Synthesis and Evaluation of 2-Chloro-3-[3-(6-Nitro-1H-Benzimidazol-2-Yl)-1H-Pyrazol-5-Yl] Quinolines as Potent Anthelmintic Agents

Introduction

Due to their diverse biological activities, heterocyclic compounds have taken a prominent place among the different classes of organic compounds. Therefore, design, synthesis and production of new molecules for crop health and human well-being have taken center stage inrecent years. Among the variety of heterocyclics studied for the development of pharmaceutically important molecules, benzimidazole played an important role in medical chemistry.¹

The synthesis of nitrogen-containing heterocyclic systems has aroused interest in the last ten years because of their benefits in various applications.² Substituted benzimidazoles are one of the most studied classes of heterocyclic compounds and are closely monitored by synthetic organic chemists because of their wide range of biological properties, such as antiviral agents, anti-cancer agents, antibacterial agents, antifungal agents and many others.^3-9

The pyrazole ring has attracted great attention since it is now easily accessible and has various properties.¹⁰Pyrazoles are one of the most active link classes with a wide range of biological activities.^11-13

Connection with benzimidazole core have been of great interest to synthetic and medicinal chemists for a long time due to their unique chemical and biological properties, especially in connection with traditional anthelmintics like Albendazole and Oxibendazole.¹⁴

As part of our search for basic information on the structural requirements for anthelmintic activity, we have synthesized a number of 2-chloro-3-[3-(6-nitro-1H-benzimidazol-2-yl)-1Hpyrazol-5-yl]quinolines as potent anthelmintic agents.

Reagents and conditions:

(i) 4N HCl, reflux with microwave irradiation, 160 min, (ii) K2 Cr2 O7 , H2 SO4 , 2 h, (iii) 2-chloro-3-formylquinolines, EtOH, 10% NaOH, 0.5 h, (iv) NH.NH2 , EtOH, CH3 COOH, reflux 4 h, (v) iodobenzene diacetate , dichloromethane, 4h.

Materials and Methods

All solvents and reagents were purchased and used as such. The fusion points are examined by open capillary tube method and are uncorrected. Using KBr pellet technology, IR spectra of compounds were observed on the Shimadzu FTIR Spectrometer 8400S. NMR spectra were recorded on Bruker Avance II from 400 NMR spectrometer and MASS spectra on a Waters, Q-TOF Microma SS spectrometer.

Synthesis of1-(6-nitro-1H-benzimidazol-2-yl) ethanol(II)

Equimolar amount of 4-nitro-O-phenylenediamine (0.01 mole) (I) and lactic acid (0.01 mole), 4N HCl arerefluxed in synthetic microwave oven with condenser at theintensity of 65 % (450 W) for 160 min. The reaction was monitoredby TLC, after the completion of the reaction; the mixturewas cooled and neutralized with sodium bicarbonate and thenthe precipitate was filtered, washed with cold water, dried andrecrystallized from absolute alcoholm.p-190ċ.^15,16,17

Synthesis of1-(6-nitro-1H-benzimidazol-2-yl) ethanone(III)

To a solution of 1- (6-nitro-1H-benzimidazol-2-yl) ethanol(II) (10.358g, 50 mmol) in 40 mL dilute H₂SO₄ (5%) was added drop wise with constant stirringa solution of K₂ Cr₂ O₇ (19.8g, 150 mmol) in 80 mL of diluteH₂SO₄ (25% v/v) at room temperature over a period of 20min. Further the reaction mixture was stirred at room temperaturefor 2 h. After completion of the reaction (monitored byTLC), the separated solution (constituting the chromium complex)was washed with water (3×10 mL), then suspended in 50 mLof water and treated with aqueous ammonia (1:1) to have a pH of 6to 6.5. The separated product was washed with water, driedand crystallized from boiling ethyl acetate to give pure productm.p- 202-2040 C.^18,19

Synthesis of3-(2-chloroquinolin-3-yl)-1-(6-nitro -1H-benzimidazol-2-yl)prop-2-en-1-one (IV)

To a solutionof1- (6-nitro-1H-benzimidazol-2-yl) ethanone(III) (2.07 g10 mmol) in 30mL aqueous NaOH (10%) was added to the respective 2-chloro-3-formylquinoline (1.91g 10 mmol) at room temperature.The reaction mixture was stirred for 0.5 h. At the end of this period, the separated solid was filtered, washed with distilledwater and dried. The crude product thus obtained was recrystallizedfrom suitable solvent.^20-22

Similarly,chalcone derivatives of 6- nitrobenzimidazole (IVa-k) were synthesized.

IVb: yield 69 %, m.p-226-228ċ; IR (KBr): 3600 (-NH str), 1600(-C=O), 1550 (C=C str), 1500(CH=CH str), 1225 (C-N Ar), 740 (C-Cl str)cm^-1; ¹ H NMR (CDCl3, 400 MHz):δ 2.77 (s, 3H, CH3), 4.91(s, 1H, NH-benzimidazole), 6.85(d, 1H, H-1-ethylene), 7.43 (d, 1H, H-1-ethylene), 7.52-7.65 (m, 3H, CH-benzimidazole),7.71-10.56 (m, 4H, CH-quinoline; δ 10.56 (s, 1H), 8.69 (s, 1H), 8.54 – 7.76 (m, 1H), 7.71 (d, J = 6.5 Hz, 1H), 7.65 (s, 1H), 7.60 (s, 1H),7.52 (t, J = 7.2 Hz, 1H), 7.43 (s, J = 16.5 Hz 1H), 6.85 (s, J = 17.0 Hz 1H), 4.91 (s, 1H), 2.77 (d, J = 7.3 Hz, 3H); MS: m/z 392 (M⁺ ), 394 (M⁺⁺2) and other important peaks are 56, 163, 177.

IVe: yield 83 %, m.p-230-232⁰ c; IR (KBr): 3550 (N-H str), 1650(-C=O), 1550 (C=C str), 1500 (CH=CHstr), 1225 (C-N Ar), 725 (C-Cl str)cm-1; 1 H NMR (400 MHz, CDCl₃ ):δ 3.95 (s, 3H, OCH₃ ) ,4.90 (s, 1H, NH-benzimidazole), 6.97 (d, 1H, H-1-ethylene), 7.18 (d, 1H, H-1-ethylene), 7.22-7.52 (m, 3H, CH-benzimidazole),7.94-10.53 (m, 4H, CH-quinoline);δ 10.53 (s, 1H), 8.62 (s, 1H), 7.96 (s, 1H), 7.94 (s, 1H), 7.52 (d, J = 2.8 Hz, 1H), 7.49 (d, J = 2.8 Hz, 1H), 7.33 – 7.22 (m, 1H), 7.18 (d, J = 16.7 Hz, 1H), 6.97 (d, J = 16.2 Hz 1H), 4.90 (s, 1H), 3.95 (s, 3H).

IVh: yield 82 %, m.p-237-239⁰c; IR (KBr): 3600 (N-H str), 1625 (-C=O), 1525 (C-C str), 1500 (CH=CH str), 1225 (C-N Ar), 850,750 (C-Cl str)cm^-1; ¹H NMR (CDCl₃ , 400 MHz):δ 4.90 (s, 1H, NH-benzimidazole), 6.86 (d, 1H, H-1-ethylene), 7.26 (d, 1H, H-1-ethylene),7.51-7.80 (m, 3H, CH-benzimidazole), 7.82-8.02 (m, 4H, CH-quinoline); δ 8.02 (s, 1H), 8.00 (s, 1H), 7.93 – 7.83 (m, 1H), 7.82 (d, J = 2.3 Hz, 1H), 7.80 (d, J = 2.3 Hz, 1H), 7.76 – 7.66 (m, 1H), 7.66 – 7.51 (m, 1H), 7.26 (d,J = 16.9 Hz, 1H), 6.86 (d, J = 16.6 Hz, 1H), 4.90 (s, 1H).

Synthesis of2-chloro-3-[3-(6-nitro-1H-benzimidazol-2-yl) -4,5-dihydro-1H-pyrazol-5-yl]quinoline(V)

To a solution of 3- (2-chloroquinolin-3-yl) -1-(6-nitro-1H-benzimidazol-2-yl)prop-2-en-1-one (IV) (3.78 g, 0.01 mole) dissolved in ethanol (40 mL) and glacial acetic acid (10 mL), Hydrazinehydrate (0.75 g, 0.015 mole) was then added and the reactionmixture refluxed for 4 h on a water bath. The solvent was reducedto half its volume. The crystalline product deposited on cooling was filtered off, washed with water, dried and crystallizedfrom ethanol.²³

Similarly, pyrazoline derivatives of 6-nitro benzimidazole (Va-k) were synthesized.

Vb : yield 62 %, m.p-105-107⁰ c; IR (KBr): 3342 (N-H str), 1693 (C=N), 1599 (C=C str), 1392(C-NO₂ ), 1205 (C-N Ar), 765 (C-Cl str)cm^-1; ¹ H NMR(CDCl₃ , 400 MHz):δ 2.03 (d, 2H, methylene), 2.23 (d, 3H, -CH₃ ), 3.72 (d, ¹H, methine), 5.35 (s, 1H, NH-benzimidazole), 6.89 (d, 1H, NH-pyrazoline), 7.22-7.48 (m, 3H, benzimidazole), 7.54-7.76 (m, 4H, quinoline);δ 7.76 (dd, J = 8.2 Hz, 1H), 7.65 (d, J = 6.6 Hz, 1H), 7.59 (d, J = 4.4 Hz, 1H), 7.54 (d, J = 6.8 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.44 (d, J = 6.7 Hz, 1H), 7.22 (d, J = 6.4 Hz, 1H),6.89 (d, J = 10.1 Hz, 1H), 5.35 (s, 1H), 3.72 (d, J = 6.8 Hz, 1H), 2.23 (d, J = 16.2 Hz, 3H), 2.03 (d, J = 16.6 Hz 2H).MS: m/z407 (M+)and other important peaks are 70, 163, 177.

Vc : yield 69 %, m.p-109-111⁰c; IR (KBr):3180 (N-H str), 1622(C=N), 1514 (C=C str), 1386(C-NO₂), 1215 (C-N Ar), 806 (C-Cl str)cm^-1; ¹ H NMR (CDCl₃, 400 MHz):δ 2.17 (d, 2H, methylene), 2.55 (d, 3H, -OCH3), 3.11 (d, 1H, methine), 4.91 (s, 1H, NH-benzimidazole),7.15 (s, 1H, NH-pyrazoline), 7.41-7.64 (m, 3H, benzimidazole), 7.77-8.36 (m, 4H, quinoline);δ 8.36 (d, J = 5.5 Hz, 1H), 8.17 (s, 1H), 7.87 (s, 1H), 7.77 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.56 (s, 1H), 7.41 (d, J = 7.9 Hz, 1H), 7.15 (d,J = 8.2 Hz 1H), 4.91 (s, 1H), 3.11 (d,J = 7.6 Hz, 1H), 2.55 (d, J = 3.5 Hz, 3H),2.17 (d, J = 13.6 Hz, 2H).

Vh: yield 72 %, m.p-106-108⁰c;IR (KBr):3190 (N-H str), 1662 (-C=N str), 1585 (C=C str), 1336 (C-NO₂ ), 1261 (C-N Ar), 825,734 (C-Cl str)cm^-1;¹H NMR(CDCl₃, 400 MHz):δ 2.04 (d, 2H, methylene), 3.42 (d, 1H, methine), 5.48 (s, 1H, NH-benzimidazole), 6.86 (d, 1H, NH-pyrazoline), 7.52-7.61 (m, 3H, benzimidazole), 7.70-8.31 (m, 4H, quinoline);δ 8.31 (s, 1H), 7.86 (d,J = 9.9 Hz, 1H), 7.72 (s, 1H), 7.70 (s, 1H), 7.61 (s, 1H), 7.59 (s, 1H), 7.52 (s, 1H), 6.86 (d, J = 8.1 Hz, 1H), 5.48 (s, 1H), 3.42 (d,J = 7.4 Hz 1H), 2.04 (d, J = 16.7 Hz, 2H).

Vk: yield 70 %, m.p-104-106⁰c; IR (KBr):3196 (N-H str), 1674 (C=N str), 1504 (C=C str), 1338 (C-NO₂ ), 1267 (C-N Ar), 1149 (C-F), 831 (C-Cl str)cm^-1; ¹ H NMR (CDCl₃ , 400 MHz):δ 2.05 (d, 2H, methylene), 3.73 (d, 1H, methine), 4.91 (s, 1H, NH-benzimidazole), 6.86 (d, 1H, NH-pyrazoline), 7.00-7.46 (m, 3H, benzimidazole), 7.51-8.02 (m, 4H, quinoline); δ 8.02 (s, 1H), 7.77 (s, 1H), 7.76 (d, J = 5.2 Hz, 1H), 7.51 (d, J = 6.4 Hz, 1H), 7.46 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.00 (s, 1H), 6.86 (d,J = 8.4 Hz 1H), 4.91 (s, 1H), 3.73 (d, J = 7.4 Hz, 1H), 2.05 (d,J = 17.1 Hz, 1H).

Synthesisof2-chloro-3-[3-(6-nitro-1H-benzimidazol-2-yl)-1H-pyrazol-5-yl]quinoline(VI)

To a stirred solution of 2-chloro -3-[3- (6-nitro-1H-benzimidazol-2-yl)-4,5-dihydro-1H-p yrazol-5-yl] (V) (0.392g, 0.001mole) in dichloromethane (20 mL) iodobenzene diacetate (0.386g, 0.0012 mole) was added at room temperature. The resulting mixture was stirred for 4 h. Dichloromethane was distilled off on a steam bath to give a gum, which was triturated with pet-ether to remove iodobenzene and then purified by recrystallization from ethanol to give the product.²⁴

Similarly, pyrazole derivatives of 6-nitrobenzimidazole (VIa-k) were synthesized.

VIc: yield 74%, m.p-81⁰c; IR (KBr): 3171 (amide N-H Str), 1581 (C=N), 1491 (Ar C=C str), 1338 (C-NO₂), 1228 (ArC-N ), 734 (Ar Cl str) cm^-1; ¹H NMR(CDCl₃, 400 MHz):δ 2.44 (d, 3H, -CH₃), 5.32 (s, 1H, -NH-benzimidazole), 6.71 (d, 1H, -CH-pyrazole ), 7.40-7.67 (m, 3H, Benzimidazole), 7.79-8.71 (m, 4H, Quinoline), 10.52 (d, 1H, -NH-pyrazole); δ 10.52 (d,J = 9.1 Hz, 1H), 8.71 (d, J = 8.5 Hz, 1H), 8.36 (d, J = 6.8 Hz, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.79 (dd, J= 7.9 Hz, 1H), 7.67 (s, 1H),7.48 (d, J = 7.4 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 6.71 (d,J = 17.8 Hz, 1H), 5.32 (s, 1H), 2.44 (d, J = 8.8 Hz, 3H).

VIf: yield 68%, m.p-83⁰c; IR (KBr): 3064 (amide N-H Str), 1543 (C=N), 1492 (Ar C=C str), 1350 (C-NO₂), 1228 (ArC-N), 746 (Ar Cl str) cm^-1; ¹ H NMR(CDCl₃ , 400 MHz):δ 3.71 (d, 3H, -OCH₃ ), 5.01 (s, 1H, -NH-benzimidazole), 6.35 (d, 1H, -CH-pyrazole ), 6.78-7.29 (m, 3H, Benzimidazole), 7.44-8.76 (m, 4H, Quinoline), 10.52 (d, 1H, -NH-pyrazole);δ 10.52 (d, J = 6.9 Hz, 1H), 8.76 (d, J = 9.6 Hz, 1H), 8.34 (s, 1H), 7.49 (s, 1H), 7.44 (s, 1H),7.29 (d, J = 2.4 Hz, 1H), 6.98 (s, 1H), 6.78 (s, 1H), 6.35 (d, J = 16.3 Hz, 1H), 5.01 (s, 1H), 3.71 (d,J = 7.4 Hz, 3H).

VIh: yield 72%, m.p-84⁰c; IR (KBr): 3070 (amide N-H Str), 1579 (C=N), 1491 (Ar C=C str), 1340 (C-NO₂), 1219 (ArC-N ), 827 (Ar Cl str) cm^-1;¹H NMR (CDCl₃, 400 MHz):δ 5.81 (s, 1H, -NH-benzimidazole), 6.86 (d, 1H, -CH-pyrazole ), 7.34-7.77 (m, 3H, Benzimidazole), 7.88-8.70 (m, 4H, Quinoline), 10.55 (d, 1H, -NH-pyrazole);δ 10.55 (d,J = 8.3 Hz, 1H), 8.70 (dd, J = 6.4 Hz, 1H), 8.17 (s, 1H), 8.01 (d, J = 9.1 Hz, 1H),7.88 (s, 1H), 7.77 (s, 1H), 7.49 (s, 1H), 7.34 (s, 1H), 6.86 (d, J = 17.6 Hz, 1H), 5.81 (s, 1H).

VIk: yield 77%, m.p-80⁰ c; IR (KBr): 3066 (amide N-H Str), 1595 (C=N), 1494 (Ar C=C str), 1340 (C-NO₂), 1273 (ArC-N), 827 (Ar Cl str) cm^-1;¹H NMR(CDCl₃,400 MHz):δ 4.89 (s, 1H, -NH-benzimidazole), 6.87 (d, 1H, -CH-pyrazole ),7.58-7.77 (m, 3H, Benzimidazole), 7.86-8.33 (m, 4H, Quinoline), 10.55 (d, 1H, -NH-pyrazole);δ 10.55 (d, J = 3.5 Hz, 1H), 8.33 (d, J = 8.1 Hz, 1H), 8.23 (d, J = 5.8 Hz, 1H), 7.94 (s, 1H), 7.86 (s, 1H), 7.77 (d, J = 5.2 Hz, 1H), 7.69 (s, 1H), 7.58 (m, 1H),6.87 (dd, J = 18.6 Hz, 1H), 4.89 (s, 1H). MS: m/z 409 (M+)and other important peaks are 70, 163, 181.

Each value represents the Mean ± SEM (n=5); aStandard drug- Albendazole (ALZ); bControlNormal Saline

Examination of all investigated substance groups at the tested concentrations of 30, 50 and 100 μg/mL showed a significant activity compared to the standard drug Albendazole.

It has been observed that as the concentrations of compounds and Albendazole increases, the time taken for paralysis and death reduces significantly. The compounds VIb, Vid, and VIf showed an excellent strong effect on the duration of paralysis and death when compared to the standard drug Albendazole. The compounds VIc, Vie, and VIk were also relatively effective with respect to the above compounds. The compounds VIh, Vii, and VIj also showed a good anthelmintic activity, but the compounds VIa and VIg have a comparatively less strong effect than other compoundstested.

A closer look at the results shows that all the testedcompounds have good anthelmintic activity at concentrations of 30 and 50µg/mL.

Results and Discussion

Scheme summarizes the synthetic pathway to obtain1-(6-nitro-1H-benzimidazol-2-yl)ethanol (II)by reacting 4-nitro-o-phenylenediamine(I) with lactic acid. This on oxidation gives1- (6-nitro-1H-benzimidazol-2-yl)ethanone(III). Further condensation with different aromatic/ heteroaromatic aldehydes gives3- (2-chloro quinolin-3-yl)-1-(6-nitro-1H- benzimidazol-2-yl) prop-2-en-1- one(IVa-k). The reaction with hydrazine hydrate in ethanol and glacial acetic acid gives 2-chloro-3-[3- (6-nitro1H-benzimidazol-2-yl) -4,5-dihydro- 1Hpyrazol-5-yl]quinoline (Va-k). Further on treating with iodobenzene diacetate in dichloromethane give the title compounds 2-chloro-3-[3-(6-nitro-1H-benzimidazol-2-yl)-1Hpyrazol-5-yl]quinoline(VIa-k).

Compound, analyzed asC20H13ClN4O3, m.p250-52ċ, exhibited 392 (M+), 394 (M+ +2) and other important peaks at 56, 163, 177in its mass spectrum. The IR spectrum of IVb showed characteristic absorption peaks at 3600 (NH), 1600 (C=O) and 1550 (C=N). The ¹H NMR spectrum of IVb showed a singlet observed at δ 2.77 and assigned to the CH₃ protons. A multiplet at 7.52-7.65 for 3H protons of benzimidazoleand also, 4H protons ofquinoline, were observed at δ 7.71-10.56 due to aromatic protons. A doublet at δ 6.85 and 7.43 each of 1H protons responsible for (CO-CH=CH) of chalcone moiety and also a singlet, 1H proton for NH-benzimidazole was observed at δ 4.91.

Compound, analyzed as C₂₀H₁₅ClN₆O₂ , m.p107-09⁰c, exhibited 407 (M+) and other important peaks are 70, 163, 177in its mass spectrum. The IR spectrum of Vbshowed characteristic absorption peaks at 3342 (N-H str), 1693 (C=N), 1599 (C=C str), 1392(C-NO₂), 1205 (C-N Ar), 765 (C-Cl str). The 1H NMR spectrum of Vb showed a doublet for CH³ proton was observed at δ 2.23. A doublet of methylene and methane observed at δ 2.03 and δ 3.72, respectively. Singlet for NH of benzimidazole and doublet for NH of pyrazoline observed at δ 5.35 and δ 6.89, respectively. A multiplet at δ 7.22-7.48 for 3H protons of benzimidazole and also, 4H protons of quinoline, were observed at δ7.54 -7.76due to aromatic protons.

Compound, analyzed as C₁₉H₁₀ClFN₆O₂ , m.p180-82⁰c, exhibited 409 (M+) and other important peaks are 70, 163, 181in its mass spectrum. The IR spectrum of VIkshowed characteristic absorption peaks at3066 (amide N-H Str), 1595 (C=N), 1494 (Ar C=C str), 1340 (C-NO₂), 1273 (Ar C-N), 827 (Ar Cl str). The 1H NMR spectrum of VIk showed a doublet at 6.87 of -CH-pyrazole. A singlet and a doublet at δ 4.89 and δ 10.55 for -NH-benzimidazole and pyrazole, respectively. A multiplet at δ 7.58-7.77 for 3H protons of benzimidazole and also, 4H protons of quinoline, were observed at δ7.86 - 8.33due to aromatic protons.

Similarly, the structures of the remaining compounds were confirmed by the spectral data. In addition, they were tested for anthelmintic activity.

Biological Evaluation

Anthelmintic activity :The synthesized compounds were tested for anthelmintic activity by in-vitro bioassay method.^25-27 The South Indian adult earthworms Pheretimaposthuma (earthworms authenticated by the Government Agricultural College, Hitnalli, Vijayapur, Karnataka) with a length of 9-11cm and a width of 0.2-0.3 cm were used for the in vitro anthelmintic bio-assay due to its anatomical and physiological similarity with the intestinal worm parasites of human beings.

The nearly equal sized earthworms (9±1cm) were randomly selected, washed thoroughly with normal saline to remove any fecal and adherent materials before being released in to petridishes containing active ingredient in 15 mL of normal saline.The worms were divided into control, standard and test groups of five earthworms each. All tested compounds and the standard drug solution were freshly prepared before starting the tests. The control group petridish contains 0.5 mL of dimethyl sulfoxide in 14.5 mL of normal saline. The standard drug Albendazole and tested compounds were prepared at a dosage of 30, 50, 100 µg/mL, by dissolving in a minimal amount, about 0.5 mL of dimethyl sulfoxide and the volume was diluted to 15 mL with normal saline andthen poured into petri dishes.

The fiveearth worms were placed in each petridish at room temperature and the time for initiation of completeparalysis and the time required for death of individual earthworms was recorded. The time when the worms were motionless and not even usedto receive normal saline was found to be the time of paralysis. The death time was determined by external stimuli, unless the individual worms were placed in warm water at 50⁰C, whichstimulate and induces the movement of worms while they are still alive.

Mean paralysis time and mean dead time wascalculated for each concentration of the compounds tested.

Conclusion

A protocol for the synthesis of new series of 2-chloro-3-[3-(6-nitro-1H-benzimidazol-2-yl)-1Hpyrazol-5-yl]quinolineswas developed under the synthetic microwave irradiation method and also according to the conventional method. It has been found that the preferred synthetic pathway shows good yield and a shortened reaction time for benzimidazole synthesis. The synthesized compounds were tested for their anthelmintic activity. Many of these products showed moderate to good activity.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgement

The authors thank the Vision Group for Science and Technology (VGST), Government of Karnataka State, for financial support.