key: cord-0985385-vxr15lvy
authors: Novikov, Mikhail S.; Babkov, Denis A.; Paramonova, Maria P.; Khandazhinskaya, Anastasia L.; Ozerov, Alexander A.; Chizhov, Alexander O.; Andrei, Graciela; Snoeck, Robert; Balzarini, Jan; Seley-Radtke, Katherine L.
title: Synthesis and anti-HCMV activity of 1-[ω-(phenoxy)alkyl]uracil derivatives and analogues thereof
date: 2013-07-15
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2013.05.009
sha: 51f9f787ef1fe06c169838b518a134458cd07893
doc_id: 985385
cord_uid: vxr15lvy

HCMV infection represents a life-threatening condition for immunocompromised patients and newborn infants and novel anti-HCMV agents are clearly needed. In this regard, a series of 1-[ω-(phenoxy)alkyl]uracil derivatives were synthesized and examined for antiviral properties. Compounds 17, 20, 24 and 28 were found to exhibit highly specific and promising inhibitory activity against HCMV replication in HEL cell cultures with EC(50) values within 5.5–12 μM range. Further studies should be undertaken to elucidate the mechanism of action of these compounds and the structure–activity relationship for the linker region.

Human cytomegalovirus (HCMV) belongs to the viral family known as Herpesviridae, and is also known as human herpesvirus-5 (HHV-5). Within Herpesviridae, HCMV belongs to the Betaherpesvirinae subfamily. 1 One characteristic feature of the herpesviruses, including HCMV, is the ability to remain latent within the body after infection. 2 Latent HCMV is present in approximately 90% of adults aged over 80 in the U.S., 3 and typically goes unnoticed in healthy people, but can be life-threatening for the immunocompromised person. In this regard, HIV-infected persons, 4,5 organ transplant recipients, 6 or newborn infants are all at high risk of infection. Transplacental HCMV transmission can lead to congenital abnormalities and stillbirth, thus represents one of the most common viral causes of birth defects. 7 HCMV infection may also cause mucoepidermoid carcinoma and possibly other malignancies. 8 Moreover, a number of studies have revealed that HCMV is associated with autoimmune diseases, 9 atherosclerosis, 10 coronary restenosis 11, 12 and increased risk of diabetes. 13, 14 To date only three anti-HCMV agents, ganciclovir, 15 cidofovir, 16 and foscarnet, 17 have been approved for clinical use. They exhibit their effects by inhibiting the activity of the HCMV polymerase, thus reducing viral replication in patients with recognized HCMV infection symptoms. The use of these drugs however has been severely limited by their toxicity. 18 In addition, due to their inherent poor oral bioavailability, they must be administered intravenously to reach appropriate therapeutic levels. The exception to this limitation is valganciclovir, the orally administered prodrug for ganciclovir. 19 Unfortunately, as is typical for many nucleoside drugs, the development of drug-resistant viral strains has emerged. [20] [21] [22] Thus, there is an urgent need for new and more effective anti-HCMV agents with improved activity and pharmacokinetic profiles.

Recently a new class of nonnucleoside HCMV polymerase inhibitors has attracted interest. 23 As a result of the BioChem Pharm screening campaign, a series of 1,6-naphthiridine-based HCMV inhibitors were identified. The highest activity was exhibited by compound 1 (Fig. 1 ) which possesses an ortho-substituted benzyl moiety. 24 Subsequent design modifications led to the macrocyclic analogues represented by 2. 25 These compounds exhibited potent activity against HCMV and other herpesviruses, however were accompanied by significant cytotoxicity. Potent anti-HCMV and anti-VZV activity was also noted for other heterocyclic-based inhibitors including imidazo[1,2-a]pyridine derivatives 3 26-28 and benzothiadiazines 4. 29, 30 Other examples of broad-spectrum herpesvirus inhibitors are represented by derivatives of fused heterocyclic systems including the quinolones, 31 (6) , all developed by Pfizer (Fig. 1) . It should be noted that all of these compounds possess a common structural feature: an N-heterocycle linked to a benzene ring.

Previously we have reported the synthesis and antiviral properties for a number of different classes of nucleobase derivatives. Specifically, the anti-HIV and anti-HCMV activity for a series of 9-[2-(phenoxy)ethyl]-and 9-[2-(benzyloxy)ethyl]-derivatives of adenine have been described. 36 The 1-{[2-(phenoxy)ethoxy]methyl}uracils were found to be moderately active against HIV, 37 while 1-[2-(2-benzoylphenoxy)ethyl]-derivatives exhibited strong inhibitory properties. 38 In addition, several derivatives of 3-benzyl-1-(cinnamyl)uracil demonstrated significant activity against HIV and HCMV replication in cell culture. 39 As a result, these observations provided strong impetus to further explore the antiviral activity spectrum of 1-[x-(phenoxy)alkyl]uracils, as well as to investigate the structural requirements for the linker between the aromatic moieties, since this scaffold is common to many known inhibitors of HCMV polymerase.

A series of novel uracil derivatives was synthesized in a similar manner as previously described by our group. 38, 40 Condensation of equimolar amounts of 2,4-bis(trimethylsilyl)pyrimidines 7 with bromides 8 was performed at 160-170°C in the absence of solvent to afford target compounds 9-28 in 70-88% yield (Scheme 1).

Compound 29 was obtained in an 81% yield by alkaline hydrolysis of 18 in refluxing water-ethanol for 8 h (Scheme 2), while reduction of compound 21 was accomplished with SnCl 2 in mild acid-free conditions 41, 42 to produce the amino-substituted compound 30 in 59% yield. 

The anti-HCMV properties of target compounds 9-30 were evaluated against HCMV (AD-169 and Davis strains) in HEL cell cultures and the results are shown in Table 1 . Most of the compounds proved to be inhibitory against HCMV. In examining the structure activity relationship for the compounds, it appears that the activity is strongly dependent upon the substituent at the para-position of the benzene core. Polar groups proved unfavorable and resulted in a loss of activity (compounds 18, 21, 29) . Activity for alkyl substituted compounds increased with the size of the substituent: EC 50 for H (9) = Me (12) < i-Pr (14) < t-Bu (15), however, introduction of a bulky phenyl group led to poor activity for 16, thus there appears to be a steric limit. The 4-cyano-(17) and 4-bromo- (20) substituted compounds were found to exhibit the highest activity, which was comparable to ganciclovir. Interestingly, the 4chloro-substituted compound 19 was essentially inactive. One possible rationale for these observations could be complementary polar interactions between the halogen-or cyano-substituents and an appropriate protein functionality, for example, a carbonyl group, since the oxygen-containing compound 29 was inactive. This assumption is partially supported by the fact that reduction of NO 2 -group in inactive compound 21 led to 4-amino substituted compound 30 which exhibited slight anti-HCMV activity.

Substitutions at position five of the uracil ring were also examined. In that regard, while thymine analogue 24 was slightly more active than uracil derivative 20, for halogen-substituted 22 and 23 activity was notably diminished. As a result, this modification was not investigated further.

Initially, 20 was selected to investigate optimal length of the linker chain between the uracil and the benzene moieties. Compounds 25-27, containing three, four and six methylenes, respectively, were found to be inactive against HCMV at subtoxic concentrations. In contrast, compound 28, which has eight methylene units, demonstrated the same level of activity as 20.

In 

A series of novel 1-[x-(phenoxy)alkyl]uracil derivatives was synthesized and shown to have promising and highly specific inhibitory properties against HCMV replication in cell culture. Notably, the substitution pattern in the benzene core appears to be of importance for activity. The EC 50 values for the most active compounds in the series (compounds 17, 20, 24 and 28) lie within the range of 5.5-12.0 lM and are accompanied with low cytotoxicity (CC 50 P 100 lM). Additional efforts are currently underway to elucidate the mechanism of action of these compounds and further explore the structure-activity relationship for the linker region.

All reagents were obtained at highest grade available from Sigma and Acros Organics and used without further purification unless otherwise noted. Anhydrous DMF and isopropanol were purchased from Sigma-Aldrich Co. Anhydrous acetone, 1,2-dichloroethane, and ethyl acetate were obtained by distillation over P 2 O 5 . NMR spectra were registered on a Bruker Avance 400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C) in DMSO-d 6 with tetramethylsilane as an internal standard. Data are reported in the following order: multiplicity (br, broad; s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet; q, quartet; qu, quintet). TLC was performed on Merck TLC Silica gel 60 F 254 plates eluted with ethyl acetate or chloroform/MeOH (10:1) and developed with UV-lamp VL-6.LC (France). Acros Organics (Belgium) silica gel (Kieselgur 60-200 lm, 60A) was used for column chromatography. Melting points were determined in glass capillaries on a Mel-Temp 3.0 (Laboratory Devices Inc., U.S.). Yields refer to spectroscopically ( 1 H and 13 C NMR) homogeneous materials. High resolution mass spectra were measured on Bruker micrOTOF II instruments using electrospray ionization (HRESIMS). The measurements were done in a positive ion mode (interface capillary voltage À4500 V) in a mass range from m/z 50-3000 Da; external or internal calibration was done with ESI Tuning Mix™ (Agilent Technologies). A syringe injection was used for solutions in acetonitrile (flow rate 3 ll/min).

Nitrogen was applied as a dry gas; interface temperature was set at 180°C. Bromides 8 were synthesized as per known procedures. 43 1H, s, H-3 ). 13 

The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK À ) HSV-1 KOS strain resistant to ACV (ACV r ), herpes simplex virus type 2 (HSV-2) strains Lyons and G, human cytomegalovirus (HCMV) (strains AD-169 and Davis), varicellazoster virus (strains OKA and YS), vaccinia virus Lederle strain, respiratory syncytial virus (RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, Parainfluenza 3, Influenza virus A (subtypes H1N1, H3N2), influenza virus B, Reovirus-1, Sindbis and Punta Toro. The antiviral assays were based on inhibition of virusinduced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa) or Madin-Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID 50 of virus (1 CCID 50 being the virus dose to infect 50% of the cell cultures) or 10 or 100 plaque forming units (PFU) (for VZV and HCMV) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC 50 or compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%.

Inhibition of HIV-1(III B )-and HIV-2(ROD)-induced cytopathicity in CEM cell cultures was measured in microtiter 96-well plates containing $3 Â 10 5 CEM cells/mL infected with 100 CCID 50 of HIV per milliliter and containing appropriate dilutions of the test compounds. After 4À5 days of incubation at 37°C in a CO 2 -controlled humidified atmosphere, CEM giant (syncytium) cell formation was examined microscopically. The EC 50 (50% effective concentration) was defined as the compound concentration required to inhibit HIV-induced giant cell formation by 50%.

All assays were performed in 96-well microtiter plates. To each well were added (5À7.5) Â 10 4 tumor cells and a given amount of the test compound. The cells were allowed to proliferate for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37°C in a humidified CO 2 -controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The IC 50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%.

-Chlorophenoxy)pentyl]uracil (19). Yield 84%, mp 168-169°C

CH 2 ), 3.67 (2H, t, J = 7.3 Hz, NCH 2 ), 3.92 (2H, t, J = 6.3 Hz, OCH 2 ), 5.55 (1H, dd

-Bromophenoxy)pentyl]uracil (20). Yield 76%, mp 125-127°C

CH 2 ), 3.66 (2H, t, J = 7.2 Hz, NCH 2 ), 3.92 (2H, t, J = 6.3 Hz, OCH 2 ), 5.55 (1H, dd

Nitrophenoxy)pentyl]uracil (21). Yield 78%, mp 197-199°C

Yield 77%, mp 160-162°C

CH 2 ), 3.69 (2H, t, J = 7.4 Hz, NCH 2 ), 3.94 (2H, t, J = 6.5 Hz, OCH 2 ), 6.89 (2H, d

93 (2H, t, J = 6.4 Hz, OCH 2 ), 6.88 (2H, d

CH 2 ), 1.62 (2H, qu, J = 7.1 Hz, CH 2 ), 1.72 (2H, qu, J = 7.6 Hz, CH 2 ), 1.75 (3H, s, CH 3 ), 3.63 (2H, t

OCH 2 ), 5.53 (4H, dd, J = 7.8 and 2.3 Hz, H-5), 6.87 (2H, d, J = 9

CH 2 ), 5.56 (1H, dd, J = 7.8 and 2.2 Hz, H-5), 6.90 (2H, d, J = 9

CH 2 ), 3.65 (2H, t, J = 7.2 Hz, CH 2 ), 3.93 (2H, t, J = 6.5 Hz, CH 2 ), 5.53 (1H, dd, J = 7.8 and 2.2 Hz, H-5), 6.89 (2H, d

92 (2H, t, J = 6.5 Hz, OCH 2 ), 5.54 (1H, dd, J = 7.8 and 2.2 Hz, H-5), 6.88 (2H, d, J = 8.9 Hz, H-3 0 , H-5 0 ), 7.41 (2H, d, J = 8.9 Hz, H-2 0 , H-6 0 ), 7.64 (1H, d, J = 7.8 Hz, H-6), 11.22 (1 H, s, H-3)

This work was supported by grant of Russian Foundation For Basic Research (13-04-01391). The antiviral work was supported by KU Leuven (GOA 10/14). The authors thank Lies Van den Heurck, Anita Camps, Steven Carmans, Leentje Persoons, Frieda De Meyer, Leen Ingels and Lizette van Berckelaer for their help in performing the antiviral and cytostatic assays.

Supplementary data (NMR spectra for all of the synthesized compounds) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.009.