Synthesis and evaluation of thiophene-based guanylhydrazones (iminoguanidines) efficient against panel of voriconazole-resistant fungal isolates

Accepted Manuscript

Synthesis and evaluation of thiophene-based guanylhydrazones (iminoguani-

dines) efficient against panel of voriconazole-resistant fungal isolates

Vladimir Ajdačić, Lidija Senerovic, Marija Vranić, Marina Pekmezovic,

Valentina Arsic-Arsnijevic, Aleksandar Veselinovic, Jovana Veselinovic,

Bogdan A. Šolaja, Jasmina Nikodinovic-Runic, Igor M. Opsenica

PII:

S0968-0896(16)30070-0

DOI:

Reference:

http://dx.doi.org/10.1016/j.bmc.2016.01.058

BMC 12801

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

7 December 2015

21 January 2016

29 January 2016

Please cite this article as: Ajdačić, V., Senerovic, L., Vranić, M., Pekmezovic, M., Arsic-Arsnijevic, V., Veselinovic,

A., Veselinovic, J., Šolaja, B.A., Nikodinovic-Runic, J., Opsenica, I.M., Synthesis and evaluation of thiophene-

based guanylhydrazones (iminoguanidines) efficient against panel of voriconazole-resistant fungal isolates,

Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.01.058

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Bioorganic & Medicinal Chemistry

Synthesis and evaluation of thiophene-based guanylhydrazones (iminoguanidines)

efficient against panel of voriconazole-resistant fungal isolates

Vladimir Ajdačić,^aLidija Senerovic,^bMarija Vranić,^aMarina Pekmezovic,^cValentina Arsic-Arsnijevic,^c

Aleksandar Veselinovic,^dJovana Veselinovic,^dBogdan A. Šolaja,^aJasmina Nikodinovic-Runic*^band Igor

M. Opsenica,*^a

^aFaculty of Chemistry, University of Belgrade, Studentski trg 16, P.O. Box 51, 11158, Belgrade, Serbia; *E-mail: igorop@chem.bg.ac.rs

^bInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia; *E-mail:

jasmina.nikodinovic@gmail.com; jasmina.nikodinovic@imgge.bg.ac.rs

^cNational Reference Medical Mycology Laboratory, Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Dr Subotica 1,

11000 Belgrade, Serbia

^dFaculty of Medicine, Department of Chemistry, University of Niš, 18000 Niš, Serbia

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

A series of new thiophene-based guanylhydrazones (iminoguanidines) were synthesized in high

yields using a straightforward two-step procedure. The antifungal activity of compounds was

evaluated against a wide range of medicaly important fungal strains including yeasts, molds, and

dermatophytes in comparison to clinically used drug voriconazole. Cytotoxic properties of

compounds were also determined using human lung fibroblast cell line and hemolysis assay. All

guanylhydrazones showed significant activity against broad spectrum of clinically important

species of Candida spp., Aspergillus fumigatus, Fusarium oxysporum, Microsporum canis and

Trichophyton mentagrophytes, which was in some cases comparable or better than activity of

voriconazole. More importantly, compounds 10, 11, 13, 14, 18 and 21 exhibited excellent

activity against voriconazole-resistant C. albicans CA5 with very low minimal inhibitory

concentration (MIC) values <2 µg mL^-1. Derivative 14, bearing bromine on the phenyl ring, was

the most effective compound with MICs ranging from 0.25 to 6.25 µg mL^-1. However, bis-

guanylhydrazone 18 showed better selectivity in terms of therapeutic index values. In-vivo

embryotoxicity on zebrafish (Danio rerio) showed improved toxicity profile of 11,14 and 18 in

comparison to that of voriconazole. Most guanylhydrazones also inhibited C. albicans yeast to

hyphal transition, essential for its biofilm formation, while 11 and 18 were able to disperse

preformed Candida biofilms. All guanylhydrazones showed the equal potential to interact with

genomic DNA of C. albicans in vitro, thus indicating a possible mechanism of their action, as

well as possible mechanism of observed cytotoxic effects. Tested compounds did not have

significant hemolytic effect and caused low liposome leakage, which excluded the cell

membrane as a primary target. On the basis of computational docking experiments using both

human and cytochrome P450 from Candida it was concluded that the most active

guanylhydrazones had minimal structural prerequisites to interact with the cytochrome P450

14α-demethylase (CYP51). Promising guanylhydrazone derivatives also showed satisfactory

pharmacokinetic profile based on molecular calculations.

Available online

Keywords:

Guanlylhydrazone

antifungal

fungicidal

voriconazole-resistant Candida

biofilm

1. Introduction

less threatening fungal skin infections were in the top 10 most

prevalent diseases worldwide in 2010, affecting 984 million

people³and posing enormous health burden globally. Given that

the majority of infections caused by C. albicans are biofilm-

related, and that this form is usually more resistant to traditional

antifungal treatment it adds to recurrence and chronicity of the

disease.^4,5

Fungal infections have received less attention over past

decades, however mortality and morbidity rate of opportunistic

fungal infections is exponentially increasing and the number of

fatal incidence due to fungi is becoming comparable with that of

tuberculosis and malaria.¹Pathogenic fungi, particularly Candida

and Aspergillus species are one of the most common diagnosed

opportunistic infections and are the causes of significant

mortality in immunocompromised patients.²On the other side,

Antifungal agents are mainly divided into four classes:

polyenes, pyrimidines, echinocandins and azoles (Fig. 1).⁶The

polyene amphotericin B was developed in 1953, and by far is the

most potent antifungal agent for almost all invasive fungal

infections. It binds to ergosterol, which is the major sterol in

fungal cell membranes, and thereby forming membrane-spanning

channels that lead to the leakage of intracellular constituents and

finally fungal cell death. The most frequent side effects

associated with amphotericin B are infusional toxicity and

nephrotoxicity. Flucytosine, pyrimidine-base antifungal was

synthesized in 1957 as a potential antitumor drug but later on it

was discovered that it had significant fungistatic activity.

Flucytosine itself has no antifungal activity but it is converted to

5-fluorouracil within fungal cell, which then inhibits DNA and

RNA synthesis. Usually, it is used in combination therapy with

amphotericin B. The echinocandin derivative caspofungin

inhibits the synthesis of β-(1,3)-d-glucan, an important

component of the fungal cell wall. The major disadvantages

associated with caspofungin are narrow spectrum of activity

(only active against Candida spp. and Aspergillus spp.) and poor

oral bioavailability. Voriconazole is a member of clinically

important triazoles that are widely used for serious invasive

fungal infections, as well as for dermal mycoses.⁷Voriconazole

has fungistatic activity against Candida spp. and Fusarium spp.

and with fungicidal activity against Aspergillus spp. The

mechanism of action of voriconazole is inhibition of fungal

cytochrome P450-mediated (14α-lanosterol demethylation, an

essential step in the synthesis of ergosterol. This eventually

causes the depletion of ergosterol and disrupts the integrity of

fungal cell membranes, eventually leading to cell lysis.^8,9The

most common side effects of voriconazole therapy include visual

disturbances, skin rashes, elevation of hepatic enzyme levels,

headache, and hallucinations.^10-12

drugs cause an urgent need for the development of novel safe and

effective antifungals.¹³Apart from lower toxicity and higher

selectivity, panfungal (broad spectrum) activity is also very

desirable property that novel antifungals should have.

Compounds containing imonoguanidine moiety have been

known for a while and explored for their wide variety of

biological activities. Antiviral,^14-16antiparasitic,^17-19and

antibacterial^20-23activities of this versatile group of compounds

have been described. However, their antifungal potential has not

been extensively explored.²⁴More importantly, to the best of our

knowledge, they have not been evaluated against human fungal

pathogens.

In a view of the general lack of novel antifungal agents and in

a

continuation of our studies towards the discovery of

biologically active heterocyclic molecules, herein we report the

synthesis and structural characterization of novel

guanylhydrazones with their subsequent in vitro biological

evaluation for antifungal activity including activity against fungal

biofilms, their cytotoxic properties, in-vitro DNA interaction

ability and in-silico analysis. In-vivo embryotoxicity on zebrafish

(Danio rerio) of the most promising derivatives has been

determined. The druglikeness of studied compounds was also

established using Lipinski’s “rule of five”.^25,26

2. Materials and methods

2.1. Instrumentation

Melting points were determined on a Boetius PMHK

apparatus and were not corrected. IR spectra were recorded on a

Thermo-Scientific Nicolet 6700 FT-IR Diamond Crystal. NMR:

¹H and ¹³C NMR spectra were recorded on a Bruker Ultrashield

Advance III spectrometer (at 500 and 125 MHz, respectively)

using tetramethylsilane (TMS) as the internal standard. The

NMR solvents are specified individually for each compound.

Chemical shifts are expressed in parts per million (ppm) on the

() scale. Chemical shifts were calibrated relative to those of the

solvents. ESI MS spectra of the synthesized compounds were

recorded on an Agilent Technologies 6210 Time-of-Flight

LC/MS instrument in positive ion mode using CH₃CN/H₂O =

1:11:1 with 0.2 % HCOOH as the carrying solvent solution. The

samples were dissolved in pure acetonitrile (HPLC grade). The

selected values were as follows: capillary voltage = 4 kV; gas

temperature = 350 °C; drying gas = 12.l min^-1; nebulizer pressure

= 45 psig; fragmentator voltage = 70 V. All yields reported refer

to isolated yields. Compounds were analyzed for purity using:

Agilent 1200 HPLC system equipped with Quat Pump

(G1311B), Injector (G1329B) 1260 ALS, TCC 1260 ( G1316A)

and Detector 1260 DAD VL+ (G1315C), and Waters 1525 HPLC

dual pump system equipped with an Alltech Select degasser

system, and a dual λ 2487 UV-VIS detector. All compounds were

> 95% pure. Spectroscopic data of compounds are listed below

(¹H and ¹³C NMR spectra are in the Supplementary material).

2.2. General procedure for Suzuki coupling reactions

5-phenylthiophene-2-carbaldehyde (3) ²⁷: To a dry glass flask

purged with argon were added Pd(OAc)₂(3.4 mg, 0.015 mmol),

PPh₃(16 mg, 0.06 mmol) and dry DME (2 mL). The resultant

solution was stirred at room temperature for 10 min, and 5-

bromo-2-thiophenecarboxaldehyde (77 L, 0.65 mmol) and

Na₂CO₃(aq) (2 M, 0.65 mL, 1.3 mmol) were added. After 5 min

a solution of phenylboronic acid (99 mg, 0.82 mmol) in ethanol

(1 mL) was added and reaction mixture was purged with argon

and refluxed for 2 h under argon. The solution was cooled to

room temperature and filtered through a pad of Celite, washed

with CH₂Cl₂and dried with anh. Na₂SO₄. The organic solvent

Figure 1. Clinically used antifungals.

The increasing number of reports of fungal infections among

immunocompromised patients and additional problems

associated with toxicity and resistance to standard antifungal

was removed under reduced pressure and the crude product was

purified by dry-flash chromatography (SiO₂: hexane/EtOAc =

19:1) to afford the title compound 3 (110 mg, 90%) as an pale-

yellow amorphous powder; mp = 88-90 C. IR (ATR): 3281w,

3099w, 1637s, 1527w, 1436s, 1388m, 1329w, 1227s, 1060m,

142.84, 137.26, 132.38, 132.01, 127.83, 124.39, 123.62 ppm.

GC/MS (m/z (%)) 267.9 (100), 192.9 (40), 158.0 (20), 115.0

(30).

4-(5-formylthiophen-2-yl)benzonitrile (8)²⁷: The general

Suzuki coupling procedure was followed. The reaction mixture

was refluxed for 18 h. The crude product was purified by dry-

flash chromatography (SiO₂: hexane/EtOAc = 3:7) to afford the

title compound 8 (131 mg, 73%) as an pale-yellow amorphous

1

993w, 905w, 814m cm^-1. H NMR (500 MHz, CDCl₃): δ 9.89 (s,

1H), 7.74 (d, 1H, J = 3.5 Hz), 7.70-7.65 (m, 2H), 7.47-7.37 (m,

4H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 182.78, 154.30,

142.48, 137.36, 133.05, 129.44, 129.20, 126.44, 124.08 ppm.

GC/MS (m/z (%)) 187.0 ([M]⁺, 100), 115.1 (55).

o

powder; mp = 166-176 C. IR (ATR): 3304w, 3099w, 3052w,

2857w, 2826w, 2762w, 2221s, 1692s, 1673s, 1604m, 1532w,

1450s, 1414m, 1332w, 1285w, 1231s, 1181w, 1129w, 1065w,

5-(4-methylphenyl)thiophene-2-carbaldehyde (4)²⁷: The

general Suzuki coupling procedure was followed. The reaction

mixture was refluxed for 18 h. The crude product was purified by

dry-flash chromatography (SiO₂: hexane/EtOAc = 19:1) to afford

the title compound 4 (112 mg, 85%) as an pale-yellow

1

837w, 802m cm^-1. H NMR (500 MHz, CDCl₃): δ 9.94 (s, 1H),

7.81-7.75 (m, 3H), 7.75-7.69 (m, 2H), 7.50 (d, 1H, J = 4.0 Hz)

ppm. ¹³C NMR (125 MHz, CDCl₃): δ 182.70, 150.98, 144.15,

137.23, 137.02, 132.97, 126.82, 125.82, 118.26, 112,66 ppm.

GC/MS (m/z (%)): 212.0 ([M]⁺, 100), 140.1 (45).

o

amorphous powder; mp = 88-91 C. IR (ATR): 3351w, 3291w,

3095w, 3026w, 2970w, 2921w, 2850w, 2818w, 2751w, 1657s,

1614w, 1581m, 1535w, 1490w, 1449s, 1383m, 1333w, 1286w,

1226s, 1141w, 1123w, 1084w, 1056m, 1038w, 957w, 901w,

5-phenylfuran-2-carbaldehyde (9)²⁷: The general Suzuki

coupling procedure was followed. The reaction mixture was

refluxed for 2 h. The crude product was purified by dry-flash

chromatography (SiO₂: hexane/EtOAc = 9:1) to afford the title

compound 9 (145 mg, 87%) as an yellow oil; IR (ATR): 3114w,

2814w, 1669s, 1520m, 1474m, 1449m, 1391m, 1255m, 1028m,

1

854w, 802s cm^-1. H NMR (500 MHz, CDCl₃): δ 9.88 (s, 1H),

7,72 (d, 1H, J = 4.0 Hz), 7.57 (d, 2H, J = 8.5 Hz), 7.36 (d, 1H, J

= 4.0 Hz), 7.24 (d, 2H, J = 8.0 Hz), 2.39 (s, 3H) ppm. ¹³C NMR

(125 MHz, CDCl₃): δ 182.73, 154.64, 141.98, 139.74, 137.47,

130.30, 129.87, 126.32, 123.57, 21.32 ppm. GC/MS (m/z (%))

202.1 ([M]⁺, 100), 173.1 (8), 129.1 (28), 115.1 (6).

1

967m, 920w, 802m, 765s cm^-1. H NMR (500 MHz, CDCl₃): δ

9.65 (s, 1H), 7.85-7.80 (m, 2H), 7.47-7.42 (m, 2H), 7.41-7.37 (m,

1H), 7.32 (d, 1H, J = 3.5 Hz), 6.84 (d, 1H, J = 4.0 Hz) ppm. ¹³C

NMR (125 MHz, CDCl₃): δ 177.20, 159.40, 152.01, 129.65,

128.92, 125.27, 123.44, 107.66 ppm. GC/MS (m/z (%)) 172.1

([M]⁺, 100), 144.1 (15), 115.1 (100), 102.1 (5), 89.1 (20), 77,1

(7), 63.1 (15), 51.1 (10).

5-(4-methoxyphenyl)thiophene-2-carbaldehyde (5)²⁷: The

general Suzuki coupling procedure was followed. The reaction

mixture was refluxed for 2 h. The crude product was purified by

dry-flash chromatography (SiO₂: hexane/EtOAc = 1:1) to afford

the title compound 5 (175 mg, 82%) as an pale-yellow

amorphous powder; mp = 120-121 ^oC. IR (ATR): 3029w, 1693w,

1644s, 1600s, 1568m, 1530m, 1507m, 1453s, 1331w, 1308w,

1287s, 1254s, 1220s, 1178s, 1112m, 1054s, 1022s, 831s, 799s

cm^-1. ¹H NMR (500 MHz, CDCl₃): δ 9.86 (s, 1H), 7.70 (d, 1H, J

= 4.0 Hz), 7.61 (d, 2H, J = 9.0 Hz), 7.29 (d, 1H, J = 4.0 Hz), 6.94

(d, 2H, J = 9.0 Hz), 3.85 (s, 3H) ppm. ¹³C NMR (125 MHz,

CDCl₃): δ 182.64, 160.71, 154.52, 141.52, 137.65, 127.80,

125.78, 122.98, 114.58, 55.42 ppm. GC/MS (m/z (%)) 218.0

(100), 203.0 (38), 175.0 (12), 147.0 (14).

2.3. General procedure for reparation of guanylhydrazones 10-

18 and 21

2-[(5-phenylthiophen-2-yl)methylidene]hydrazine

carboximidamide hydrochloride (10) To a solution of aldehyde

(65 mg, 0.35 mmol) in absolute ethanol (12 mL) was added

aminoguanidine hydrochloride (38 mg, 0.35 mmol). The resultant

solution was stirred at room temperature for 5 min, and solution

of concentrated HCl in absolute EtOH (39 µL, 1:25 v/v) was

added. The reaction mixture was heated to reflux for 18 h and

allowed to cool to room temperature. The solvent was removed

under reduced pressure, and the crude product was washed with

CH₂Cl₂and then crystallized from EtOH/hexane (9:1) to provide

the title compound 10 (98 mg, 100%) as a pale-yellow solid; mp

= 184-187 ^oC. IR (ATR): 3818w, 3418m, 3307w, 3220w, 3133s,

3086m, 2848w, 1676m, 1643s, 1608m, 1462w, 1433w, 1253w,

1154w, 1108w, 1068w, 1005w, 925w, 802w, 757m, 688m cm^-1.

¹H NMR (500 MHz, CD₃OD): δ 8.26 (s, 1H), 7.72-7.67 (m, 2H),

7.45-7.38 (m, 4H), 7.37-7.32 (m, 1H) ppm. ¹³C NMR (125 MHz,

CDCl₃): δ 156.97, 149.62, 144.35, 138.20, 135.09, 134.24,

130.35, 129.79, 127.09, 125.04 ppm. (+)ESI-HRMS (m/z): [M +

H]⁺245.08616 (error 2.52 ppm). HPLC purity: Method A: RT

6.274, area 99.28%; method B: RT 7.407, area 99.88%.

5-(4-fluorophenyl)thiophene-2-carbaldehyde

(6)²⁷:

The

general Suzuki coupling procedure was followed. The reaction

mixture was refluxed for 2 h. The crude product was purified by

dry-flash chromatography (SiO₂: hexane/EtOAc = 4:1) to afford

the title compound 6 (145 mg, 72%) as an pale-brown amorphous

o

powder; mp = 114-116 C. IR (ATR): 3092m, 2855w, 1633s,

1594s, 1528m, 1502m, 1441s, 1413s, 1384m, 1327m, 1306m,

1

1231s, 1158s, 1100s, 1058s, 959m, 832s, 803s cm^-1. H NMR

(500 MHz, CDCl₃): δ 9.89 (s, 1H), 7.73 (d, 1H, J = 4.0 Hz), 7.68-

7.61 (m, 2H), 7.34 (d, 1H, J = 4.0 Hz), 7.17-7.10 (m, 2H) ppm.

¹³C NMR (125 MHz, CDCl₃): δ 182.71, 163.40 (d, J = 248.2

Hz), 153.03, 142.53, 137.38, 129.28 (d, J = 3.5 Hz), 128.29 (d, J

= 8.1 Hz), 124.06, 116.31 (d, J = 21.8 Hz) ppm. GC/MS (m/z

(%)) 206.0 (100), 178.0 (10), 133.0 (80), 120.0 (10), 107.0 (10).

2-{[5-(4-methylphenyl)thiophen-2-yl]methylidene}

hydrazinecarboximidamide hydrochloride (11) Following the

general procedure for guanylhydrazone formation, 11 was

obtained as a pale-yellow solid (85 mg, 59%). Mp = 244-245 ^oC.

IR (ATR): 3938w, 3428m, 3364m, 3320m, 3220m, 3093s,

2979m, 2916m, 2833m, 2773m, 1672s, 1617s, 1539w, 1511w,

1463m, 1432w, 1311w, 1239w, 1185w, 1123m, 1059w, 928m,

5-(4-bromophenyl)thiophene-2-carbaldehyde (7) The general

Suzuki coupling procedure was followed. The reaction mixture

was refluxed for 18 h. The crude product was purified by dry-

flash chromatography (SiO₂: hexane/EtOAc = 9:1) to afford the

title compound 7 (125 mg, 46%) as an pale-yellow amorphous

o

powder; mp = 94-97 C. IR (ATR): 3309w, 3103w, 2829w,

1

819w, 795m, 712w, 662w, 581w cm^-1. H NMR (500 MHz,

2802w, 2742w, 1662s, 1585m, 1530m, 1491w, 1448s, 1399m,

1324w, 1269w, 1226s, 1117w, 1079w, 1055m, 1004w, 959w,

CDCl₃): δ 8.25 (s, 1H), 7.57 (d, 2H, J = 8.5 Hz), 7.39 (d, 1H, J =

3.5 Hz), 7.35 (d, 1H, J = 4.0 Hz), 7.23 (d, 2H, J = 7,5 Hz), 2.36

(s, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 159.94, 149.90,

144.42, 140.08, 137.62, 134.29, 132.33, 130.93, 127.01, 124.47,

1

823s, 804s cm^-1. H NMR (500 MHz, CDCl₃): δ 9.90 (s, 1H),

7.73 (d, 1H, J = 4.0 Hz), 7.60-7.51 (m, 4H), 7.39 (d, 1H, J = 4.0

Hz) ppm. ¹³C NMR (125 MHz, CD₃CD): δ 182.68, 152.71,

o

21.29 ppm. (+)ESI-HRMS (m/z): [M + H]⁺259.10187 (error 2.59

ppm). HPLC purity: Method A: RT 6.377, area 97.25%; method

B: RT 7.543, area 99.87%.

yellowish solid (180 mg, 100%). Mp = 66-69 C. IR (ATR):

3901w, 3882w, 3621w, 3361s, 3148s, 2962s, 2861s, 2786m,

1676s, 1633s, 1545m, 1479m, 1448m, 1336w, 1278w, 1220w,

1147m, 1074w, 1624w, 975w, 924w, 793w, 754m, 689w, 665w,

2-{[5-(4-methoxyphenyl)thiophen-2-yl]methylidene}

638w cm^-1. H NMR (500 MHz, CD₃CD): δ 8.04 (s, 1H), 7.84-

1

hydrazinecarboximidamide hydrochloride (12) Following the

general procedure for guanylhydrazone formation, 12 was

obtained as a pale-yellow solid (140 mg, 72%). Mp = 260-264

^oC. IR (ATR): 3392s, 3312m, 3285m, 3222m, 3136s, 3130s,

3000m, 2970m, 2939m, 2916m, 2848m, 1668s, 1639s, 1603s,

1563m, 1541w, 1507m, 1465m, 1431m, 1351m, 1289m,

1250s,1180m, 1158m, 1119w, 1025m, 922w, 829w, 796m, 629w

7.78 (m, 2H), 7.43-7.40 (m, 2H), 7.37-7.31 (m, 1H), 7.03 (d, 1H,

J = 3.5 Hz), 6.94 (d, 1H, J = 3.5 Hz) ppm. ¹³C NMR (125 MHz,

CD₃CD): δ 158.12, 157.07, 149.62, 138.83, 131.26, 130.12,

129.78, 125.61, 118,.44, 108.85 ppm. (+)ESI-HRMS (m/z): [M +

H]⁺229.10821 (error -0.76 ppm). HPLC purity: Method A: RT

6.238, area 99.40%; method B: RT 7.318, area 98.69%.

cm^-1. H NMR (500 MHz, d-DMSO): δ 8.34 (s, 1H), 7.76-7.55

5-(pyridin-4-yl)thiophene-2-carbaldehyde²⁷The general

Suzuki coupling procedure was followed. The reaction mixture

was refluxed for 18 h. The crude product was purified by dry-

flash chromatography (SiO₂: hexane/EtOAc = 1:1) to afford 5-

(pyridin-4-yl)thiophene-2-carbaldehyde (125 mg, 50%) as an

1

(m, 6H), 7.50 (d, 1H, J = 4.0 Hz), 7.43 (d, 1H, J = 4.0 Hz), 7.06-

6.97 (m, 2H), 3.80 (s, 3H) ppm. ¹³C NMR (125 MHz, d-DMSO):

δ 159.99, 155.10, 147.14, 142.88, 135.87, 133.72, 127.46,

126.10, 123.49, 115.11, 55.70 ppm. (+)ESI-HRMS (m/z): [M +

H]⁺275.09530 (error -2.95 ppm). HPLC purity: Method A: RT

6.302, area 99.10%; method B: RT 7.392, area 99.91%.

o

pale-brown amorphous powder; mp = 107-110 C. IR (ATR):

3445s, 2361m, 1646s, 1543m, 1447m, 1415m, 1228m, 805m,

754w, 704m cm^-1. H NMR (500 MHz, CDCl₃): δ 9.95 (s, 1H),

1

2-{[5-(4-fluorophenyl)thiophen-2-yl]methylidene}

8.68 (d, 2H, J = 6.0 Hz), 7.80 (d, 1H, J = 4.0 Hz), 7.58 (d, 1H, J

= 4.0 Hz), 7.54 (d, 2H, J = 6.0 Hz) ppm. ¹³C NMR (125 MHz,

CDCl₃): δ 182.73, 150.74, 150.14, 144.22, 140.03, 136.86,

126.01, 120.23 ppm. (+)ESI-HRMS (m/z): [M + H]⁺190.03231

(error, +1.04 ppm).

hydrazinecarboximidamide hydrochloride (13) Following the

general procedure for guanylhydrazone formation, 13 was

obtained as a pale-yellow solid (145 mg, 100%). Mp = 195-200

^oC. IR (ATR): 3387s, 3278s, 3221s, 3167s, 2989m, 2855m,

2362w, 1690s, 1619s, 1538m, 1505s, 1463w, 1434w, 1350w,

1317w, 1230m, 1154m, 1103m, 1058m, 1009m, 916m, 830m,

791m, 658m, 578m cm^-1. NMR (500 MHz, CD₃OD): δ 8.26 (s,

1H), 7.75-7.69 (m, 2H), 7.41 (d, 1H, J = 4.0 Hz), 7.37 (d, 1H, J =

4.0 Hz), 7.20-7.13 (m, 2H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ

164.45 (d, J = 246.4 Hz), 156.98, 148.35, 144.27, 138.30, 134.26,

131.60 (d, J = 2.6 Hz), 129.12 (d, J = 8.1 Hz), 125.17, 117.18 (d,

J = 21.8 Hz) ppm. (+)ESI-HRMS (m/z): [M + H]⁺263.07710

(error 3.72 ppm). HPLC purity: Method A: RT 6.293, area

98.89%; method B: RT 7.413, area 99.59%.

2-{[5-(pyridin-4-yl)thiophen-2-yl]methylidene}hydrazine

carboximidamide dihydrochloride (17) Following the general

procedure for guanylhydrazone formation, 17 was obtained as a

o

pale-yellow solid (27 mg, 32%). Mp = 272-276 C. IR (ATR):

3311s, 3090s, 2992s, 2846s, 1678m, 1631s, 1535m, 1560s,

1462w, 1376w, 1326m, 1241w, 1202w, 1145w, 1698w, 1004w,

799w, 626w, 581w, 551w cm^-1. H NMR (500 MHz, D₂O): δ

1

8.59-8.55 (m, 2H), 8.11-8.09 (m, 1H), 8.09-8.07 (m, 2H), 7.82 (d,

1H, J = 4.5 Hz), 7.39 (d, 1H, J = 4.0 Hz) ppm. ¹³C NMR (125

MHz, D₂O): δ 154.66, 149.52, 143.66, 141.73, 141.26, 139.39,

133.22, 131.54, 122.06 ppm. (+)ESI-HRMS (m/z): [M + 2H]²⁺

123.54429 (error 2.09 ppm). HPLC purity: Method A: RT 6.184,

area 99.51%; method B: RT 6.273, area 96.37%.

2-{[5-(4-bromophenyl)thiophen-2-yl]methylidene}

hydrazinecarboximidamide hydrochloride (14) Following the

general procedure for guanylhydrazone formation, 14 was

obtained as a pale-yellow solid (72 mg, 99%). Mp = 236-240 ^oC.

IR (ATR): 3466s, 3423m, 3277s, 3139s, 2862m, 1678s, 1611s,

1526m, 1491w, 1463w, 1440w, 1398w, 1347w, 1314w, 1243w,

1153w, 1111w, 1079w, 1007w, 954w, 928w, 826w, 803m cm^-1.

¹H NMR (500 MHz, CD₃CD): δ 8.27 (s, 1H), 7.63-7.54 (m, 4H),

7.44-7.39 (m, 2H) ppm. ¹³C NMR (125 MHz, CD₃CD): δ 156.93,

147.93, 144.14, 138.73, 134.23, 134.18, 133.44, 128.70, 125.62,

123.43 ppm. (+)ESI-HRMS (m/z): [M + H]⁺322.99565 (error -

1.27 ppm). HPLC purity: Method A: RT 6.406, area 95.31%;

method B: RT 7.581, area 96.53%.

5-(4-formylphenyl)thiophene-2-carbaldehyde To a dry glass

flask purged with argon were added Pd(OAc)₂(2.9 mg, 0.013

mmol), PPh₃(14 mg, 0.050 mmol) and dry DME (2 mL). The

resultant solution was stirred at room temperature for 10 min, and

5-bromo-2-thiophenecarboxaldehyde (30 µL, 0.26 mmol) and

Na₂CO₃(aq) (2 M, 0.3 mL, 0.6 mmol) were added. After 5 min a

4-formylphenylboronic acid (49 mg, 0.33 mmol) was added and

reaction mixture was purged with argon and refluxed for 3 h

under argon. The solution was cooled to room temperature and

filtered through a pad of Celite, washed with CH₂Cl₂and dried

with anh. Na₂SO₄. The organic solvent was removed under

reduced pressure and the crude product was purified by dry-flash

chromatography (SiO₂: hexane/EtOAc = 7:3) to afford 5-(4-

formylphenyl)thiophene-2-carbaldehyde (54 mg, 96%) as an

2-{[5-(4-cyanophenyl)thiophen-2-yl]methylidene}

hydrazinecarboximidamide hydrochloride (15) Following the

general procedure for guanylhydrazone formation, 15 was

obtained as a pale-yellow solid (68 mg, 65%). Mp = 190-195 ^oC.

IR (ATR): 3936w, 3313s, 3182s, 2364w, 2233m, 1679s, 1637s,

1599s, 1504m, 1466m, 1412w, 1318m, 1271w, 1247m, 1177m,

1154m, 1013w, 832w, 800w cm^-1. ¹H NMR (500 MHz, CD₃OD):

δ 8.29 (s, 1H), 7.88-7.84 (m, 2H), 7.79-7.74 (m, 2H), 7.59 (d, 1H,

J = 4.0 Hz), 7.47 (d, 1H, J = 4.0 Hz) ppm. ¹³C NMR (125 MHz,

CDCl₃): δ 157.00, 146.65, 143.86, 140.43, 139.42, 134.21,

134.18, 127.59, 127.35, 119.64, 112.66 ppm. (+)ESI-HRMS

(m/z): [M + H]⁺270.08137 (error 2.12 ppm). HPLC purity:

Method A: RT 6.216, area 98.83%; method B: RT 7.217, area

98.44%.

o

pale-yellow amorphous powder; mp = 130-132 C. IR (ATR):

3078w, 3047w, 2844w, 2757w, 1693s, 1658s, 1601s, 1566m,

1506w, 1447s, 1396m, 1318w, 1291w, 1224s, 1183m, 1059w,

961w, 840m, 806m cm ^-1. H NMR (500 MHz, CDCl₃): δ 10.06

1

(s, 1H), 9.95 (s, 1H), 7.99-7.94 (m, 2H), 7.88-7.83 (m, 2H), 7.80

(d, 1H, J = 4.0 Hz), 7.55 (d, 1H, J =4.0 Hz) ppm. ¹³C NMR (125

MHz, CDCl₃): δ 191.20, 182.75, 151.85, 143.95, 138.52, 137.08,

136.48, 130.52, 126.84, 125.72 ppm. GC/MS (m/z (%)): 214.9

([M]⁺, 100).

2-[(5-{4-[(2-carbamimidoylhydrazinylidene)methyl]

2-[(5-phenylfuran-2-yl)methylidene]hydrazine

carboximidamide hydrochloride (16) Following the general

procedure for guanylhydrazone formation, 16 was obtained as a

phenyl}thiophen-2-yl)methylidene]hydrazinecarboximidamide

dihydrochloride (18) Following the general procedure for

guanylhydrazone formation, 18 was obtained as a pale-yellow

solid (37 mg, 100%). Mp = 248-250 ^oC. IR (ATR): 3352s, 3275s,

The minimum inhibitory concentration (MIC) of

guanylhydrazones to six human clinical fungal strains was

determined using a reference method for testing antimicrobial

agents for yeasts and moulds in a 96-well microtitre plate assay

(EUCAST 2012; EUCAST 2014).^29,30All tested compounds

were initially dissolved in DMSO in stock concentration of 50

mg mL^-1. Further dilutions were made in RPMI 1640 medium

(Roswell Park Memorial Institute 1640) with 2% (w/v) glucose

in the concentration range of 250-0.02 µg mL^-1. Additionally,

MIC of commercial antifungal voriconazole was determined for

each isolate (concentration range 16-0.001 µg mL^-1; Sigma

Aldrich, Munich, Germany). Time-kill dependence was

monitored spectrophotometrically at 600 nm over 12 h period at

37 °C for selected guanylhydrazones (11, 14 and 18) and

voriconazole using sub-MIC concentrations (70% of MIC value

determined for the planktonic growth) on Tekan Infinite 200 Pro

multiplate reader (Tecan Group Ltd., Männedorf, Switzerland).

3153s, 2872m, 1668s, 1612s, 1536m, 1437m, 1350w, 1237m,

-1

1181w, 1141m, 1011w, 829w cm

.

¹H NMR (500 MHz,

DO₃OD): δ 8.30 (s, 1H), 8.15 (s, 1H), 7.86 (d, 2H, J = 8.0 Hz),

7.78 (d, 2H, J = 8.0 Hz), 7.52 (d, 1H, J = 4.0 Hz), 7.45 (d, 1H, J

= 4.0 Hz) ppm. ¹³C NMR (125 MHz, DO₃OD): δ 157.26, 156.96,

148.75, 148.28, 144.14, 139.14, 137.25, 134.81, 134.29, 129.67,

127.32, 126.04 ppm. (+) ESI-HRMS (m/z): [M + H]⁺329.12823

(error: -2,77 ppm). HPLC purity: Method C: RT 6.322, area

99.52%; method D: RT 2.543, area 96.86%.

4,4'-thiene-2,5-diyldibenzaldehyde (20)²⁸: A reaction tube

containing a stirrer bar was evacuated and backfilled with argon.

The tube was then charged with Pd(OAc)₂(16 mg, 0.070 mmol),

PPh₃(73 mg, 0.28 mmol) and dry DME (7 mL) under argon. The

resultant solution was stirred at room temperature for 10 min, and

2,5-dibromothiophene (150 mg, 0.620 mmol) and Na₂CO₃(aq) (2

M, 1.2 mL, 2.4 mmol) were added. After

5 min, 4-

cyanophenylboronic acid (225 mg, 1.54 mmol) was added, the

tube was returned under argon and capped. The reaction mixture

was heated with stirring for 2 h at 80 °C in a MW reactor. The

solvent was then removed under reduced pressure, and the

reaction mixture was suspended in CH₂Cl₂, transferred to a

separation funnel, and washed well with saturated Na₂CO₃

solution (2×25 mL) containing 5 mL NH₃. The organic layer was

collected, dried with anh. Na₂SO₄, and the solvent was removed

under reduced pressure. The crude product was purified by dry-

flash chromatography (SiO₂: CH₂Cl₂) to afford the 4,4'-thiene-

2,5-diyldibenzonitrile (160 mg, 90%) as an pale-yellow

amorphous powder; mp = 282-284 ^oC. IR (KBr): 3432m, 2222s,

Pathogenic strains and preparation of inocula: Human

clinical fungal strains were obtained from collection of National

Reference Medical Mycology Laboratory (Institute of

Microbiology and Immunology, Faculty of Medicine, University

of Belgrade, Belgrade) and included: yeasts isolated from blood

(Candida albicans CA5 and C. parapsilosis CA27); non-

dermatophyte mould isolated from upper respiratory tract

(Fusarium oxysporum AB18); and dermatophyte moulds isolated

from skin derivates (Trichophyton mentagrophytes DMT2 and

Microsporum canis DMT4). The reference strains C. albicans

ATCC10231, C. krusei ATCC34135 and Aspergillus fumigatus

ATCC13073 were obtained from American Type Culture

Collection (ATCC). Stock culture of each isolate was maintained

on Potato dextrose agar (PDA; Hi Media Laboratories Mumbai,

India) at 4 °C. Prior to testing, working cultures were obtained by

preculturing stock cultures onto PDA in Petri dishes and

incubating for 7 days at 30 °C. The inocula were prepared by

resuspending fungal conidia from PDA working cultures in 4 mL

of sterile distilled water (for yeasts) or in 0.9% (w/v) NaCl with

0.1% (v/v) Tween 20 (for moulds). Conidial suspensions were

adjusted to 2-5 ×10⁶CFU mL^-1by using Neubauer

haemocytometer and diluted in RPMI 1640 medium with 2%

glucose in a ratio of 1:10 (2-5 × 10⁵CFU mL^-1).

1

1599s, 1491w, 1410w, 1278w, 1176w, 838m cm ^-1. H NMR

(500 MHz, DMSO): δ 7.97-7.87 (m, 8H), 7.84 (s, 2H) ppm.

To a stirred suspension of di-nitrile (20 mg, 0.070 mmol) in

PhMe (2.5 mL) was added DIBAH (0.45 mL, 1 M in toluene) at

o

0 C. After stirring at 0 C under Ar atmosphere for 1 h the

reaction was quenched with 5% H₂SO₄(0.60 mL), and stirring

was continued for 1 h at r.t. The reaction mixture was transferred

into a separation funnel as an emulsion in EtOAc (50 mL). The

organic layer was separated, and the aqueous layer was

additionally extracted with CH₂Cl₂(3×25 mL). The combined

organic layers were washed with brine (15 mL) and dried over

anh. Na₂SO₄. The organic solvent was removed under reduced

pressure and the crude product 20 (20 mg, 98%) was obtained as

a pale-yellow solid and was used for the next reactions without

further purification. Mp = 149-151 ^oC. IR (ATR): 3349w, 3198w,

3069m, 2923m, 2854m, 2764w, 1694s, 1599s, 1568m, 1498w,

1451w, 1423w, 1397w, 1346w, 1310w, 1280m, 1216m, 1167m,

1116w, 838m cm ^-1. ¹H NMR (500 MHz, CDCl₃): δ 10.03 (s, 2H),

7.93 (d, 4H, J = 8.5 Hz), 7.80 (d, 4H, J = 8.5 Hz), 7.50 (s, 2H)

ppm.

Volume of 100 µL of prepared inocula was incubated together

with 100 µL of different concentrations of guanylhydrazones and

voriconazole at 37 °C (for yeasts) or at 30 °C (for moulds) and

the growth inhibition was assessed over time (24 h for Candida

spp.; 48 h for A. fumigatus and F. oxysporum and 96 h for M.

canis and T. mentagrophytes). Controls containing solvents were

carried out in each assay. MIC was defined as the lowest

concentration of compound at which no evident fungal growth

was observed. The assay was repeated two times in triplicates.

(2,2'-{thiene-2,5-diylbis[benzene-4,1-diylmethylylidene]}

dihydrazinecarboximidamide dihydrochloride (21) Following

the general procedure for guanylhydrazone formation, 21 was

2.5. Microscopic studies

C. albicans ATCC10231 overnight culture was diluted to

OD600=1 and incubated with MIC of compounds 11 and 18. At

time points 0 min and 2 h the cell viability and membrane

integrity were assessed after staining with 1 µg mL^-12-(4-

amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI,

Sigma Aldrich), 2.5 µM propidium iodide (PI, Sigma Aldrich)

and 50 µg mL^-1Concanavalin A conjugated with FITC (Sigma

Aldrich) in PBS using fluorescence microscope (Olympus BX51,

Applied Imaging Corp., San Jose, USA) at 40× magnification.

o

obtained as a yellow solid (20 mg, 48%). Mp = 266 C. IR

(ATR): 3362m, 3332m, 3160m, 2917m, 2850m, 1737m, 1673s,

1619s, 1539m, 1511m, 1456w, 1148w cm ^-1. ¹H NMR (500 MHz,

DO₃OD): δ 8.12 (s, 2H), 7.88-7.84 (m, 4H), 7.80-7.76 (m, 4H),

7.54 (s, 2H) ppm. ¹³C NMR (125 MHz, DMSO): δ 155.39,

146.25, 142.60, 135.11, 132.82, 128.44, 126.26, 125.36 ppm. (+)

ESI-HRMS (m/z): [M + H]⁺405.16006 (error: -0,94ppm). HPLC

purity: Method C: RT 6.342, area 99.06%; method D: RT 2.761,

area 95.73%.

2.6. Aniproliferative assay on human fibroblast cell line

2.4. Broth dilution method for antifungal susceptibility testing

Antiproliferative activity was tested by (3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

assay on human lung fibroblasts (MRC5; ATCC collection). The

cells were incubated in the media containing test compounds at

concentrations ranging from 0.1 to 250 µg mL^-1and the cell

viability was measured after 48 h. MRC5 cell line was cultured in

RPMI-1640 medium supplemented with 100 µg mL^-1

streptomycin, 100 U mL^-1penicillin and 10% (v/v) fetal bovine

serum (FBS) (all from Sigma, Munich, Germany). Cells were

maintained as a monolayer (1 x 10⁴cells per well) in RPMI-1640

and grown in humidified atmosphere of 95% air and 5% CO₂at

37 °C.

the concentration of DNA were estimated by measuring UV

absorbance with a NanoVue Plus spectrophotometer (GE

Healthcare, Freiburg, Germany). The ability of guanylhydrazones

to bind gDNA from C. albicans was examined by using agarose

gel electrophoresis.³²Briefly, gDNA (500 ng) was treated with

the compounds (25 μg mL-1) in phosphate buffer (pH 7.4), and

the contents were incubated for 12 h at 37 °C, then subjected to

gel electrophoresis on 0.8% (w/v) agarose gel containing 0.1

μg/mL of ethidium bromide in TAE buffer (40 mM Tris acetate/1

mM EDTA, pH 7.4) buffer at 60 V for 2 h. Gels were visualized

and analyzed using the Gel Doc EZ system (Bio-Rad, Life

Sciences, Hercules, USA), equipped with the Image Lab™

Software.

The extent of MTT reduction was measured

spectrophotometrically at 540 nm using Tekan Infinite 200 Pro

multiplate reader (Tecan Group Ltd., Männedorf, Switzerland)

and the cell survival was expressed as percentage of the control

(untreated cells). The percentage viability values were plotted

against the log of concentration and a sigmoidal dose response

curve was calculated by non-linear regression analysis using

Graphpad Prism software version 5.0 for Windows (Graphpad

Software, CA, USA). Cytotoxicity is expressed as the

concentration of the compound inhibiting growth by 50% (IC₅₀).

2.10. The effect of guanylhydrazones on C. albicans yeast to

hyphe transition using Spider medium

Morphological changes of C. albicans in the presence and

absence of guanylhydrazones and voriconazole in subinhibitory

concentrations (MIC_70;70% of MIC value determined for the

planktonic growth) was observed upon C. albicans growth on

Spider medium as previously described.³³

2.7. Hemolytic assay

2.11. The effect of guanylhydrazones on Candida biofilm

formation and disruption

Sheep red blood cells in PBS (1% v/v, Torlak, Belgrade,

Serbia) were treated for 1 h with concentration of compounds

that corresponded to determined MIC and IC50 values at 37°C.

Hemoglobin absorbance was measured at 405 nm (Tekan Infinite

200 Pro multiplate reader (Tecan Group Ltd., Männedorf,

Switzerland). The hemolysis percentage was calculated using the

following equation: hemolysis (%) = 100[(Abs_405nm(treated) -

Abs_405nm(non treated) / (Abs_405nm(0.1% Triton X-100 lysed) -

Abs_405nm(non treated)].

The biofilm disruption ability of guanylhydrazones was tested

by determining minimal biofilm eradication concentration

(MBEC), defined as the lowest concentration of an antimicrobial

agent required to completely eradicate biofilm. Candida biofilms

were developed in flat bottom 96-well clear microtitre plates as

described previously.³⁴Briefly, harvested from the overnight

grown liquid C. albicans ATCC10231, C. krusei ATCC34135

and C. parapsilosis CA27 cultures were washed twice and

2.8. Liposomes leakage assay

collected by centrifugation (3000

× g) in sterile PBS,

resuspended in RPMI-1640 with 2% glucose and adjusted to cell

density of 1 × 10⁶cells mL^-1using Neubauer haemocytometer.

Biofilms were formed by pipetting 100 μL of the prepared cell

suspension into wells of a microtiter plate and incubated for 48 h

at 37°C. The established biofilms were rinsed with PBS to

remove non-adherent cells and subsequently treated with two-

fold serial dilutions of guanylhydrazones (concentration range

1000-25 µg mL^-1). The resultant biofilm biomass was quantified

using the CV assay and was compared with untreated controls.

The bioassay was performed in six replicates of each strain.

Mixtures containing L-α-phosphatidylcholine (PC, from egg

yolk) (Sigma Aldrich, Munich, Germany) and L-a-

phosphatidylserine (PS, from bovine brain) (Sigma Aldrich,

Munich, Germany) (PC/PS=8:2), or PC, PS and ergosterol

(E)(Sigma Aldrich, Munich, Germany) (PC/PS/E=11/4/5) were

dissolved in chloroform and dried under a stream of argone

followed by vacuum drying. Large unilamellar vesicles (LUVs)

with a diameter of 0.4 mm containing 50 mM calcein (Molecular

Probes, Invitrogen) were prepared using an extruder (Avestin

Europe GmbH., Mannheim, Germany) according to Hope et al.³¹

The non-encapsulated dye was removed from the liposome

suspension by ultracentrifugation. The osmolarity of the solution

(calcein and buffer) was measured with an Osmomat 030,

Gonotec GmbH (Berlin, Germany).

To study the effect of selected guanylhydrazones (11 and 18)

on C. albicans biofilm formation, overnight fungal culture grown

in RPMI medium supplemented with 2 % glucose and 10 % FCS

was diluted to optical density OD600 of 0.1 and the biofilms

were grown on plastic cover slips in the presence of MIC of

guanylhydrazones or DMSO. After 24 h growth at 37 °C,

biofilms were washed with PBS, stained with 2.5 µM propidium

iodide (PI, Sigma Aldrich) and 50 µg mL^-1Concanavaline A

conjugated with Fluorescence isothiocyanate (FITC; Sigma

Aldrich) in PBS, and observed by microscopy.

Liposome leakage assay was performed in 96-well plate with

50 nmol LUV suspension added per well and calcein release was

recorded by measuring the fluorescence at excitation and

emission wavelengths of 485 and 518 nm, respectively at room

temperature using Tekan Infinite 200 Pro multiplate reader

(Tecan Group Ltd., Männedorf, Switzerland). The percentage of

fluorescence F_tat time t is defined as:

To examine the biofilm disruption activity of the same

compounds, 48 – 72 h pre-formed C. albicans biofilms were

washed with PBS and then incubated with MIC of 11 and 18 or

DMSO in RPMI for 24 h followed by DAPI, PI and

Concanavaline A staining as above. Biofilms and cells were

examined using fluorescence microscope (Olympus BX51,

Applied Imaging Corp., San Jose, USA) at 20× magnification.

% F_t=(I_t-I₀/I_f-I₀)x100;

where I₀was the initial fluorescence obtained after the dilution of

the vesicles in the appropriate buffer, I_fwas the total fluorescence

observed after addition of Triton X-100, and I_twas the

fluorescence at time t corrected for the dilution.

2.12. Molecular docking studies

2.9. In vitro DNA binding by gel electrophoresis assay

Glide module incorporated in the Schrödinger Molecular

Modeling Package (Maestro Version 10.1.013, free trial) was

Genomic DNA (gDNA) from C. albicans was purified with a

DNeasy tissue kit (Qiagen, Hilden, Germany). The quality and

used for performing molecular docking studies. For estimating

protein-ligand binding affinities Glide Extra-Precision (XP)

scoring function was adopted in the presented study. In this

scoring function force field-based parameters like solvation and

repulsive interactions, lipophilic, hydrogen bonding interactions,

metal-ligand interactions as well as contributions from coulombic

and van der Waals interaction energies are incorporated in the

empirical energy functions calculations. Up to this day there is no

reported X-ray structure of sterol 14α-demethylase (CYP51)

from C. albicans. For this reason homology model was used for

docking study.³⁵The X-ray structure of human CYP51 (PDB

code: 3JUV) was obtained from the RCSB Protein Data Bank

(PDB), http://www.rcsb.org/pdb. Protein preparation wizard with

the application of the OPLS-2005 force field was applied for

preparing protein-inhibitor complexes. Hydrogen atoms were

added to the structure corresponding to pH 7.0 with the

consideration of the appropriate ionization states for both the

acidic and basic amino acid residues and any crystallographic

water, if present was eliminated. Further, prepared structure was

subjected to energy minimization until the average root

meansquare deviation (r.m.s.d.) reached 0.3 Ǻ. The 3D structures

of the studied molecules were built in the Maestro Suite of the

module and subsequently optimized using the LigPrep module in

the Schrödinger Suite. Partial atomic charges were computed

using the OPLS-2005 force-field and possible ionization states

were generated at a pH of 7.0. The ligand structures thus obtained

were further optimized by energy minimization using the LBFGS

method until a gradient of 0.01 kcal mol^-1Å^-1was achieved.

2.13. Zebrafish embryotoxicity of selected guanylhydrazones

The assessment of toxicity (lethality and teratogenicity) of

selected guanylhydrazones (11, 14 and 18) and voriconazole on

zebrafish embryos was performed following general rules of the

OECD Guidelines for the Testing of Chemicals.³⁶All

experiments involving zebrafish were performed in compliance

with the European directive 86/609/EEC and the ethical

guidelines of Guide for Care and Use of Laboratory Animals of

Institute for Molecular Genetics and Genetic Engineering,

University of Belgrade.

Adult zebrafish (Danio rerio, wild type) were maintained in

the fish medium (2 mM CaCl₂, 0.5 mM MgSO₄, 0.7 mM

NaHCO₃, 0.07 mM KCl) at 27 ± 1°C and 14 h light/10 h dark

cycle, and regularly fed twice daily with commercially dry flake

food supplemented with Artemia nauplii (TetraMin^TMflakes;

Tetra Melle, Germany). Eggs at 6 hours post fertilization (hpf)

were treated with five different concentrations (2.5, 5, 10, 25, and

50 μg mL^-1) and 0.15% DMSO as negative control. Embryos

were then individually transferred into 24-well plates containing

1000 μl test solution, 10 embryos per well, and incubated at

28°C. Experiments were repeated three times, using 30 embryos

per concentration.

Apical endopoints (Table S1) for toxicity evaluation were

recorded at 24, 48, 72, and 96 hpf using an inverted microscope

(CKX41; Olympus, Tokyo, Japan. At 96 hpf, the embryos were

anesthetized by addition of 0.1% (w/v) tricaine solution (Sigma-

Aldrich, St. Louis, MO), photographed and killed by freezing at

−20°C for ≥ 24 h.

Molinspiration tool (Molinspiration Cheminformatics-2013)

was used for calculating physic-chemical properties of

investigated guanylhydrazones. For calculating of logP fragment-

based contributions and correlation factors were used.

o

.

Scheme 1 Synthesis of the target compounds 10-16. Conditions: (i) ArB(OH)₂, Pd(OAc)₂, PPh₃, DME, Na₂CO₃aq, EtOH, 90 C; (ii) H₂NNHC(NH)NH₂HCl,

EtOH, HCl, 90 ^oC.

Scheme 2 Synthesis of the target compounds 17 and 18. Conditions: (i) (a) ArB(OH)₂, Pd(OAc)₂, PPh₃, DME, Na₂CO₃aq, EtOH, 90 ^oC; (b)

.

H₂NNHC(NH)NH₂HCl, EtOH, HCl, 90 ^oC; (ii) (a) ArB(OH)₂, Pd(OAc)₂, PPh₃, DME, Na₂CO₃aq, 90 ^oC; (b) H₂NNHC(NH)NH₂HCl, EtOH, HCl, 90 ^oC

o

Scheme 3 Synthesis of the target compound 21. Conditions: (i) (a) ArB(OH)₂, Pd(OAc)₂, PPh₃, DME, Na₂CO₃aq, 80 C, µW; (b) DIBAL, PhMe, 0 C; (ii)

.

H₂NNHC(NH)NH₂HCl, EtOH, HCl, 90 ^oC

and F. oxysporum AB18) exhibited increased resistance towards

standard antifungal compounds, including resistance to

voriconazole. The results of in vitro antifungal screening (MIC

values: minimal inhibitory concentrations that causes fungicidal

effect) and cytotoxicity (IC₅₀values) of the test compounds are

given in Table 1.

3. Results and discussion

3.1. Chemistry

The synthesis of guanylhydrazone derivatives 10-16 was

accomplished using a simple two-step procedure described in

Scheme 1. The first step involved Suzuki cross-coupling reaction

between commercially available thiophene 1 and furan 2

derivatives and arylboronic acids.²⁷Subsequent condensation of

aldehydes 3-9 with aminoguanidine hydrochloride afforded

compounds 10-16 in moderate to excellent yields.

All synthesized guanylhydrazones showed considerable

antifungal activity against type strains, as well as human clinical

isolates (Table 1). Aldehyde 3 did not show significant antifungal

activity with MIC values of 250 µg mL^-1and 125 µg mL^-1against

A. fumigatus, which indicated that the guanylhydrazone group

was essential for the antifungal activity of the tested compounds.

Thiophene-based guanylhydrazone 10 showed higher inhibitory

activity than corresponding furan analog 16 against all screened

fungi, so all further examined derivatives contained thiophene

substructure.

Following the similar two-step procedure guanylhydrazone

derivative 17 and bis-guanylhydrazone 18 were obtained

(Scheme 2).

Preparation of the bis-guanylhydrazone 21 was accomplished

by reaction of dialdehyde 20²⁸with aminoguanidine

hydrochloride (Scheme 3).

Nine out of ten guanylhydrazones exhibited promising activity

against C. albicans type strain with MICs ranging from 0.9 to 7.8

µg mL^-1, with derivative 11 containing methyl group in the para-

position of the phenyl ring being the most active one. It is

noteworthy that seven out of ten derivatives showed excellent

activity against voriconazole resistant C. albicans CA5 with

MICs ranging from 0.25 to 3.9 µg mL^-1. In the case of this

clinical isolate, bis-guanylhydrazone 21 exhibited the best

activity with MIC in submicromolar range (MIC = 0.25 µg mL^-1).

Additionally, desirable high selectivity towards C. albicans CA5

strain in comparison to human lung fibroblasts (SI = 20) was

observed for compound 21 (Table 1). Similarly, 20-fold ratio was

observed for another bis-guanylhydrazone derivative 18 in the

case of C. albicans CA5 strain, suggesting lower cytotoxicity of

bis-derivatives.

3.2. Antifungal activity and cytotoxic properties of

guanylhydrazones

The aldehyde 3 and guanylhydrazones 10-18 and 21 were

evaluated for their in vitro antifungal activity against Candida

albicans ATCC10231, Candida albicans CA5, Candida krusei

ATCC34135, Candida parapsilosis C27, Aspergillus fumigatus

ATCC13073, Fusarium oxysporum AB18, Microsporum canis

DMT2 and Trichophyton mentagrophytes DMT4, where

voriconazole was used as reference drug. The antifungal activity

on well characterized clinical isolates (C. albicans CA5, C.

parapsilosis C27, F. oxysporum AB18, M. canis DMT2 and

T. mentagrophytes DMT4) was carried out at National Reference

Medical Mycology Laboratory, Institute of Microbiology and

Immunology, Faculty of Medicine, University of Belgrade,

Serbia. Clinical isolates (C. albicans CA5, C. parapsilosis C27

Table 1 Antifungal activity and cytotoxic properties of guanylhydrazones 10-21 in comparison to aldehyde 3 and

voriconazole (VOR)

Cytotox.

(µg mL^-1)^a

Minimal inhibitory concentrations (MICs, µg mL^-1)

Compd

Candida

albicans

Candida

albicans

CA5

Candida

parapsilosis

C27

Aspergillus

fumigatus

Fusarium Microsporum

Trichophyton

mentagrophytes

DMT4

Candida krusei

oxysporum

canis

MRC5^b

ATCC34135

ATCC10231

ATCC 13073

AB18

DMT2

3

10

250

1.95

0.90

7.80

3.90

3.12

3.90

7.80

15.6

3.90

0.50

250

1.95

0.50

3.9

>250

6.25

3.12

6.25

1.56

25

>250

6.25

3.90

6.25

3.90

12.5

50

125

25

250

62.5

7.8

250

1.56

0.97

0.25

0.97

1.56

125

250

3.12

1.56

3.12

1.56

0.97

6.25

25

50

5

11

6.25

25

2.5

5

12

>250

3.9

13

1.95

1.56

7.80

15.6

125

0.50

0.25

R^d

25

4

14

6.25

25

2

15

>250

125

5

16

50

125

250

25

20

25

10

5

17

>250

25

>250

50

>250

R

18

0.97

0.25

0.50

3.90

0.25

21

50

25

VOR^c

0.06

R

0.06

40

^aIC₅₀; SD values ±2-5%. ^bhuman lung fibroblasts. ^cControl drug: VOR as a voriconazole. ^dResistance.

Guanylhydrazones 11 and 14 with methyl- and bromo-

substituent in para-position on the phenyl ring, respectively,

showed broad spectrum activity. Of particularly interest was their

activity against voriconazole resistant C. parapsilosis C27 (MIC

= 3.9 µg mL^-1) and F. oxysporum AB18 (11: MIC = 7.8 µg mL^-1;

14: MIC = 3.9 µg mL^-1) strains. However, they both exhibited

potent in vitro antirpoliferative effect on human fibroblasts

(Table 1). Nevertheless, in the case of 11, MIC concentrations

were 5 and 2.7 times lower in comparison to IC₅₀value for C.

albicans CA5 and ATCC10231 strains, respectively. With

respect to dermatophyte M. canis, compounds 14 and 21 showed

improved activity (MICs = 0.25 µg mL^-1) in comparison to the

standard drug voriconazole (MIC 0.5 = µg mL^-1). The MIC

values for novel guanylhydrazones were comparable or better to

recently reported novel triazole derivatives containing the 1,2,3-

triazole group against Candida strains.³⁷

Antifungal activity of new guanylhydrazones 10-17 was not

apparently dependent on the electronic effect of substituent on

phenyl ring. However, based on the activity of the compounds 11

and 14, hydrophobic effect could be important for the

pronounced antifungal activity. Interestingly, although deemed

essential for antifungal activity, an additional guanylhydrazone

substructure did not have an additive effect on the antifungal

activity of compound 18 in comparison to parent compound 10

against most of the tested strains. On the other side, bis-

guanylhydrazone 18 was 4 times more potent than 10 against

voriconazole resistant C. albicans CA5 human clinical isolate.

Based on the obtained results, compounds 11, 14 and 18 were

identified as promising candidates and were further evaluated.

Antifungal properties and time-kill dependence of selected

guanylhydrazones were examined and followed further using

subinhibitory concentrations of 11, 14 and 18 and compared to

activity of voriconazole (Fig. 2A).

Figure 3. A) Hemolytic effect of guanylhydrazones using MIC (black

bars) and IC₅₀(light grey bars) concentrations. B) Release of calcein from

PC/PS and PC/PS/E liposomes in the presence of selected guanylhydrazones

and voriconazole (VOR) and nystatin (NYS) was monitored for 30 min at 518

nm. C) In-vitro DNA interaction ability of guanylhydrazones with C.albicans

DNA (M-molecular marker λ DNA/HinDIII digest; C-DMSO treatment).

Within 12 h of incubation, voriconazole-treatred cells reached

the growth of the control that was treated with DMSO, while 11

and 14 treated cells reached 50% of the control growth. On the

other side, compound 18 exhibited pronounced fungistatic effect

even after 12 h, not allowing the growth of C. albicans culture.

However, within 24 h all cultures reached the full growth (results

not shown). It can be concluded, that under conditions tested, 11

and 14 showed better fungistatic effect then voriconazole, but not

as good as 18. Next, we examined the effect of 11 and 18 on C.

albicans membranes by the combined concavaline/PI/DAPI cell

staining assay. PI is membrane impermeable dye and binds to

nucleic acid only in the dead cells yielding fluorescence in the

red wavelength region. DAPI easily passes the membrane and

strongly binds to DNA of both living and dead cells (giving blue

color). C. albicans was treated with 11 and 18 MIC

concentrations for 2 h and showed that treatment with compound

18 induced the membrane damage and the cell death in only

about 20 % cells according to red PI staining (Fig. 2 D). The cell

death was not observed in Candida sample treated with 11, as PI

staining of these cells (Fig. 2C) was comparable to non-treated

control (Fig. 2B). None of the treatments induced apoptosis,

since the DAPI staining appeared light blue and homogenous in

all samples indicating the absence of nuclei condensation (Fig.

2C and D).

Figure 2. Antifungal effect of selected guanylhydrazones on C. albicans

growth. A) Time-kill effect of subinhibitory concentrations of 11 (diamond),

14 (circle), 18 (black square), and voriconazole (triangle) on C. albicans

growth measured by optical density in comparison to DMSO treated contol

(white square). Effect of MIC concentrations of 11 (C) and 18 (D) on culture

of C. albicans (OD600=1) after 2 h in comparison to DMSO treated culture

(B) revealed by fluorescent microscopy using Concavaline-FITC/PI/DAPI

staining.

We also studied the effect of compounds on the membrane

integrity of sheep red blood cells (RBC) to determine their ability

to cause hemolysis and to examine their effect on cell membrane

(Fig. 3A). We have monitored the release of haemoglobin from

RBCs treated with concentrations of compounds corresponding

to the lowest MIC against fungal strains and IC₅₀values (Table 1)

and showed that generated guanylhydrazones mostly did not

exhibit membrane disturbances (up to 10% hemolysis), except

for 17 and 18 at 25 µg mL^-1and 10 µg mL^-1respectively, causing

about 20% hemolysis (Fig. 3A).

Given that polyenes, such as clinically used nystatin, with

known membrane disturbance mode of action,^38,39caused 35-

40% hemolysis at 40 µg mL^-1(results not shown) under same

conditions, we concluded that membrane may not be the primary

target of novel guanylhydrazones. In addition, the ability of

selected guanylhydrazones (11, 14, 18 and 21) to disrupt

unilamellar liposomes was assessed (Fig. 3B). Two type of

liposomes were included in the leakage assay: ones containing L-

α-phosphatidylcholine and L- α -phosphatidylserine (PC:PS) and

the ones mimicking fungal membrane consisting of L-α-

phosphatidylcholine, L- α -phosphatidylserine and ergosterol

(PC:PS:E) in the ratio 11:4:5. At MIC concentrations, only 18

induced 8% PC:PS liposome leakage in comparison to that of 12

and 21 induced by voriconazole and nystatin, respectively.

However, low leakage of liposomes containing ergosterol and

mimicking fungal membrane of 7-12% was induced by all

selected guanylhydrazones at MIC concentrations (Fig. 3B).

Therefore, membrane disruption was excluded as primary mode

of action of these compounds.

We also examined in-vitro interaction of these molecules with

C. albicans genomic DNA using gel electrophoresis assay after

prolonged incubation (12 h) of compounds (25 µg/mL) with

purified DNA. This study was prompted by ability of the

guanylhydrazone groups to bind to DNA.⁴⁰The basicity of the

guanylhydrazone groups open possibilities that this functionality

bind to the phosphate groups of the DNA molecule. Furthermore,

intercalating properties might be also envisaged due to possible

near planar geometry of guanylhydrazone group and aromatic

ring.

Figure 4. Effect of subinhibitory amounts (MIC₇₀) of guanylhydrazones

derivatives on C. albicans hyphal growth.

planktonic growth; MIC₇₀) showed complete inhibition of

C. albicans filamentous growth on the hyphal inducing Spider

medium (Fig. 4). Guanylhydrazone 16 almost completely

inhibited the formation of hyphe, while aldehyde 3 and

compound 14 showed the inhibition to lesser extent in

comparison to DMSO control. On the other hand, 21 that showed

excellent activity against C. albicans CA5, apparently induced

higher level of hyphal formation in comparison to the control

(Fig. 4).

Under tested conditions, all guanylhydrazones, apart from

aldehyde 3, showed the excellent and comparable ability to

competitively intercalate double stranded fungal genomic DNA,

which resulted in the inability of ethidum bromide to intercalate

and emit under UV exposure (Fig. 3C). Ethidium bromide is one

of the most sensitive fluorescence probes for DNA binding:

intercalation of a substrate into DNA leads to a decrease in the

fluorescence intensity of the ethidium bromide-DNA complex.

Significant and comparable DNA interaction properties of all

tested guanylhydrazones lead to the conclusion that this may be a

primary cause of their observed cytotoxicity, as well as one of the

possible mechanisms of antifungal action (Table 1).

Combined results for compounds 11 and 18 supported further

screening for their ability to inhibit C. albicans biofilm formation

and to disrupt pre-formed mature biofilms. C. albicans was

cultured in the absence or in the presence of MIC concentrations

of compounds 11 and 18, and biofilms were allowed to form on

plastic coverslips for 24 h. Fluorescence isothiocyanate

conjugated Concanavalin A-FITC staining of Candida cells

demonstrated that, comparing to non-treated control, both

compounds completely prevented the hyphal growth which is an

essential feature for biofilm formation (Fig. 5A). Observed result

was in accordance to obtained effects on the Spider medium (Fig.

4) and showed that both compounds have an outstanding

antifungal properties in terms of preventing Candida biofilms

formation. Obtained result is very important, having in mind that

C. albicans usually very readily forms biofilms on indwelling

medical devices and mucosal tissues, which then serve as

infectious reservoir that is difficult to eradicate and influence

drug response.⁴⁶

3.3. Activity of guanylhydrazones against Candida biofilms

C. albicans can grow in three different morphologies: yeast,

pseudohyphae and hyphae. In particular, the hyphal form plays

an important role during infection process and virulence⁴¹and it

is as critical in biofilm formation. Biofilms represent complex

community of microorganisms surrounded by extracellular

polymeric matrix that are attached to a surface, such as plastics

and catheters.^42,43Biofilms protect microorganisms from host

human immune system and antimicrobial agents.⁴⁴Candida spp.

biofilms are difficult to eradicate because they are resistant to

most antifungal drugs, including the azoles and amphotericin B,

but are susceptible to the echinocandins.⁴⁵Therefore, we have

examined the effect of guanylhydrazones on Candida yeast to

hypae transition, and biofilm formation and dispersion.

When mature Candida biofilms were pre-formed for 48 h and

then exposed to MIC concentrations of the same compounds,

Concanavalin A-FITC staining showed that 11 was more

effective in biofilm disruption (Fig. 5B) versus 18. However,

minimal biofilm eradication concentration (MBEC) determined

Compounds 10-13, 15, 17 and 18, as well as voriconazole at

sub-MIC concentrations (70% of MIC value determined for the

Figure 5. Activity of 11 and 18 on C. albicans biofilms. A) inhibition of biofilms formation and B) disruption of preformed Candida biofilms (Scale bar=10 µm)

Figure 6. Effects of 11, 14 and 18 on development of zebrafish embryos in comparison to Vor and DMSO treated control at 96 hpf. LC₅₀represent compound

concentration estimated to cause lethal effect in 50% of embryos. This data is from three repeat experiments. 30 embryos were tested for each treatment.

Developmental embryos’ malformation such as pericardial edema (arrowhead), unresorbed egg yolk (asterisk), lordosys (dashed arrow), tail tissue disintegration

(arrow), small head (bracket) and reduced otoliths (boxed) are denoted.

.

against mature biofilms of three strains C. albicans ATCC10231,

C. krusei ATCC34135 and C. parapsilosis CA27 were all much

higher than MIC values and in the range above 125 µg mL^-1

(Table S2) suggesting that these compounds could not be used

for efficient biofilm eradication.

rerio embryos were examined at concentrations close to MIC and

IC₅₀values determined in antifungal and cytotoxicity assessment

(Table 1). Strikingly, 18 showed no lethal or adverse

developmental effects up to 50 μg mL^-1, while LC₅₀values of 11

and 14 were comparable and 2.6- and 4.5-fold higher in

comparison to that of Vor, respectively (Fig 6). None of tested

guanylhydrazones, applied at concentration of 2.5 μg mL^-1,

showed lethal or adverse developmental effects on treated

embryos until 96 hpf (Fig. 6). Survived embryos upon Vor

treatment, found only at 2.5 and 5 µg mL^-1, were reduced in

growth and exhibited a serious skeletal deformities, whereas at 5

µg mL-1 embryos suffered of cardiac dysfunctions such as

3.4. In-vivo embryotoxicity assessment

The zebrafish allows high-quality in-vivo validation of drug

leads early in drug discovery, well before clinical trials serving as

a very useful bioassay platform for toxicology studies.⁴⁷

Therefore, the effects of the most potent guanylhydrazones 11, 14

and 18 and those of voriconazole on the development of Danio

pericardial edemas, low heart beating and impaired caudal

circulation (Fig. 6). Embryos treated with compounds 11 and 14

did not developed scoliosis like those upon Vor treatment, and

suffered only of relatively small pericardial edemas (Fig. 6). At

the highest concentration of 11 (10 µg mL^-1) and 14 (25 µg mL^-1)

where alive embryos were still detected, some tissue

disintegration in tail region was observed. At 5 and µg mL^-1of

11, embryos were reduced in growth, had smaller head and

otoliths reduced in size (Fig. 6).

according to Docking Score are presented in Table 2. In the case

of C. albicans CYP51, the highest interaction was calculated for

bis-guanylhydrazone 18, followed by 16, 12 and 3. In the case of

human CYP51, compound 18 still had the highest calculated

score, followed by 16, 17, and 13. The analysis of the Glide

docking scores showed that these compounds docked to the

active site of the cytochrome P450 sterol 14α-demethylase, so the

antifungal activity of studied compounds could be reflected by

their interaction with the heme cofactor as well as amino acids in

the CYP51 active site, where the guanylhydrazones side chain

plays very important role (Fig. 7).

Embryotoxicity data showed that at tested concentrations 11,

14 and 18 were less embryotoxic and teratogenic than

voriconazole. To the best of our knowledge there are no studies

that evaluated voriconazole in zebrafish embryos model, however

toxic side effects of this triazole drug have been recently reported

such as visual disturbances through inhibition of TRPM1 and

TRPM3 cation channels or the effect on renal function.^48,49

Furthermore, the interaction of these compounds at the active

site of CYP51 may also be influenced by the heterocyclic ring

fused to the main scaffold along with the substituents borne by it.

It has to be noted that in presented study the real interaction

profile for the synthesized compounds discussed herein has not

been establish experimentally. However, it is interesting to

discuss the predicted interactions for the best docking poses,

since two and three compounds, respectively to studied enzyme,

showed better activity in comparison to that of voriconazole.

Surprisingly, one of the most active compounds 11, did not rank

amongst the first five in docking analysis while inactive aldehyde

3 did (Table 2).

3.5. Molecular docking and evaluation

In order to further evaluate possible mode of antifungal action

of studied compounds, we have decided to examine one of the

most studied molecular targets of antifungal compounds, namely

lanosterol 14α-demethylase (CYP51) from the ergosterol

biosynthetic pathway, by in silico molecular docking. CYP51 is a

member of the cytochrome P450 super family and is implicated

in the biosynthesis of fungal ergosterol, by catalyzing the

oxidative removal of the 14α-methyl group of lanosterol to give

the ∆^14,15- unsaturated key intermediates.⁵⁰Given that up to date

the crystal structure of CYP51 enzyme for Candida spp. has not

been obtained we have used appropriate homology model for

computation analysis. It is reported that typical CYP51 inhibitors

fit in the putative active site of CYP51 by a combination of heme

co-ordination, hydrogen bonding, π-π stacking and hydrophobic

interactions.^51,52These interactions at the active site of CYP51,

provide the basis for the design of novel and potential antifungal

agents with a broad antifungal spectrum and possibly with less

potential to develop drug resistance. On the another hand, most

commonly used antifungal drugs show undesirable side effect by

reacting with human cytochrome P450 super family enzymes.⁵³

Therefore, we have carried out comparative binding study of

synthesized guanylhydrazones with CYP51 and human

cytochrome P450 to examine the possibility if synthesized

guanylhydrazones have the minimal structural prerequisites to

interact with these targets.

Docking mode of the best compounds poses, according to

docking score, in the active site of CACYP51 is presented in

Figure 6. There are many amino acid residues in the active site of

cytochrome P450 sterol 14α-demethylase that can participate in

close van der Waals and coulombic interactions with the

inhibitor, when bound to it. For this reason the active site of

cytochrome P450 sterol 14α-demethylase can be considered as

highly conserved. Another important interaction of ligand with

enzyme active site is the formation of hydrogen bond with amino

acids. The amino acid THR-311 is most prominently involved in

the interaction in the case of most ligands. Comparison of

docking scores and other studied functions presented at Table 2

revealed that compound 18 (Fig. 7A) possibly has better affinity

for CACYP51 in comparison to human enzyme. Overall, good

correlation between docking scores and antifungal activity was

observed (Table 1, Table 2).

Beside bis-guanylhydrazone 18, guanylhydrazones 11 and 14

also showed pronounced antifungal activity (Table 1) and it was

important to examine these two molecules closer through

molecular docking study. Both compounds 11 and 14 have

comparable binding energy (-37.549 kcal mol^-1and -42.449 kcal

The docking scores of all studied compounds were calculated

and top four studied compounds for both investigated enzymes

Table 2. Molecular docking data

Docking Glide evdw

Glide ecoul

(kcal mol^-1)

Glide Energy

(kcal mol^-1)

Glide Emodel

(kcal mol^-1)

Hbond

(kcal mol^-1)

Compd

Score

(kcal mol^-1)

C. albicans CYP51

18

16

12

3

-6.953

-5.958

-5.431

-5.38

-46.632

-33.459

-30.357

-23.664

-2.688

-49.32

-38.846

-73.003

-47.43

-1.765

-1.304

0

-5.388

-2.822

-2.992

-33.179

-50.543

-35.746

-26.656

-0.7

Human CYP51

-56.438

18

16

17

13

-6.44

-6.166

-6.076

-5.617

-39.031

-36.826

-30.717

-33.981

-17.407

-9.226

-15.666

-9.564

-92.105

-67.781

-63.977

63.41

-2.308

-1.87

-46.052

-46.384

-2.525

-0.991

-43.545

.

Figure 7. Docking mode of A) 18 (top panel) and B) 11 (left) and 14 (right) in the active site of CACYP51.

mol^-1, respectively) and glide emodel was similar between two of

them (-47.964 kcal mol^-1and -55.012 kcal mol^-1). These two

parameters for aldehyde 3 were much lower (Table 2 and Table

S3). Docking mode of compounds 11 and 14 inside the active site

of CACYP51 revealed strong interactions with amino acids PRO-

375 and MET-508 (Fig. 7B), which could indicate their

importance in enzyme inhibition and can be correlated with their

experimentally establish activity.

investigated guanylhydrazones. For calculating of logP fragment-

based contributions and correlation factors were used. Calculated

physicochemical properties of investigated compounds are shown

in Table S4. Data indicate that all guanylhydrazones except 18

and 21 do not violate the “Rule of five”. For all investigated

compounds clogP are below 4 and therefore they have favourable

physicochemical profiles for oral bioavailability. Topological

polar surface area (TPSA) is a good descriptor for the drug

transport properties, drug absorption, including intestinal

absorption, bioavailability, Caco-2 permeability. TPSA can be

defined as a sum of the surface areas occupied by the oxygen and

nitrogen atoms and the hydrogen atoms attached to them and

represent the hydrogen bonding capacity of the molecules.

Molecules with TPSA < 140 Å²have good intestinal absorption,

while those with TPSA < 60 Å²show good blood–brain barrier

penetration. All investigated compounds except 18 and 21

satisfied the criterion for good intestinal absorption (Table S4).

Docking studies of compounds 11 and 14 with human CYP51

revealed that among all studied compounds they have the lowest

energy interactions with enzyme represented as docking score (-

4.609 for compound 11 and -4.295 for compound 14), and low

values of binding energy (-44.330 kcal mol^-1and -28.262 kcal

mol^-1, respectively; -48.185 kcal mol^-1and -45.407 kcal mol^-1for

glide emodel, respectively; Table S3), suggesting lower affinity

towards human CYP51. Nevertheless, calculated low affinity

towards human cytochrome P450 was not mirrored in

antiproliferative cytotoxic experiments (Table 1). Overall, in

silico analysis and docking study using CYP51s supported the

hypothesis that inhibition of CACYP51 could be one of the

mechanisms of antifungal activity of guanylhydrazone

compounds. However, discrepancies between experimentally

obtained data and predicted parameters from in silico analysis

indicate that other modes of action could also be involved.

The number of H-bond donors and acceptors can be used for

establishing hydrogen bonding capacity of the investigated

compounds. An important factor for oral bioavailability, as well

as for the efficient bonding to receptors and channels, is the

conformational flexibility of the molecules described by the

number of rotatable bonds. Sufficient oral bioavailability is

expected for molecules with 10 rotatable bonds or fewer. In all

investigated compounds there is at least one rotatable bond, but

no more than 8 (compound 21).

Many problems in the drug development are the result of

pharmacokinetic disadvantages such as poor absorption, first pass

effect, a high degree of binding to the protein, therefore,

establishing druglikeness is still a very important step in the

search for new potential drug candidates. Druglikeness allows the

assessment of the pharmacokinetic profile of the tested molecules

based on the prediction of their absorption and distribution.

Based on Lipinski’s “Rule of five”, the critical limit for

acceptable drug-likeness is that no more than one violation of the

rule exists in molecule.^25,26

Summarizing the physicochemical properties of investigated

compounds, conclusion can be made that they all meet criteria for

good solubility and permeability and can be considered as

potential drug candidates.

4. Conclusions

A new therapeutic options and better understanding of their

mechanisms of action is essential to improving the outcome of

fungal infections. A novel thiophene-based guanylhydrazones

(iminoguanidines) were obtained in high yields using a simple

Molinspiration tool (Molinspiration Cheminformatics-2013)

was used for calculating physicochemical properties of

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29. European Committee for Antimicrobial Susceptibility Testing

(EUCAST). Document E.DEF 7.2: Method for the determination

of broth dilution of antifungal agents for fermentative yeasts;

March, 2012.

30. European Committee for Antimicrobial Susceptibility Testing

(EUCAST). Document E.DEF 9.2: Method for the determination

of broth dilution minimum inhibitory concentrations of antifungal

agents for conidia forming moulds; July, 2014.

31. Hope, M. J.; Bally, M.; Webb, G.; Cullis, P. R., Biochim. Biophys.

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two-step procedure. In vitro antifungal activity assays showed

their potent, antifungal effect against yeasts, molds and

dermaptophytes, the most common human and animal clinical

isolates. Compounds 11, 18 and 21 exhibited excellent activity

against voriconazole resistant C. albicans CA5 with MIC <1 µg

mL^-1having selectivity index in comparison to human fibroblasts

from 5 to 20. Additionally, bis-guanylhydrazone 18 showed

better embryotoxicity profile, as well as better fungistatic effect

in comparison to clinically used voriconazole and very efficiently

inhibited Candida biofilm formation. Interaction with genomic

DNA of C. albicans in vitro, indicated a possible mechanism

of their activity. From the hemolysis experiments cell membrane

was excluded as the primary target of these compounds, whereas

in silico analysis and docking study using CYP51s supported the

hypothesis that inhibition of CACYP51 could be one, but not the

only mechanism of antifungal activity. For all compounds it has

been calculated to have satisfying pharmacokinetic profile based

on Lipinski’s “rule of five”. This study provides strong evidence

that selected guanylhydrazone derivatives are good structural

platform for further derivatization and possible application in

antifungal therapy.

Acknowledgments

This research was financially supported by the Ministry of

Education, Science and Technological Development of Serbia

(Grant No. 172008 and 173048) and the Serbian Academy of

Sciences and Arts. Research Grant 2015 by the European Society

of Clinical Microbiology and Infectious Diseases (ESCMID) to

JNR is also acknowledged.

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Supplementary data related to this article is available

including: ¹H NMR, ¹³C NMR and HPLC analyses of

guanylhydrazones, Tables S1-S4.

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Synthesis and evaluation of thiophene-based

guanylhydrazones (iminoguanidines)

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resistant fungal isolates

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Vladimir Ajdačić,^aLidija Senerovic,^bMarija Vranić,^aMarina Pekmezovic,^cValentina Arsic-Arsnijevic,^c

Aleksandar Veselinovic,^dJovana Veselinovic,^dBogdan A. Šolaja,^aJasmina Nikodinovic-Runic*^band Igor M.

Opsenica,*^a

^aFaculty of Chemistry, University of Belgrade, Studentski trg 16, P.O. Box 51, 11158, Belgrade, Serbia

^bInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000

Belgrade, Serbia

^cNational Reference Medical Mycology Laboratory, Institute of Microbiology and Immunology, Faculty of

Medicine, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia

^dFaculty of Medicine, Department of Chemistry, University of Niš, 18000 Niš, Serbia

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Synthesis and evaluation of thiophene-based guanylhydrazones (iminoguanidines) efficient against panel of voriconazole-resistant fungal isolates

DOI: 10.1016/j.bmc.2016.01.058

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Article abstract of DOI:10.1016/j.bmc.2016.01.058

Full text of DOI:10.1016/j.bmc.2016.01.058

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