Bioorganic & Medicinal Chemistry 22 (2014) 813–826

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Synthesis and antifungal activity of substituted 2,4,6-pyrimidinetrione carbaldehyde hydrazones Donna M. Neumann a,b,⇑, Amy Cammarata a, Gregory Backes a, Glen E. Palmer c, Branko S. Jursic d,e a

Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112, United States Department of Ophthalmology, LSUHSC, New Orleans, United States c Department of Microbiology, Immunology and Parasitology, LSUHSC-New Orleans, United States d Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United States e STEPHARM, LLC., P.O. Box 24220, New Orleans, LA 70184, United States b

a r t i c l e

i n f o

Article history: Received 21 October 2013 Revised 25 November 2013 Accepted 4 December 2013 Available online 12 December 2013 Keywords: Antifungal Hydrazine Carbaldehyde Pyrimidinetrione Hydrazones

a b s t r a c t Opportunistic fungal infections caused by the Candida spp. are the most common human fungal infections, often resulting in severe systemic infections—a significant cause of morbidity and mortality in at-risk populations. Azole antifungals remain the mainstay of antifungal treatment for candidiasis, however development of clinical resistance to azoles by Candida spp. limits the drugs’ efficacy and highlights the need for discovery of novel therapeutics. Recently, it has been reported that simple hydrazone derivatives have the capability to potentiate antifungal activities in vitro. Similarly, pyrimidinetrione analogs have long been explored by medicinal chemists as potential therapeutics, with more recent focus being on the potential for pyrimidinetrione antimicrobial activity. In this work, we present the synthesis of a class of novel hydrazone-pyrimidinetrione analogs using novel synthetic procedures. In addition, structure–activity relationship studies focusing on fungal growth inhibition were also performed against two clinically significant fungal pathogens. A number of derivatives, including phenylhydrazones of 5-acylpyrimidinetrione exhibited potent growth inhibition at or below 10 lM with minimal mammalian cell toxicity. In addition, in vitro studies aimed at defining the mechanism of action of the most active analogs provide preliminary evidence that these compound decrease energy production and fungal cell respiration, making this class of analogs promising novel therapies, as they target pathways not targeted by currently available antifungals. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Opportunistic fungal infections caused by the Candida spp. represent the most common fungal infections of humans.1 Candida spp. infections can result in a broad spectrum of clinical manifestations, ranging from superficial mucocutaneous infections to the more severe invasive systemic fungal infections, and these invasive fungal infections (IFI’s) are a significant cause of morbidity and mortality in at-risk populations, particularly transplant recipients, cancer patients and those infected with HIV and AIDS.2,3 IFI’s present further diagnostic and therapeutic challenges due to the fact that they are difficult to diagnose early and are associated with high resistance rates to currently marketed antifungal agents.4 Finally, the incidence of candidiasis caused by non-albicans Candida spp. is increasing, with Candida glabrata and Candida krusei most frequently isolated in clinical settings, in addition to Candida albicans.5,6 Currently available clinical therapies for both cutaneous and systemic candidiasis

⇑ Corresponding author. Tel.: +1 504 568 3179. E-mail address: [email protected] (D.M. Neumann). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.12.010

include the first-line treatments of azoles. However, the development of clinical resistance to azoles by the Candida spp. occurs through multiple mechanisms and limits azole efficacy. For example, resistance to azoles by C. albicans has been shown to be largely due to both the overexpression of efflux pumps1 and point mutations in ERG11 gene.7 The opportunistic yeast pathogen C. glabrata is also recognized for its ability to acquire resistance during prolonged treatment with azole antifungals.8 For these reasons, there is a continuous demand for the discovery of novel therapeutics to treat fungal infections, particularly Candida spp. infections. Hydrazine derivatives have recently begun to emerge in the literature as novel classes of antifungal agents with therapeutic potential against numerous Candida spp., including species commonly resistant to azole antifungals. For example, it was shown that (4-aryl-thiazol-2-yl)hydrazines possessed potent antifungal activities against a number of clinically relevant Candida spp.9 Analogs from derivatives of the C2 and C4 positions of this hydrazine skeleton also yielded a number of promising antifungal agents that had synergistic effects when used in combination with an azole, while maintaining low mammalian cell toxicity. Finally, the hydrazine pharmacophore with substitutions of N1, together with

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4-substituted phenyls at the C4 of a thiazole nucleus produced a number of potent and selective hydrazine derivatives that possessed antifungal activity in the lM range.9 Other literature reports have shown that hydrazone derivatives have also emerged as compounds with the ability to potentiate antifungal activities in vitro. For example, the ability of hydrazone derivatives to inhibit the growth of Candida spp. was recently explored by Altintop et al.10 Hydrazone derivatives bearing 5-thio-1-methyl 1H tetrazole moiety were synthesized and evaluated for potential antifungal activity and mammalian cell toxicity, with a number of compounds showing potential for further development as antifungal agents.10 Finally, pyrimidinetrione analogs have long been explored by medicinal chemists as not only psychotropic compounds, but as anti-seizure, anticancer and antimicrobial compounds as well. For example, pyrazole and isoxazole derivatives have gained importance as potential chemotherapeutics that have applications as antimicrobials and are active against a number of different fungal species,11 while other pyrimidinetrione derivatives, including bisoxadiazolyl and bisthiadiazolyl pyrimidinetriones have use as antibiotic and antifungal therapies.12 In this manuscript, we present the synthesis of an extensive collection of substituted pyrimidinetrione derivatives using novel synthetic procedures. In addition, structure–activity relationship studies focusing on fungal growth inhibition were also performed against two clinically significant fungal pathogens, namely C. albicans and C. glabrata A number of derivatives, including phenylhydrazones of 5-acylpyrimidinetrione exhibited potent growth inhibition at or below 10 lM with minimal mammalian cell toxicity. In addition, in vitro studies aimed at defining the mechanism of action of the most active analogs provide preliminary evidence that these compound decrease energy production and fungal cell respiration, making this class of analogs promising novel therapies, as they target pathways not targeted by currently available antifungals. 2. Results 2.1. Synthesis 2.1.1. Preparation of 1,3-substituted 2,4,6-pyrimidinetriones The pyrimidinetrione building blocks used for all subsequent syntheses reported here were first prepared by the condensation of diethyl malonate with substituted urea in the presence of sodium ethoxide and ethanol following the classic Dickey–Gray procedure.13 If the appropriate substituted urea was not commercially available, then the desired substituted urea was prepared from the corresponding amines and phenyl chloroformate by following the procedure outlined in Scheme 1.14 Using this method, a small library of 1,3-di and mono-substituted pyrimidinetrione derivatives were generated, and then used to further synthesize all substituted pyrimidinetrione analogs presented in this work. 2.1.2. Synthesis of 5-acyl-2,4,6-pyrimidinetriones Selecting the optimal preparation method for 5-acyl pyrimidinetriones depends on both the nature of the substituted pyrimidinetrione moiety as well as the acyl moiety. Previously, we prepared

R1 NH 2

C6 H 5OCOCl THF/H2 O/0o C 10-30 minutes

R 1NHOCOC6 H5

a number of derivatives, including 5-formyl-1,3-dimethylpyrimidinetrione using a modified Reimer–Tiemann reaction.15,16 However, the isolated yields using this method were 90% (Scheme 5). 2.1.6. Synthesis of substituted hydrazines, hydrazides, and benzosulfonohydrazides The majority of hydrazides and semicarbazides used in the preparation of the Schiff bases were not available commercially and had to be prepared. The preparation of these hydrazides started with readily available acid esters. A methanol solution of an ester with hydrazine hydrate (10 equiv) was refluxed for one hour, followed by the distillation of methanol at atmospheric pressure. In this way, the amount of hydrazine was gradually increased to facilitate the reaction. After all the methanol was distilled from the reaction mixture, the remaining residue was mixed with water and extracted in ethyl acetate.24 In the case of semicarbazides, there are many different methods for the preparation of substituted semicarbazides. However, the two most commonly used

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D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826

R1 N

R1 N

O R4

N

O

O N R2

R3

Schiff Bases

O 1

R

O N N H R3

N O N R2

R

6

R1 N

R NH r H 2N n ol o d a aci h t me et ic ac

O O

O R3

N 6 R2 OR C H or ol NN H 2 th an a ci d m e c eti c a

H2 N NH SO me 2R 7 than ac e o tic a l or c id

O

R5 N N H R3

N R2 O Hydrazones and PhenylHydrazones

5

H me 2 N - R 4 t ace hanol tic acidor

O

O

R1 N

O

O

O S R7

N N

O

H N

R3

R2 O Benzenesulfonohydrazones

O

Acylhydrazones

Scheme 5. Preparation of Schiff bases an hydrazones of 5-acyl-2,4,6-pyrimidinetriones. Reagents and conditions: R1 = H, CH3, (CH2)3CH3, C6H5, 4-O2NC6H4, 2-HOC6H4, 3-HOC6H4, 4-HOC6H4, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl; R2 = H, CH3, (CH2)3CH3, C6H5, p-HOC6H4, CH2CO2H, (CH2)4CO2H; R3 = H, CH3, CH3(CH2)4, C6H5, 2-HOC6H4, 3-HOC6H4, 4-HOC6H4, 4-(CH3)2NC6H4, 4-CH3OC6H4, 4-CH3C6H4, 4-HO2CC6H4, 2-O2NC6H4, 3-O2NC6H4, 4-O2NC6H4, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl; R4 = (CH2)5CH3, (CH2)2CO2H, (CH2)5CO2H, C6H5, 2-HOC6H4, 3-HOC6H4, 4-HOC6H4, 4-CH3OC6H4, 4-O2NC6H4, 4-HO2CC6H4, 4-NCC6H4; R5 = CH3, (CH2)5CH3, CH2C6H5, C6H5, 4-CH3C6H4, 4-CH3OC6H4, 4-O2NC6H4, 2,4-(O2N)2C6H3, 4-HO2CC6H4; R6 = CH3, (CH2)5CH3, C6H5, 2-HOC6H5, 3-HOC6H4, 4-HOC6H4, 4-(CH3)2NC6H4, 4-CH3OC6H4, 3-O2NC6H4, 4-O2NC6H4, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl; R7 = C6H5, 4-CH3C6H4.

NH 2

HCl/H2 O/NaNO2/0ºC

X

R 1CO2 C2 H 5

Y

H 2NNH2 C2 H5 OH, 70ºC SO2 Cl

NHNH2

Na2 SO3 /H 2O r.t.

X

R 1CONHNH2

H 2NNH2 THF/H2 O/0ºC

Y

LiAlH4 Ether, r.t.

R 1CH2 NHNH2

SO 2NHNH 2

Scheme 6. Preparation of substituted hydrazines, hydrazides, and benzosulfonohydrazides. Reagents and conditions: X = H, 4-CH3, 4-CH3O; Y = H, 4-CH3O, 4-O2N; R3 = C5H9, C6H5, 4-O2NC6H4.

are (a) from substituted urea and hydrazine hydrate25 and (b) from substituted isocyanate.26 We have further developed a very efficient synthetic procedure for the preparation of semicarbazides that starts with the corresponding amine, phenyl chloroformate and hydrazine14 (Scheme 6). 2.2. Antifungal activity The antifungal activity of the 2,4,6-pyrimidinetrione analogs shown in Schemes 2–6 were evaluated in vitro using C. albicans (ATCC no. 10231) and C. glabrata (ATCC no. 48435). All assays were done in accordance with NCCLS reference documents.27 The results of these screenings are summarized in Tables 1–9 as the minimal inhibitory concentrations that inhibited more than 80% fungal growth as compared to the positive controls in 1% DMSO and HEPES buffered RPMI media. All MIC screens were done using a visual scoring method as opposed to spectroscopic methods of analysis, due to the physical properties of many of the compounds altering their absorbance spectra relative to the positive, negative and drug controls. Of the analogs tested, none of the 5-acyl-2,4,6-pyrimidinetriones, 5-alkylated pyrimidinetriones or the 5,5-dialkylated pyrimidinetriones showed antifungal activity through 125 lg/mL, regardless of the nature of the acyl or alkyl group attached at the 5-position of the pyrimidinetrione ring (Tables 1, 3 and 4). In contrast, however, the addition of an arylidene moiety to the pyrimidinetrione led to increased growth inhibition. Specifically, the 5-arylidene derivatives, such as BA40, BA41, BA42, BA43 and

BA53 (shown in Table 2) modestly inhibited fungal growth of either C. albicans or C. glabrata, indicating that extended conjugation of the pyrimidinetrione ring may be required for growth inhibition. The possibility that extended conjugation of the 2,4,6-pyrimidinetrione ring is a prerequisite for antifungal activity was an intriguing prospect. To explore this possibility more thoroughly, we synthesized and analyzed the antifungal activity (by growth inhibition) of a number of Schiff base derivatives of 5-acylpyrimidinetriones, including several analogs with substituted and unsubstituted phenyl groups, all of which would extend conjugation through the aromatic ring system. Making these modifications to the pyrimidinetrione ring yielded a number compounds that had inhibitory activity of C. albicans and/or C. glabrata (compounds BA47 and BA48, Table 2 and BA93 and BA94, Table 5). Interestingly, all of these compounds had a hydroxyl group in the 2-position of the aromatic ring. Substitution of the aromatic system with an ortho hydroxyl group would position the non-bonded electron pair of the hydroxyl close to the carbon–nitrogen double bond of BA93 and BA94, further extending and stabilizing the conjugation of the system. In contrast, compounds with a hydroxyl in the 3position (BA95 and BA96) or in the 4-position (BA79–81, Table 5; BA44–46, Table 2) of the aromatic ring lack that extended conjugation and stabilization, and were subsequently found to have no inhibition of fungal growth. Collectively, these data indicate that the contribution from extended conjugation of molecule through the presence of the non-bonding electron pair on the OH in the 2 position might be integral for antifungal activity.

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D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826 Table 1 5-Acyl-2,4,6-pyrimidinetriones synthesized following procedures in Scheme 2*18,15

R1 N

O O

O R3

N R2

*

O

Compound

R1

R2

R3

MIC80 C. albicans (lg/mL)

MIC80 C. glabrata (lg/mL)

BA1 BA2 BA3 BA4 BA5 BA6 BA7 BA8 BA9 BA10 BA11 BA12 BA13 BA14 BA15 BA16 BA17 BA18 BA19 BA20 BA21

H H CH3 H CH3 H CH3 H CH3 H CH3 H H H CH3 H CH3 H H H H

H CH3 CH3 H CH3 H CH3 H CH3 H CH3 C4H9 C6H5 H CH3 C4H9 CH3 H CH3 H C6H5

C6H5 C6H5 C6H5 3-O2NC6H4 3-O2NC6H4 4-O2NC6H4 4-O2NC6H4 3,5-(O2N)2C6H3 3,5-(O2N)2C6H3 4-HOC6H4 4-HOC6H4 4-HOC6H4 4-HOC6H4 4-CH3OC6H4 4-CH3OC6H4 4-CH3OC6H4 3-Pyridinyl H H CH3 CH3

— — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — —

Compounds previously synthesized.

Table 2 5-Arylidene-2,4,6-pyrimidinetriones synthesized following procedures in Scheme 3

R1 N

O

O R3

N 2

R

O

Compound

R1

R2

R3

MIC80 C. albicans (lg/mL)

MIC80 C. glabrata (lg/mL)

BA39 BA40 BA41 BA42 BA43 BA44 BA45 BA46 BA47 BA48 BA50 BA51 BA52 BA53 BA54 BA55 BA56

H H H H CH3 H CH3 H H CH3 CH3 CH3 CH3 H CH3 H H

H H H H CH3 H CH3 C6H5 H CH3 CH3 CH3 CH3 H CH3 H H

C6H5 2-Naphthyl (C10H7) 1-Naphthyl (C10H7) [email protected] [email protected] 4-HOC6H4 4-HOC6H4 4-HOC6H4 2-HOC6H4 2,4-(HO)2C6H3 4-CH3OC6H4 2,3,4-(CH3O)3C6H4 2,4,6-(CH3O)3C6H4 4-(CH3)2NC6H4 4-(CH3)2NC6H4 3-Furanyl (C4H3O) 2-Furanyl (C4H3O)

— 125 125 62 62 — — — — — — — — 62 — — —

— — —

Functionally and structurally, derivatives of 2,4,6-pyrimidinetriones with hydrazone moieties share similar characteristics to compounds BA93 and BA94, namely extended conjugation through the hydrazone moiety. To test whether this structural component was important in antifungal function, hydrazones of 5-acylpyrimidinetriones and phenylhydrazones of 5-acylpyrimidinetriones synthesized in Schemes 5 and 6 were evaluated for antifungal activity. We found minimal inhibition resulting from treatment with 5-acylpyrimidinetrione hydrazones, even though extended conjugation existed through the C–N and N–N double bonds (Table 6). Similar results were observed in the case of N-acylhydrazones of 5-acylpyrimidinetriones and sulfonohydrazones (Tables 8

125 — — — 8 2 — — — — — — —

and 9). However, in the cases where the substitution to 5-acylpyrimidinetriones was a phenylhydrazone, growth inhibition significantly increased, and over 80% inhibition could be observed through lower dilutions, typically in the range of 1–4 lg/mL (

Synthesis and antifungal activity of substituted 2,4,6-pyrimidinetrione carbaldehyde hydrazones.

Opportunistic fungal infections caused by the Candida spp. are the most common human fungal infections, often resulting in severe systemic infections-...
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