Structure was solved by the SHELXS-97 program and refined by full-matrix least-squares on F² using the SHELXL-97 program [18]. The non-H atoms were refined anisotropically. The H-atom positions were calculated from geometry and the ‘riding’ model for the C–H bonds was used in the refinement. The N-bonded H-atoms were located in the difference maps. Crystal data are listed in Table 2. The experimental details and final atomic parameters for 1 and 3b have been deposited with the Cambridge Crystallographic Data Centre as supplementary material. Copies of the data can be obtained free of charge on application to The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-1223-336-033; E-Mail: [email protected] or www: http://www.ccdc.cam.ac.uk).

Cell-based assays

Compounds were dissolved in DMSO at 100 mM and then diluted in culture medium.

Cell lines were purchased from American Type Culture Collection (ATCC). The absence of mycoplasma contamination was checked periodically by the Hoechst staining method. Cell lines supporting the multiplication of RNA viruses were the following: CD4⁺ human T-cells containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK); Baby Hamster Kidney (BHK-21) and Monkey kidney (Vero 76) cells.

Cytotoxicity Assays

For cytotoxicity tests, run in parallel with antiviral assays, MDBK, BHK and Vero 76 cells were resuspended in 96 multiwell plates at an initial density of 6 × 10⁵, 1 × 10⁶ and 5 × 10⁵ cells/mL, respectively, in maintenance medium, without or with serial dilutions of test compounds. Cell viability was determined after 48–120 hrs at 37 °C in a humidified CO₂ (5%) atmosphere by the MTT method. The cell number of Vero 76 monolayers was determined by staining with the crystal violet dye.

For cytotoxicity evaluations, exponentially growing cells derived from human haematological tumors [CD4⁺ human T-cells containing an integrated HTLV-1 genome (MT-4)] were seeded at an initial density of 1 × 10⁵ cells/mL in 96 well plates in RPMI-1640 medium, supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin G and 100 µg/mL streptomycin. Cell cultures were then incubated at 37 °C in a humidified, 5% CO₂ atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability was determined after 96 hrs at 37 °C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method [18].

Antiviral assays

Activity of compounds against Human Immunodeficiency virus type-1 (HIV-1) was based on inhibition of virus-induced cytopathogenicity in MT-4 cells acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly, RPMI (50 μL) containing 1 × 10⁴ MT-4 were added to each well of flat-bottom microtitre trays containing RPMI (50 μL), without or with serial dilutions of test compounds. Then, an HIV-1 suspension (20 μL) containing 100 CCID₅₀ were added. After a 4-day incubation, cell viability was determined by the MTT method.

Activity of compounds against Yellow fever virus (YFV) and Reo virus type-1 (Reo-1) was based on inhibition of virus-induced cytopathogenicity in acutely infected BHK-21 cells. Activities against Bovine viral diarrhoea virus (BVDV), in infected MDBK cells, were also based on inhibition of virus-induced cytopathogenicity.

BHK and MDBK cells were seeded in 96-well plates at a density of 5 × 10⁴ and 3 × 10⁴ cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in growth medium at 37 °C in a humidified CO₂ (5%) atmosphere. Cell monolayers were then infected with 50 µL of a proper virus dilution (in serum-free medium) to give an m.o.i = 0.01. One hr later, MEM Earle’s medium (50 µL), supplemented with inactivated foetal calf serum (FCS), 1% final concentration, without or with serial dilutions of test compounds, were added. After 3-4 days incubation at 37 °C, cell viability was determined by the MTT method.

Activity of compounds against Coxsackie virus, B-2 strain (CVB-2), Polio virus type-1 (Polio-1), Sabin strain, Vesicular Stomatitis Virus (VSV), Vaccinia Virus (VV) and Herpes Virus 1 (HSV-1), in infected Vero 76 cells, was determined by plaque reduction assays in Vero 76 cell monolayers. To this end, Vero 76 cells were seeded in 24-well plates at a density of 2 × 10⁵ cells/well and were allowed to form confluent monolayers by incubating overnight in growth medium at 37 °C in a humidified CO₂ (5%) atmosphere. Then, monolayers were infected with appropriate virus dilutions (250 µL) to give 50−100 PFU/well. Following removal of unadsorbed virus, Dulbecco’s modified Eagle’s medium (500 µL), supplemented with 1% inactivated FCS and 0.75% methyl cellulose, without or with serial dilutions of test compounds, were added. Cultures were incubated at 37 °C for 2 (Sb-1 and VSV) or 3 (CVB-2, VV and HSV-1) and then fixed with PBS containing 50% ethanol and 0.8% crystal violet, washed and air-dried. Plaques were then counted. 50% effective concentrations (EC₅₀) were calculated by linear regression technique.

AZT (3’-azido-thymidine), NM 108 (2’-ß-methyl-guanosine), NM 176 (2’-ethynyl-D-citidine), M 5255 (Mycophenolic Acid) and ACG (acycloGuanosine) were used as reference inhibitors of ssRNA+, ssRNA- and DNA viruses, respectively.

Conclusions

A series of twenty arylpiperazine derivatives of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione have been prepared. These derivatives were tested in vitro with the aim of identifying novel lead compounds active against emergent and re-emergent human and cattle viral infectious diseases. Compounds 2c, 2g and 3d showed activity against CVB-2. Molecular structure of starting imide 1 and one of the propyl-piperazine derivatives, 3b, have been determined by an X-ray crystallography study.

References

Tan, S.L.; Pause, A.; Shi, Y.; Sonenberg, N. Hepatitis C therapeutics: Current status and emerging strategies. Nat. Rev. Drug. Discov. 2002, 1, 867–881. [Google Scholar] [CrossRef] [PubMed]
Westaway, E.G. Flavivirus replication strategy. Adv. Virus Res. 1987, 33, 45–90. [Google Scholar] [PubMed]
Chambers, T.J.; Hahn, C.S.; Galler, R.; Rice, M.C. Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 1990, 44, 649–688. [Google Scholar] [CrossRef] [PubMed]
Monath, T.P.; Heinz, F.X. Flaviviruses. In Fields Virology, 3rd ed.; Lippincott-Raven Publishers: Philadelphia, PA, USA, 1996; Vol. 1, pp. 961–1034. [Google Scholar]
Nettelton, P.E.; Entrican, G. Ruminant pestiviruses: A review. Br. Vet. J. 1995, 151, 615–642. [Google Scholar] [CrossRef]
Rice, M.C. Flaviviridae: The Viruses and Their Replication. In Fields Virology, 3rd ed.; Lippincott-Raven Publishers: Philadelphia, PA, USA, 1996; Vol. 1, pp. 931–960. [Google Scholar]
Wang, M.; Ng, K.K.S.; Cherney, M.M.; Chan, L.; Yannopoulos, C.G.; Bedard, J.; Morin, N.; Nguen-Ba, N.; Alaoui-Ismaili, M.H.; Bethell, R.C.; James, M.N.C. Non-nucleoside analogue inhibitors bind to an allosteric site on HCV NS5B polymerase. J. Biol. Chem. 2003, 278, 9489–9495. [Google Scholar] [CrossRef] [PubMed]
Hayashi, P.H.; Di Bisceglie, A.M. The progression of hepatitis B- and C-infections to chronic liver disease and hepatocellular carcinoma: Epidemiology and pathogenesis. Med. Clin. North. Am. 2005, 89, 371–389. [Google Scholar] [CrossRef] [PubMed]
Echevarria-Mayo, J.M. Etiology and pathogenesis of viral hepatitis. Enferm. Infecc. Microbiol. Clin. 2006, 24, 45–56. [Google Scholar] [PubMed]
Bosch, F.X.; Ribes, J.; Cleries, R.; Diaz, M. Epidemiology of hepatocellular carcinoma. Clin. Liver Dis. 2005, 9, 191–211. [Google Scholar] [CrossRef] [PubMed]
Strauss, J.H.; Strauss, E.G. Viruses and Human Disease; Academic Press. A Division of Harcourt, Inc.: London, UK, 2002. [Google Scholar]
Wong, K.T. Enterovirus-associated neurological disease with special reference to enterovirus 71. Neurol. J. Southest Asia 2000, 5, 47–49. [Google Scholar]
Chern, I.H.; Shia, K.S.; Shih, S.R.; Hsu, T.A.; Tai, C.L. Anti-enterovirus compounds. US Pat. 6815444, 2004. [Google Scholar]
Chern, J.H.; Shia, K.S.; Hsu, T.A.; Tai, C.L.; Chung-chi, L.; Lee, C.C.; Lee, Y.C.; Chang, C.S.; Tseng, S.N.; Shih, S.R. Design, synthesis, and structure-activity relationships of pyrazolo[3,4-d]pyrimidines: A novel class of potent enterovirus inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 2519–2525. [Google Scholar] [CrossRef] [PubMed]
Romero, D.L.; Morge, R.A.; Genin, M.J.; Biles, C.; Busso, M.; Resnick, L.; Althaus, I.W.; Reusser, F.; Thomas, R.C.; Tarpley, W.G. Bis(heteroaryl)piperazine (BHAP) reverse transcriptase inhibitors: structure-activity relationships of novel substituted indole analogues and the identification of 1-[(5-methanesulfonamido-1H-indol-2-yl)-carbonyl]-4-[3-[(1-methylethyl)-amino]-pyridinyl]piperazine monomethanesulfonate (U-90152S), a second-generation clinical candidate. J. Med. Chem. 1993, 36, 1505–1508. [Google Scholar] [PubMed]
Gulick, R.M.; Su, Z.; Flexner, C.; Hughes, M.D.; Skolnik, P.R.; Wilkin, T.J.; Gross, R.; Krambrink, A.; Coakley, E.; Greaves, W.L.; Zolopa, A.; Reichman, R.; Godfrey, C.; Hirsch, M.; Kuritzkes, D.R. Phase 2 study of the safety and efficacy of vicriviroc, a CCR5 inhibitor, in HIV-1-Infected, treatment-experienced patients: AIDS clinical trials group 5211. J. Infect. Dis. 2007, 196, 304–312. [Google Scholar] [CrossRef] [PubMed]
Peri, C.A. New polychlorinated derivatives containing the ring of 1,4-endomethylenecyclohexane. Gazz. Chim. Ital. 1955, 85, 1118–1140. [Google Scholar]
Sheldrick, G.M. Program for a crystal structure solution. Acta Cryst. 2008, A64, 112–122. [Google Scholar] [CrossRef] [PubMed]
Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.; Desmyster, J.; De Clercq, E. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J. Virol. Meth. 1998, 20, 309–321. [Google Scholar] [CrossRef]

Sample Availability: Samples of the compounds 1,2,3, 2a–2j and 3a–3j are available from authors.

Scheme 1. Compound Synthesis.

Figure 1. Molecular structures of compounds 1 and 3b.

Table 1. Physical and analytical data of compounds given in Scheme 1.

Comp.	M.p. (°C)	Yield (%)	Formula	Analysis Calculated/Found			¹H-NMR, δ_H (J in Hz), solvent–CDCl₃
	M.p. (°C)	Yield (%)	Formula	%C	%H	%N	¹H-NMR, δ_H (J in Hz), solvent–CDCl₃
2	oil	72	C₁₄H₁₄Cl₅N O₄	36.32 36.23	3.25 3.58	2.82 3.09	3.64 (s, 3H, OCH₃); 3.62 (s, 3H, OCH₃); 3.44–3.37 (m, 6H, CH, CH₂); 2.03–2.0 (m, 2H, CH₂); 1.87–1.81 (m, 1H, CH₂); 1.67–1.57 (m, 1H, CH₂)
3	oil	74	C₁₅H₁₆BrCl₄NO₄	38.43 38.39	3.23 3.34	3.20 3.21	3.65 (s, 3H, OCH₃); 3.63 (s, 2H, CH); 3.59 (s, 3H, OCH₃); 3.60–3.59 (m, 2H, CH₂); 3.53–3.50 (t, 2H, CH₂, J = 6.4); 1.97–1.91 (t, 2H, CH₂, J = 10.2)
2a	233	85	C₂₆H₃₂Cl₅N₃O₅	48.50 48.37	5.01 5.25	6.53 6.34	13.3 (s, 1H, HCl); 8.17–8.15 (d, 1H, CH_arom., J = 8); 7.48–7.44 (t, 1H, CH_arom., J = 8.6); 7.09–7.03 (m, 2H, CH_arom.); 5.03–4.98 (m, 2H, CH₂N); 4.35–4.33 (m, 2H, CH₂N); 4.06 (s, 3H, OCH₃); 3.73 (s, 2H, CH); 3.65 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.65–3.56 (m, 2H, CH₂N); 3.23–3.2 (m, 4H, CH₂N); 1.96–1.92 (m, 2H, CH₂); 1.65–1.61 (m, 2H, CH₂)
2b	256	86	C₂₃H₂₈Cl₅N₅O₄	44.86 44.67	4.58 4.87	11.37 11.13	13.04 (s, 1H, HCl); 8.36 (s, 2H, CH_arom.); 6.64 (s, 1H, CH_arom.); 4.89–4.85 (m, 2H, CH₂N); 3.87–3.91 (m, 2H, CH₂N); 3.70 (s, 2H, CH); 3.65 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.46–3.43 (m, 2H, CH₂N); 3.0–2.99 (m, 2H, CH₂N); 2.79–2.77 (m, 2H,CH₂N); 1.96–1.92 (m, 2H, CH₂); 1.60–1.58 (m, 2H, CH₂)
2c	243	88	C₂₅H₃₀Cl₅N₃O₄	48.92 48.79	4.93 5.0	6.85 6.81	12.78 (s, 1H, HCl); 7.34–7.30 (s, 2H, CH_arom.); 7.02–7.04 (s, 3H, CH_arom.); 3.76 (s, 2H, CH); 3.65 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.65–3.56 (m, 6H, CH₂N); 3.46–3.44 (m, 2H, CH₂N); 3.07–2.0 (m, 2H,CH₂N); 1.97–1.95 (m, 2H, CH₂); 1.63–1.60 (m, 2H, CH₂)
2d	248	76	C₂₄H₂₉Cl₅N₄O₄	46.89 46.78	4.75 4.95	9.11 9.02	13.01 (s, 1H, HCl); 8.01 (s, 2H, CH_arom.); 7.13–7.06 (s, 1H, CH_arom.); 4.67–4.60 (m, 2H, CH₂N); 4.21–4.19 (m, 2H, CH₂N); 3.73 (m, 2H, CH₂N); 3.65 (s, 2H, CH); 3.65 (s, 3H, OCH₃); 3.57 (s, 3H, OCH₃); 3.27–3.15 (m, 4H, CH₂N); 1.94–1.92 (m, 2H, CH₂); 1.63–1.61 (m, 2H, CH₂)
2e	206	79	C₂₅H₂₉Cl₅FN₃O₄	47.53 47.25	4.63 4.64	6.65 6.51	13.25 (s, 1H, HCl); 7.61–7.6 (m, 2H, CH_arom.); 7.15–7.12 (m, 2H, CH_arom.); 4.39–4.35 (m, 2H, CH₂N); 3.85–3.83 (m, 2H, CH₂N); 3.72 (s, 2H, CH); 3.65 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.65–3.56 (m, 4H, CH₂N); 3.49–3.41 (m, 2H, CH₂N); 1.95–1.92 (m, 2H, CH₂); 1.65–1.63 (m, 2H, CH₂).
2f	211	68	C₂₆H₃₂Cl₅N₃O₄	49.74 49.56	5.14 5.41	6.69 6.34	12.96 (s, 1H, HCl); 7.66–7.63 (m, 2H, CH_arom.); 7.48–7.45 (m, 3H, CH_arom.); 4.26–4.24 (m, 2H, CH₂); 4.02–4.0 (m, 4H, CH₂N); 3.72 (s, 2H, CH); 3.64 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.46–3.41 (m, 4H, CH₂N); 3.16–3.14 (m, 2H, CH₂N); 1.89–1.85 (m, 2H, CH₂); 1.69–1.62 (m, 2H, CH₂)
2g	230	87	C₂₅H₂₉Cl₆N₃O₄	46.32 46.26	4.51 4.81	6.48 6.68	13.23 (s, 1H, HCl); 7.58–7.56 (m, 2H, CH_arom.); 7.44–7.42 (s, 2H, CH_arom.); 4.44–4.38 (m, 2H, CH₂N); 3.85–3.83 (m, 2H, CH₂N); 3.72 (s, 2H, CH); 3.65 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.65–3.56 (m, 2H, CH₂N); 3.50–3.47 (m, 2H, CH₂N); 3.19–3.17 (m, 2H, CH₂N); 1.95–1.92 (m, 2H, CH₂); 1.65–1.63 (m, 2H, CH₂)
2h	231	86	C₂₆H₃₂Cl₅N₃O₅	48.5 48.42	5.01 5.23	6.53 6.56	12.91 (s, 1H, HCl); 7.25–7.23 (m, 1H, CH_arom.); 6.72–6.63 (s, 3H, CH_arom.); 3.98–3.92 (m, 2H, CH₂N); 3.80 (s, 3H, OCH₃); 3.76 (s, 2H, CH); 3.65 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.65–3.56 (m, 4H, CH₂N); 3.3–3.28 (m, 2H, CH₂N); 1.96–1.92 (m, 2H, CH₂); 1.63–1.6 (m, 2H, CH₂)
2i	213	84	C₂₆H₃₂Cl₅N₃O₄	49.74 49.72	5.14 5.18	6.69 6.44	12.97 (s, 1H, HCl); 7.71–7.7 (m, 1H, CH_arom.); 7.36–7.3 (s, 3H, CH_arom.); 4.68–4.46 (m, 4H, CH₂N); 3.83 (s, 2H, CH); 3.74–3.65 (m, 2H. CH₂N); 3.65 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.50–3.48 (m, 2H, CH₂N); 3.24–3.22 (m, 2H, CH₂N); 2.74 (s, 3H, CH₃); 1.96–1.924 (m, 2H, CH₂); 1.66–1.63 (m, 2H, CH₂)
2j	215	76	C₂₅H₂₉Cl₅FN₃O₄	47.53 47.49	4.63 4.78	6.65 6.55	12.72 (s, 1H, HCl); 7.1–6.96 (m, 4H, CH_arom.); 4.77–4.74 (m, 2H, CH₂N); 3.76 (s, 2H, CH); 3.72–3.65 (m, 2H, CH₂N); 3.65 (s, 3H, OCH₃); 3.58 (s, 3H, OCH₃); 3.5–3.4 (m, 2H, CH₂N); 3.06–3.05 (m, 2H, CH₂N); 1.97–1.96 (m, 2H, CH₂); 1.63–1.61 (m, 2H, CH₂)
3a	185	87	C₂₅H₃₀Cl₅N₃O₅	50.61 50.60	4.93 4.91	7.08 7.09	7.02–6.99 (m, 1H, CH_arom.); 6.93–6.91 (s, 2H, CH_arom.); 6.89–6.85 (m, 1H, CH_arom.); 3.86 (s, 3H, OCH₃); 3.64 (s, 5H, CH, OCH₃); 3.59 (s, 3H, OCH₃); 3.54–3.52 (m, 2H, CH₂N); 3.16–3.14 (m, 4H, CH₂N); 2.74–2.7 (m, 4H, CH₂N); 2.54–2.52–1.92 (m, 2H, CH₂N); 1.78–1.76 (m, 2H, CH₂)
3b	244	78	C₂₂H₂₆Cl₅N₅O₄	46.74 46.52	4.46 4.61	12.39 12.28	11.17 (s, 1H, HCl); 8.44–8.43 (d, 2H, CH_arom., J = 4.8); 6.77–6.74 (t, 1H, CH_arom., J = 4.6); 3.81 (m, 2H, CH₂N); 3.60 (s, 2H, CH); 3.49–3.47 (m, 6H, CH₂N); 3.41 (s, 6H, OCH₃); 3.06–3.2.97 (m, 4H, CH₂N); 1.87–1.83 (m, 2H, CH₂)
3c	246	75	C₂₄H₂₈Cl₅N₃O₄	48.06 48.03	4.71 4.59	7.01 6.8	12.89 (s, 1H, HCl); 7.33–7.29 (m, 2H, CH_arom.); 7.01–6.99 (m, 3H, CH_arom.,); 3.74–3.72 (m, 6H, CH₂N); 3.65 (s, 8H, CH, OCH₃); 3.59–3.54 (m, 2H, CH₂N); 3.5–2.45 (m, 4H, CH₂N); 2.18–2.16 (m, 2H, CH₂)
3d	248	78	C₂₃H₂₇Cl₅N₄O₄	45.98 45.58	4.53 4.94	9.33 9.02	13.04 (s, 1H, HCl); 8.21 (d, 1H, CH_arom., J = 4); 7.6–7.58 (m, 2H, CH_arom.); 6.8–6.77 (s, 2H, CH_arom.); 4.34 (m, 2H, CH₂N); 3.90–3.89 (m, 2H, CH₂N); 3.8 (s, 2H CH); 3.65 (s, 6H, OCH₃); 3.63–3.37 (m, 4H, CH₂N);3.0–2.86 (m, 4H, CH₂N); 2.17–2.16 (m, 2H, CH₂)
3e	237	80	C₂₄H₂₇Cl₅FN₃O₄	46.66 46.22	4.41 4.73	6.80 6.51	12.83 (s, 1H, HCl); 7.09–7.03 (m, 4H, CH_arom.); 3.76–3.45 (m, 8H, CH₂N); 3.65 (s, 5H, CH, OCH₃); 3.58 (s, 3H, OCH₃); 3.15–3.1 (m, 4H, CH₂N); 2.18–2.14 (m, 2H, CH₂)
3f	241	64	C₂₅H₃₀Cl₅N₃O₄	48.92 48.72	4.93 4.85	6.85 6.72	7.64–6.61 (m, 2H, CH_arom.); 7.47–7.45 (s, 3H, CH_arom.); 4.23–4.21 (m, 2H, CH₂); 3.69–3.67 (m, 4H, CH₂N); 3.65–3.44 (m, 6H, CH₂N); 3.65 (s, 5H, CH, OCH₃); 3.57 (s, 3H, OCH₃); 3.11–3.08 (m, 2H, CH₂N); 2.03–2.0 (m, 2H, CH₂)
3g	250	85	C₂₄H₂₇Cl₆N₃O₄	45.45 45.36	4.29 4.35	6.63 6.46	13.13 (s, 1H, HCl); 7.33–7.26 (m, 4H, CH_arom.); 4.05–4.0 (m, 2H, CH₂N); 3.74–3.71 (m, 2H, CH₂N); 3.65 (s, 5H, CH, OCH₃); 3.59 (s, 3H, OCH₃); 3.65–3.58 (m, 4H, CH₂N); 3.42–3.4 (m, 2H, CH₂N); 3.14–3.12 (m, 2H, CH₂N); 2.16–2.14 (m, 2H, CH₂)
3h	219	81	C₂₅H₃₀Cl₅N₃O₅	47.68 47.82	4.80 5.04	6.67 6.52	12.9 (s, 1H, HCl); 7.23–7.21 (m, 1H, CH_arom.); 6.68–6.59 (m, 3H, CH_arom.); 3.9 (s, 3H, OCH₃); 3.89–3.74 (m, 4H, CH₂N); 3.65 (s, 5H, CH, OCH₃); 3.58 (s, 3H, OCH₃); 3.64–3.57 (m, 4H, CH₂N); 3.18–3.1 (m, 4H, CH₂N); 2.17–2.14 (m, 2H, CH₂).
3i	229	75	C₂₅H₃₀Cl₅N₃O₄	48.92 48.82	4.93 5.13	6.85 6.75	12.83 (s, 1H, HCl); 7.16–7.12 (m, 1H, CH_arom.); 6.34–6.32 (m, 3H, CH_arom.); 3.93–4.0 (m, 2H, CH₂N); 3.75–3.71 (m, 2H, CH₂N); 3.65 (s, 5H, CH, OCH₃); 3.59 (s, 3H, OCH₃); 3.58–3.31 (m, 4H, CH₂N); 3.15–3.12 (m, 2H, CH₂N); 2.43–2.4 (m, 2H, CH₂N); 2.17–2.14 (m, 2H, CH₂)
3j	224	73	C₂₄H₂₇Cl₅FN₃O₄	46.66 46.70	4.41 4.66	6.80 6.57	12.92 (s, 1H, HCl); 7.08–7.04 (m, 4H, CH_arom.); 3.74–3.72 (m, 2H, CH₂N); 3.69–3.67 (m, 2H, CH₂N); 3.65 (s, 5H, CH, OCH₃); 3.58 (s, 3H, OCH₃); 3.57–3.45 (m, 4H, CH₂N); 3.08–3.04 (m, 4H, CH₂N); 17–2.14 (m, 2H, CH₂)

Table 2. Crystal data and refinement parameters for 1 and 3b.

		1	3b
Empirical formula		C₁₁H₉Cl₄NO₄	C₂₂H₂₆Cl₅N₅O₄ × (C₂H₅O)_0.5
Formula weight		360.99	638.79
Crystal system, space group		triclinic, P-1	monoclinic, P2₁/c
Unit cell dimensions	a (Å)	8.304(2)	16.230(3)
b (Å)		13.554(3)	8.642(2)
c (Å)		14.642(3)	22.153(4)
α (°)		111.97(3)	90
β (°)		96.28(3)	104.03(3)
γ (°)		103.63(3)	90
Volume (Å³)		1449.2(6)	3014.5(10)
Z, Calculated density (g cm^–3)		4, 1.655	4, 1.408
F(000)		728	1324
Absorption coefficient (mm^-1)		7.548	4.725
Crystal size (mm)		0.32 × 0.25 × 0.18	0.42 × 0.19 × 0.11
Absorption corr.; T min, max		0.054, 0.151	0.145, 0.245
θ range for data collection (°)		5.6–72.9	5.5–75.8
Limiting indices		-10 ≤ h ≤ 0, -15 ≤ k ≤ 15, -16 ≤ l ≤ 17	-20 ≤ h ≤ 19, 0 ≤ k ≤ 10, 0 ≤ l ≤ 27
Reflections collected / unique / observed [I > 2σ (I)]		5960 / 5596 / 2773 [R_int = 0.0424]	6295 / 6162 / 2021 [R_int = 0.0354]
Data / parameters		5596 / 366	6162 / 365
Goodness-of-fit on F²		1.074	0.944
Final R indices [I > 2σ (I)]		R₁ = 0.0452, wR₂ = 0.1370	R₁ = 0.0620, wR₂ = 0.1515
Max and min Δρ (e Å^-3)		0.43 and -0.32	0.59 and -0.39
Extinction coeff.		0.0018(3)	0.0019(3)
CCDC No		713452	713453

Table 3. Cytotoxicity and antiviral activity of 4-substituted a arylpiperazine derivatives of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione.

Compd.	^aMT-4 CC₅₀ [µM]	^bHIV-1 EC₅₀ [µM]	^cMDBK CC₅₀ [µM]	^dBFDV EC₅₀ [µM]	^eBHK-21 CC₅₀ [µM]	^fYFV EC₅₀ [µM]	^fReo-1 EC₅₀ [µM]
1	>100	>100	-	-	-	-	-
2a	24	>24	86	>86	13	>13	>13
2b	>100	>100	>100	>100	>100	>100	>100
2c	45	>45	>100	>100	>100	>100	>100
2d	36	>36	>100	>100	>100	>100	>100
2e	16	>16	>100	>100	64	>64	>64
2g	11	>11	>100	>100	>100	>100	>100
2h	18	>18	>65	>65	11	>11	>11
2i	17	>17	>14	>14	7	>7	>7
2j	24	>24	>100	>100	30	>30	>30
3a	32	>32	73	>73	22	>22	>22
3c	30	>30	100	>100	29	>29	>29
3d	53	>53	>100	>100	>100	>100	>100
3e	34	>34	89	>89	36	>36	>36
3i	20	>20	24	>24	9	>9	>9
Compd.	^gVero-76 CC₅₀ [µM]	^hHSV-1 EC₅₀ [µM]	^hVV EC₅₀ [µM]	^hVSV EC₅₀ [µM]	^hCVB-2 EC₅₀ [µM]	^hSB-1 EC₅₀ [µM]
1	-	-	-	-	-	-
2a	80	>80	>80	>80	>80	>80
2b	95	>95	>95	>95	>95	>95
2c	84	>84	>84	>84	13	>84
2d	95	>95	>95	>95	>95	>95
2e	60	>60	>60	>60	>60	>60
2g	95	>95	>95	>95	10	>95
2h	70	>70	>70	>70	>70	>70
2i	45	>45	>45	>45	>45	>45
2j	52	>52	>52	>52	>52	>52
3a	60	>60	>60	>60	>60	>60
3c	70	>70	>70	>70	>70	>70
3d	82	>82	>82	>82	17	>82
3e	75	>75	>75	>75	>75	>75
3i	34	>34	>34	>34	>34	>34

^a Compd. concn. (Mμ) required to reduce the viability of mock-infected MT-4 (CD4+ Human T-cells containing an integrated HTLV-1 genome) cells by 50%, as determined by the colorimetric MTT method; ^b Compound concentration (Mμ) required to achieve 50% protection of MT-4 cells from the HIV-1-induced cytopathogenicity, as determined by the MTT method; ^c Compd. concn. (Mμ) required to reduce the viability of mock-infected MDBK (Bovine normal kidney) cells by 50%, as determined by the MTT method; ^d Compd. concn. (Mμ) required to achieve 50% protection of MDBK cells from the BVDV (Bovine Viral Diarrhea Virus)-induced cytopathogenicity, as determined by the MTT method; ^e Compd. concn. (Mμ) required to reduce the viability of mock-infected BHK (Hamster normal kidney fibroblast) monolayers by 50%, as determined by the MTT method; ^f Compound concentration (Mμ) required to achieve 50% protection of BHK (Kidney fibroblast) cells from the YFV (Yellow Fever Virus) and Reo (Reovirus 1)-induced cytopathogenicity, as determined by the MTT method; ^g Compd. concn. (μM) required to reduce the viability of mock-infected VERO-76 (Monkey normal kidney) monolayers by 50%, as determined by the MTT method; ^h Compd. concn. (μM) required to reduce the plaque number of HSV-1 (Herpesvirus 1), VV (Vaccinia Virus), VSV (Vesicular Stomatitis Virus), CVB-2 (Coxsackievirus B2), Sb-1 (Poliovirus 1) and RSV (Respiratory Syncytial Virus) by 50% in VERO-76 monolayers

© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Share and Cite

MDPI and ACS Style

Kossakowski, J.; Pakosinska-Parys, M.; Struga, M.; Dybala, I.; Koziol, A.E.; La Colla, P.; Marongiu, L.E.; Ibba, C.; Collu, D.; Loddo, R. Synthesis and Evaluation of in Vitro Biological Activity of 4-Substituted Arylpiperazine Derivatives of 1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione. Molecules 2009, 14, 5189-5202. https://doi.org/10.3390/molecules14125189

AMA Style

Kossakowski J, Pakosinska-Parys M, Struga M, Dybala I, Koziol AE, La Colla P, Marongiu LE, Ibba C, Collu D, Loddo R. Synthesis and Evaluation of in Vitro Biological Activity of 4-Substituted Arylpiperazine Derivatives of 1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione. Molecules. 2009; 14(12):5189-5202. https://doi.org/10.3390/molecules14125189

Chicago/Turabian Style

Kossakowski, Jerzy, Magdalena Pakosinska-Parys, Marta Struga, Izabela Dybala, Anna E. Koziol, Paolo La Colla, Laura Ester Marongiu, Cristina Ibba, David Collu, and Roberta Loddo. 2009. "Synthesis and Evaluation of in Vitro Biological Activity of 4-Substituted Arylpiperazine Derivatives of 1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione" Molecules 14, no. 12: 5189-5202. https://doi.org/10.3390/molecules14125189

APA Style

Kossakowski, J., Pakosinska-Parys, M., Struga, M., Dybala, I., Koziol, A. E., La Colla, P., Marongiu, L. E., Ibba, C., Collu, D., & Loddo, R. (2009). Synthesis and Evaluation of in Vitro Biological Activity of 4-Substituted Arylpiperazine Derivatives of 1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione. Molecules, 14(12), 5189-5202. https://doi.org/10.3390/molecules14125189

Article Menu

Synthesis and Evaluation of in Vitro Biological Activity of 4-Substituted Arylpiperazine Derivatives of 1,7,8,9-Tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione

Abstract

Introduction

Results and Discussion

Experimental

General

General procedure for the preparation of arylpiperazine derivatives 2a–2j and 3a–3j

General procedure for the preparation of hydrochloride salts of compounds 2a–2j and 3a–3j

X-Ray crystal structure analysis of 1 and 3b diethyl ether hemisolvate

Cell-based assays

Cytotoxicity Assays

Antiviral assays

Conclusions

References

Share and Cite

Article Metrics

Article Access Statistics

Further Information

Guidelines

MDPI Initiatives

Follow MDPI