Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds

Article abstract of DOI:10.1515/chem-2020-0017

Sixteen new isoxazole tagged indole compounds have been synthesized via copper (I) catalyzed click chemistry of the aryl hydroxamoyl chloride and an indole containing alkyne moiety. The chemical structure of the synthesized compounds has been established using various physicochemical techniques. X-ray single crystal analysis of Ethyl 1-((3-phenylisoxazol-5-yl) methyl)-1H-indole-2-carboxylate (8a) has been analyzed. All compounds were tested for their antibacterial and anticancer activities. The activities for the new compounds were weak against both bacterial strains and the cancer cell lines.

Full text of DOI:10.1515/chem-2020-0017

Open Chem., 2020; 18: 138–148
Research Article
Raed A. Al-Qawasmeh*, Louy A. Al-Nazer, Sarah A. Dawlat-Kari, Luay Abu-Qatouseh, Salim S.
Sabri, Murad A. AlDamen, Mutasem Sinnokrot*
Design, Synthesis and Characterization of Novel
Isoxazole Tagged Indole Hybrid Compounds
https://doi.org/10.1515/chem-2020-0017
received October 22, 2018; accepted October 2, 2019.
[12]. In view of the importance of the indole containing
compounds, there is an ever-increasing demand to
Abstract: Sixteen new isoxazole tagged indole introduce new compounds based on indole moiety.
compounds have been synthesized via copper (I) catalyzed
Isoxazole and their derivatives, on the other hand,
click chemistry of the aryl hydroxamoyl chloride and an have received comparable attention to the indole
indole containing alkyne moiety. The chemical structure containing moieties. This is due to their diverse biological
of the synthesized compounds has been established activities, which include, but are not limited to, their role
using various physicochemical techniques. X-ray single as antiplatelet agents [13], antiviral and anti-HIV [14,15],
crystal analysis of Ethyl 1-((3-phenylisoxazol-5-yl) anti-diabetic [16] anti-Alzheimer, anti-cancer and anti-
methyl)-1H-indole-2-carboxylate (8a) has been analyzed. inflammatory agents [17,18].
All compounds were tested for their antibacterial and
The nature and the fast pace of chemical research
anticancer activities. The activities for the new compounds forced scientists to cope by creating reaction conditions
were weak against both bacterial strains and the cancer that subjugate to its nature. Hence, the relatively new
cell lines.
“click chemistry” reactions have been originated by
Sharpless and coworkers in 2001 [19,20]. Many reactions
were found to satisfy the “click” criteria, the most
notable of which are the cycloaddition reactions [21]. The
superiority of the click methodology over the traditional
Huisgen thermal process is the introduction of metal
catalysts such as Cu(I), with the possibility of using other
metal catalysts (Ru, Ni, Pt, Pd), within the reaction system
Keywords: isoxazole; indole; click chemistry,1,3-dipolar
cycloaddition, Alkyne, Copper (I).
1 Introduction
Indole derivatives, either synthetic or natural, are at room temperature, rather than resorting to elevated
considered as privileged scaffolds in medicinal chemistry temperatures. This introduction led to an enhancement
[1–4]. Many of them are the soul of an important class of of the regioselectivity of the product with respect to the
therapeutic agents, including anticancer [5], antioxidant Huisgen thermal process [22].
[6], antirheumatoidal [7], and anti-HIV [8–10] agents. They
Inspired by the above facts, and in pursuit of our
can also play a vital role in drugs targeting the immune research group theme, which targets research in the
system [11] and as tubulin polymerization inhibitors realms of drug design and discovery, we report herein, the
synthesis of new hybrid scaffolds combining indole and
substituted isoxazole based on the well-established “click
chemistry” methodology. We are hereby successfully
reporting potent antimicrobial agents based on the indole
moiety hybridization with other heterocyclic systems, such
as triazine [23] and imidazole [24]. The latter-mentioned
scaffolds have been reported to be active against various
bacterial strains as well as cancer cell lines. (Figure 1)
*Corresponding authors: Raed A. Al-Qawasmeh, College of
Sciences, Department of Chemistry, University of Sharjah, 27272
Sharjah, UAE; Department of Chemistry, the University of Jordan,
Amman 11942-Jordan, E-mail: ralqawasmeh@sharjah.ac.ae;
Mutasem Sinnokrot, Department of Chemistry, College of Arts and
Sciences, Khalifa University of Science and Technology, Abu Dhabi
127788, UAE, E-mail: mutasem.sinnokrot@ku.ac.ae
Louy A. Al-Nazer, Sarah A. Dawlat-Kari, Salim S. Sabri,
Murad A. AlDamen, Department of Chemistry, the University of
Jordan, Amman 11942-Jordan
Luay Abu-Qatouseh, Medical Microbiology and Immunology,
University of Petra, Amman-Jordan
Open Access. © 2020 Raed A. Al-Qawasmeh et al., published by De Gruyter.
Attribution alone 4.0 License.
This work is licensed under the Creative Commons

Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds
ꢀ139
O
O
O
O
ii, iii
i
N
N
N
OH
OEt
H
H
(2)
(3)
(1)
Scheme 1: Synthesis of the click precursors (2) and (3); i- Propargyl bromide, K2CO3/DMF, 0
̊
bromide, NaH/DMF, 0
̊
H
O
Cl
i
ii
OH
OH
Ar
H
Ar
N
Ar
N
Ar = C
Ar = C
Ar = C
4a
4b
4c
4d
4e
4f
6a
6b
6c
6d
6e
6f
5a
6H
Ar = 4-F-5C
6H
Ar = 4-F-5C
6H4
6H5
Ar = 4-F-C
5b
5c
5d
5e
5f
6H4
6H4
Ar = 4-Cl-C
6H4
Ar = 4-Br-C
Ar = 4-Cl-C
Ar = 4-Cl-C
Ar = 4-Br-C
6H4
Ar = 4-Br-C
6H4
6H4
H4
6H4
H4
6H4
H4
Ar = 4-CN-C
Ar = 4-CN-C
Ar = 4-CN-C
Ar = 4-Me-C 6
Ar = 4-Me-C 6
Ar = 4-Me-C 6
H4
-6C
6H4
Ar = 4-OM2e-C
6H4
H4
-6C
6H4
Ar = 4-OM2e-C
6H4
H4
-6C
6H4
Ar = 4-OM2e-C
6H4
Ar = 4-NO
Ar = 4-NO
Ar = 4-NO
4g
4h
6g
6h
5g
5h
Scheme 2: Synthesis of second click precursor the hydroxamoyl chloride 6a-h; i- Hydroxylamine hydrochloride, Ar-aldehyde, H2O/EtOH, 50
% NaOH, 1h, HCl (conc.). ii- oxime, DMF/NCS 2-6 h’s.
Ar
it with sodium hydride in dry DMF, and thereafter with
Ar
1
13
propargyl bromide. Both H and C-NMR analyses for (2)
and (3) clearly depict the characteristic features of the
propargyl moiety along with the indole part.
N
NH
H
N
N
N
N
Having the first click precursor in hand, the second
precursor was synthesized via a two-step process. Initially,
a selected set of aldehydes (4a-h) were condensed with
hydroxyl amine [25]. This was followed by a reaction
with N-chlorosuccinimide in dry DMF to give the pure
hydroxyimoyl chloride (6a-h), as shown in Scheme 2.
1,3-dipolar cycloaddition was then realized with the
dipolarophile (2) and (3) with the nitrile oxide generated
in situ from (6a-h) upon the action of base directly before
the reaction. This will serve to prevent dimerization to
furoxans. Clicking both precursors shown in Scheme
1 produced the desired new compounds (7a-h) and
(8a-h) (Scheme 3). All the new compounds were fully
Ar
N
H
Ar
(I)
(II)
Figure 1: Structure of indole hybridized with triazine (I) and
imidazole (II).
2 Results, Experimental Procedure,
and Discussion
The newly-designed compounds were synthesized from characterized using 1H, 13C and 2D-NMR techniques along
the commercially available 1-H-indole-2-carboxylic acid with HRMS (ESI). In series (7) the H-NMR shows a clear
1
(1). In detail, we utilized both the carboxylic acid and the singlet for 2H resonating around δ = 6.0 ppm assigned for
NH of the indole moiety to introduce our click precursors the methylene CH2 protons, where a 0.5-0.7 down field shift
(2) and (3), as shown in Scheme 1. The precursor (2) was were observed for all the synthesized compounds from
synthesized in a quantitative yield through the reaction the propargylated starting compound (2). Additionally,
with propargyl bromide in the presence of potassium the new singlet signal assigned for CH in the isoxazole
carbonate in dry DMF. For precursor (3) the carboxylic appeared around 6.9-7.5 ppm which was correlated with
13
acid group was protected with ethyl ester before reacting the signal for the carbon at 100-112 ppm in the C-NMR

140ꢀ
Raed A. Al-Qawasmeh et al.
O
O
N
N
H
O
(2)
(3)
i
Cl
Ar
7a-h
+
OH
Ar
N
O
N
OEt
O
N
Ar
8a-h
Scheme 3: Synthesis of compound (7a to h) and (8a to h); i- CuI, toluene/Et3N overnight.
spectrum. In the other series, the compounds show two connected to the indole via the CH2 moiety with angle
CH2 signals, the first signal belongs to the methylene N1C12C13 of 110.7(3)°. An insight into intermolecular
protons of the ester (OCH2CH3) at about 4.3 ppm, while interactions can be obtained by electrostatic potential
the second one belongs to the methylene protons of the map and two-dimensional fingerprint plots mapped in
carbon attached to indole nitrogen (NCH2-isoxazole) at the Hirschfeld surface, analyzed by CrystalExplorer 17.5
about 6.0 ppm. It was also observed that the CH of the [26–28]. The electrostatic potential of compound (8a)
isoxazole resonated in the same region as in the first series (Figure 3a) shows that the isoxazole plane is almost
for both proton and carbon.
negative towards the oxygen atoms while the indole
plane is positive. This makes the molecule neutral with
HRMS-ESI analysis gave exact molecular ions for all non-favorable intermolecular interactions. The two
the compounds. The structures of the new derivatives dimensional fingerprint plots and the contributions of
were determined from their corresponding 1H and individual interatomic contacts toward the overall crystal
13C-NMR spectra. The formation of the isoxazole moiety packing are shown in Figure 3b. Several directional
was characterized via the methine proton signal, that contacts can be observed, such as C–C (1.8%), C–H
resonates as a singlet at δ = 7.07-7.43 ppm. The singlet at (16.4%), N–H (4.2%), O–H (7.8%) along with H–H (66.7%).
δ = 7.39 ppm was assigned to H-3 of the indole moiety. These intermolecular atom-atom contacts contribute to
All compounds show the features of a para-substituted the stability of the crystal packing of compound (8a).
aromatic system along with the aromatic signals of the
Ethical approval: The conducted research is not
13
indole. C-NMR, on the other hand, clearly indicates all related to either human or animal use.
the features of the synthesized compounds. HMQC, HMBC
and DEPT experiments allow us to fully assign all protons
and carbons for the new compounds. The NH protons in 2.1 Biological Activities
compounds (3) and (7a-h) of the indole moiety appeared
at 11.90-12.00 ppm for all the synthesized compounds.
Indole derivatives have shown various antibacterial and
X-ray structure determination was performed to anticancer activities. In regards to the antimicrobial
further confirm the indole- hybrid system, with compound activity, this study reported weak in vitro antibacterial
(8a) selected as a representative example of this new class activity against both Gram positive and Gram negative
of hybrid system (Figure 2). The molecule crystallizes in bacteria. In addition, no significant activity was reported
a monoclinic chiral space group P21. A summary of data against pathogenic yeast and fungi. By the initial
collection and refinement parameters are given in Table screening against selected pathogenic bacteria including
1. Figure 2 of compound (8a) shows that the indole along Staphylococcus aureus, Escherichia coli and Pseudomonas
with the carboxylate moieties is in one plane with the aeruginosa, the minimal inhibitory concentrations of the
CH2-isoxazole plane. Additionally, the isoxazole ring is tested compounds were higher than 1 mg / ml.

Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds
ꢀ141
Table 1: Crystal data and structure refinement for compound 8a.
For the anticancer activity testing of the synthetic
compounds, the inhibitory concentrations and percentage
of killing of the tested compounds were measured using
three human cancer cell lines: Leukemic cells (Hela),
breast cancer cells (MCF-7), and epiglottis cancer cells
(Hep-2) in addition to a non-human Chinese hamster
ovary (CHO) cell lines. A concentration range of 100-500
ug/mL was used for all cells, and cell viability assay MTT
was performed. A weak anticancer activity was observed
for the tested compounds. Furthermore, Hela cell lines
showed the highest sensitivity to all chemicals (% of
killing equals to 20-34), whereas Hep-2 and MCF-7 cells
were the least sensitive (% of killing equals to 5-11).
Empirical formula
Formula weight
Appearance
Crystal system
Space group
Temperature
A/å
C21H18N2O3
346.37
Block, colorless
Monoclinic,
P21
291 K°
6.7129 (4)
B/å
12.5657 (8)
C/å
1610.8955 (9)
β/°
104.802 (7)°
Volume/å3
888.57 (11)
2.2 Experimental
Z
2
1H-NMR spectra were recorded in hexadeuterodimethyl
sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) on
a Bruker Avance III-300 MHz and III-500 MHz Spectrometer
Ρcalcmg/mm3
µ/mm-1
1.295
0.09
13
F(000)
364
with TMS as an internal standard. C-NMR spectra were
obtained at 75 MHz and 125 MHz on the same instruments,
respectively. Chemical shifts were reported as δ-values in
ppm. High resolution Mass spectra (HRMS) were recorded
in positive ion mode by Electro Spray Ionization (ESI)
using a Bruker Daltonics Apex IV, 7.0 T Ultra Shield Plus.
The samples were dissolved in chloroform, diluted in spray
solution (methanol / water 1:1 v/v + 0.1% formic acid) and
infused using a syringe pump with a flow rate of 2 µL /
min. External calibration was conducted using arginine
cluster in a mass range m/z 175-871. For all HRMS data,
mass error falls in the range of 0.00–0.50 ppm. Melting
points (m.p.) were determined on an Electrothermal
Melting Point Apparatus and were uncorrected in °C.
Solvents used in this study were obtained from Scharlau,
Θ range for data collection
Index ranges
Reflections collected
Independent reflections
3.2–29.4°
-29 ≤ h ≤ 27, -28 ≤ k ≤ 29, -21 ≤ l ≤ 10
4257
2979 [R(int) 0.017]
Data/restraints/parameters 3501/0/209
Goof on f2
1.063
Final r indexes
[i>=2σ (i)]
R1 = 0.038
, wR2 = 0.1029
Final r indexes [all data]
Largest diff. Peak/hole/eå-3
R1 = 0.0487, wR2 = 0.1097*
3.08 /-1.28
Fluka, Acros, and Aldrich. All reactions were monitored by group P21. A summary of the crystallographic data and
thin layer chromatography (TLC) using Merck aluminum structure refinement parameters is given in Table 1.
plates pre-coated with silica gel PF254; 20 × 20 × 0.25 mm,
Single-crystal X-ray diffraction data were collected
and detected by visualization of the plate under UV lamp using an Oxford Diffraction XCalibur, equipped with (Mo)
(λ = 254 or 365 nm ). Spots were also detected by spraying X-ray Source (λꢀ=ꢀ0.71073ꢀÅ) at room temperature (293(2)
with anisaldehyde- sulphuric acid in ethanol, followed by K). Data collection, reduction, and cell refinement were
heating to 140°C [35].
performed using the software package CrysAlisPro [29].
Analytical absorption corrections were applied using
spherical harmonics implemented in SCALE3 (ABSPACK)
scaling algorithm. Crystal structure was solved by direct
methods, using the program OLEX2, followed by Fourier
2.2.1 X-Ray Crystallography
Compound (8a) was recrystallized from hot DMF/benzene synthesis, and refined on F2 with SHELXL implemented
after cooling to room tempetaure. Small amount of ice was in OLEX2 [30,31]. Anisotropic least-squares refinement of
added to produce a fine colorless block crystalline solid. non-H atoms was applied. All crystallographic plots were
The compound crystallized in monoclinic chiral space obtained using the CrystalMaker program [32].

142ꢀ
Raed A. Al-Qawasmeh et al.
Figure 2: Molecular structure and atom numbering scheme of 8a. Thermal ellipsoids are drawn at 30% probability level.
Figure 3: a) Compound (8a) mapped over the Hirschfeld surface with a scale of -0.07 a.u. (red) through 0.0 (white) to +0.04 a.u. (blue); b)
Two-dimensional full fingerprint plots and decomposed fingerprint plots over the Hirschfeld surface for various intermolecular atom–atom
contacts in compound (8a). The numbers in brackets indicate the percentage contributions of each contact.
2.2.2 Prop-2-yn-1-yl 1H-indole-2-carboxylate (2)
mixture. After two hours, TLC confirmed the completion
of the reaction; which was poured over crushed ice.
1H-indole-2-carboxylic acid (1) 1.0 g (6.20 mmol) was The white precipitate that formed was collected and
dissolved in 10 ml DMF, and 5 equivalent of K2CO3, 4.28 g recrystallized from hot ethyl acetate. The product was
(31 mmol), were added at 0˚C. After 30 minutes, propargyl produced with a high yield of 90.0%, and a melting point
bromide (1.5 ml, 19 mmol) was added to the reaction range of 136-138˚C.

Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds
ꢀ143
2.2.3 .Ethyl-1-(prop-2-yn-1-yl)-1H-indole-2-carboxylate (3) over MgSO4, and the solvent was removed using rotary
evaporator. All hydroxamoyl chloride compounds (6a-h)
1-H-indole-2-carboxylic acid (1) was transformed into were used in situ directly without further purification.
ethyl 1-H-indole-2-carboxylate using general method
of esterification, where the indole acid was refluxed in
ethanol in the presence of concentrated sulfuric acid 2.2.6 General Procedure for the Synthesis of Series (7)
(H2SO4) to produce indole ester with a high yield of 93.5% as and (8)
a white solid after work up with water. The reaction of the
ethyl 1-H-indole-2-carboxylate (3) with propargyl bromide A mixture of compound 3 (1.0 mmol) with 0.019 g copper
was performed under an inert gas mixture (N2, Ar) in the (I) Iodide, CuI, (10 mmol) in the presence of Et3N (4.07
presence of sodium hydride (NaH) and DMF as a solvent, to mmol) was stirred for 30 minutes. After that, 5-7 ml of
produce the ethyl-1-(prop-2-yn-1-yl)-1H-indole-2-carboxylate toluene was added and the solution was stirred for an
as a white solid. This white solid was then recrystallized additional 30 minutes. To this solution, 1.3 mol equivalent
from hot EtOH. The product was obtained with a high yield of the corresponding hydroxamoyl chloride (7a-h) was
of 80.7% and melting point range of 65-67˚C.
added. The reaction mixture was stirred for 17-24 h. and
monitored by TLC. After completion, 25 ml of EtOAc was
added with stirring and the copper salt was removed by
filtration. The filtrate was extracted and the combined
organic layer was dried over Na2SO4. The solvent was
evaporated and the product was purified using normal thin
2.2.4 General Procedure for the Synthesis of Aryl
Oximes (5a-h)
The designed oximes were prepared according to the layer chromatography with Hexane/ethyl acetate as mobile
general procedure described herein. Hydroxylamine phase (the ratio depended on the product polarity). Figure
hydrochloride (0.55 mol) was added to a mixture of aryl 4 shows the numbering system adopted in this study with
aldehyde (5a-h) (0.25 mol) dissolved in 30 ml H2O/EtOH compounds 7a and 8a used as representatives for this order.
(1:1) and 30 ml of ice. Afterwards, 0.63 mol of 50.0%
NaOH was added dropwise and the reaction was stirred carboxylate (7a)
at room temperature for approximately one hour, and
Yield: 57%; white solid; m.p: 196 - 198°C. 1H NMR (500
(3-phenylisoxazol-5-yl)methyl
1H-indole-2-
monitored by TLC in chloroform (CHCl3). After completion MHz, DMSO-d6) δ 5.53 (s, 2H, H-9), 7.05 (pseudo t, H-5), 7.19
of the reaction, neutralization was accomplished using a (s, H-4’), 7.24 (s, H-3), 7.25 (pseudo t, H-6), 7.43 (d, J = 8.3
concentrated HCl solution. Solid oximes were collected Hz, H-7), 7.47 (m, H-4’’), 7.48 (m, H-3’’, H-5’’ ),7.65 (d, J = 8.1
by suction filtration while liquid oximes were extracted Hz, H-4), 7.86 (m, H-2’’, H-6’’), 11.90 (s, NH). 13C NMR (125
with CHCl3 (20mL×2), dried over MgSO4. The solvent was MHz, DMSO-d6): 57.1 (C-9), 103.0 (C-4’), 109.4 (C-3), 113.1
evaporator; the aryl oximes were produced with high (C-7), 120.8 (C-5), 122.7 (C-4), 125.5 (C-6), 126.6 (C-2), 126.8
yields (74 – 93%) [27].
(C-3a), 127.1 (C-2’’, C-6’’), 128.7 (C-1’’), 129.6 (C-3’’, C-5’’),
130.9 (C-4’’), 138.2 (C-7a), 161.0 (C-5’), 163.0 (C-3’), 168.2
(C-8). HRMS (ESI) m/z: Calcd. For C19H14N2O3Na [M + Na]+
341.09021. Found: 341.08966.
2.2.5 General Procedure for the Synthesis of
Hydroxamoyl Chloride (6a-h)
(3-(4-fluorophenyl)isoxazol-5-yl)methyl 1H-indole-2-
carboxylate (7b)
To a stirred solution of 1.0 g of oxime (6a-h) in 15 ml DMF
Yield: 52%; White solid; mp: 155 - 157°C. 1H NMR (500
at room temperature, 1.3 mol equivalent of NCS was MHz, DMSO-d6): δ 5.58 (s, 2H, H-9), 7.10 (pseudo t, H-5),
added. The initial addition (about 1/10 of the mass) results 7.27 (s, H-4’), 7.29 (s, H-3), 7.29 (pseudo t, H-6), 7.37 (pseudo
in a slight increase of temperature. In case that does not t, H-2’’, H-6’’), 7.48 (d, J = 8.3 Hz, H-7), 7.68 (d, J = 8.1 Hz,
happen, then the solution is heated to 45°C to initiate the H-4), 7.99 (dd, J
= 8.6 Hz , J
= 14.2 Hz, H-3’’, H-5’’),
H-H
H-F
13
reaction. Once the reaction is initiated, the rest of NCS was 11.99 (s, NH). C NMR (125 MHz, DMSO-d6): δ 57.0 (C-9),
added portion wise and the temperature was kept under 102.9 (C-4’), 109.3 (C-3), 113.1 (C-7), 116.7 (2J C-F = 21.9 Hz,
35°C. The mixture was stirred for 3-6 hours, and monitored C-3’’, C-5’’), 120.5 (C-5), 122.4 (C-4), 125.2 (C-6), 126.6 (C-3a),
by TLC (in CHCl3). After completion of the reaction, the 127.1 (C-2), 129.5 (C-1’’), 129.7 (3J C-F = 8.7 Hz, C-2’’, C-6’’),
mixture was poured over crushed ice and the resultant 138.1 (C-7a), 160.9 (C-5’), 161.6 (C-3’), 163.8 (1J C-F = 247.6 Hz,
product was filtrated as a solid. In the case of liquid C-4’’), 168.5 (C-8). HRMS (ESI) m/z: Calcd. For C19H12FN2O3
products, they were extracted using CHCl3 (20 mL×2) dried [M - H]+ 335.08320. Found: 335.08374.

144ꢀ
Raed A. Al-Qawasmeh et al.
4
7
4
7
3
3a
3
3a
7a
O
O
5
6
5
6
O
2
8
2
8
N
3'
9
5'
N
7a
O
N
OCH2CH3
O
H
4'
1''
2''
9
N
3'
3''
4''
5'
6''
4'
1''
2''
5''
3''
4''
(8a)
6''
(7a)
5''
Figure 4: structure and numbering of compounds (7a) and (8a).
(3-(4-chlorophenyl)isoxazol-5-yl)methyl 1H-indole-2-
carboxylate (7c)
Yield 54%; White solid; mp: 180 - 182°C. 1H NMR (500
(3-(p-tolyl)isoxazol-5-yl)methyl
carboxylate (7f)
Yield 35%; White solid; mp: 165 - 167°C. 1H NMR (500
1H-indole-2-
MHz, DMSO-d6): δ 9.53 (bs, 1H), 7.77 (bd, J = 8Hz, 1H), 7.69 MHz, DMSO-d6): δ 2.38 (s, 3H, CH3), 5.57 (s, 2H, H-9), 7.10
(m, 1H), 7.48-7.60 (m, 9H); 7.34-7.37 (m, 1H), 7.19-7.22 (m, 1H). (pseudo t, H-5), 7.21 (s, H-4’), 7.29 (s, H-3), 7.29 (pseudo t,
13C NMR (125 MHz, DMSO-d6): δ 57.0 (C-9), 102.9 (C-4’), 109.4 H-6), 7.34 (d, J = 7.9 Hz, H-3’’, H-5’’),7.48 (d, J = 8.2 Hz, H-7),
(C-3), 113.1 (C-7), 120.8 (C-5), 122.6 (C-4), 125.5 (C-6), 126.5 7.68 (d, J = 8.0 Hz, H-4), 7.79 (d, J = 7.9 Hz, H-2’’, H-6’’), 11.90
(C-3a), 127.1 (C-2), 127.5 (C-1’’), 128.6 (C-3’’, C-5’’), 129.4 (C-2’’, (s, NH). 13C NMR (125 MHz, DMSO-d6): δ 21.4 (CH3), 57.1
C-6’’), 135.5 (C-4’’), 138.1 (C-7a), 160.9 (C-5’), 161.7 (C-3’), (C-9), 102.8 (C-4’), 109.3 (C-3), 113.1 (C-7), 120.8 (C-5), 122.6
168.5 (C-8). HRMS (ESI) m/z: Calcd. for C19H13ClN2O3Na [M (C-4), 125.5 (C-6), 125.9 (C-2), 126.5 (C-3a), 127.0 (C-2’’, C-6’’),
+ Na]+ 375.05124. Found: 375.05069.
(3-(4-bromophenyl)isoxazol-5-yl)methyl 1H-indole-2- 160.9 (C-5’), 162.4 (C-3’), 168.0 (C-8). HRMS (ESI) m/z:
127.1 (C-4’’), 130.1 (C-3’’, C-5’’), 138.1 (C-7a), 140.5 (C-1’’),
carboxylate (7d)
Yield 46%; White solid; mp: 165 - 168°C. 1H NMR (500
MHz, DMSO-d6): δ 5.59 (s, 2H, H-9), 7.10 (pseudo t, H-5), 7.29 carboxylate (7g)
(s, H-3), 7.29 (pseudo t, H-6), 7.29 (s, H-4’), 7.48 (d, J = 8.2 Hz,
Calcd. For C20H15N2O3 [M - H]+ 333.12392. Found: 333.12337.
(3-(4-nitrophenyl)isoxazol-5-yl)methyl
1H-indole-2-
1
Yield 22%; Off-white solid; mp: 150 - 153°C. H NMR
H-7), 7.68 (d, J = 8.0 Hz, H-4), 7.74 (d, J = 8.4 Hz, H-2’’, H-6’’), (500 MHz, DMSO-d6): δ 5.62 (s, 2H, H-9), 7.10 (pseudo t,
7.87 (d, J = 8.4 Hz, H-3’’, H-5’’), 11.99 (s, NH). 13C NMR (125 H-5), 7.29 (s, H-3), 7.29 (pseudo t, H-6), 7.43 (s, H-4’), 7.48
MHz, DMSO-d6): δ 57.1 (C-9), 103.0 (C-4’), 109.4 (C-3), 113.1 (d, J = 8.3 Hz, H-7), 7.68 (d, J = 8.0 Hz, H-4), 8.20 (d, J = 8.7
(C-7), 120.8 (C-5), 122.7 (C-4), 124.3 (C-4’’), 125.5 (C-6), 126.5 Hz, H-2’’, H-6’’), 8.37 (d, J = 8.7 Hz, H-3’’, H-5’’), 12.00 (s,
(C-3a), 127.1 (C-2), 127.9 (C-1’’), 129.2 (C-2’’, C-6’’), 132.6 (C-3’’, NH). 13C NMR (125 MHz, DMSO-d6): δ 21.4 (CH3), 57.1 (C-9),
C-5’’), 138.1 (C-7a), 160.9 (C-5’), 161.7 (C-3’), 168.6 (C-8).
102.8 (C-4’), 109.3 (C-3), 113.1 (C-7), 120.8 (C-5), 122.6 (C-4),
(3-(4-cyanophenyl)isoxazol-5-yl)methyl 1H-indole-2- 125.5 (C-6), 125.9 (C-2), 126.5 (C-3a), 127.0 (C-2’’, C-6’’), 127.1
carboxylate (7e)
(C-4’’), 130.1 (C-3’’, C-5’’), 138.1 (C-7a), 140.5 (C-1’’), 160.9
Yield 34%; White solid; mp: 162 - 164°C. 1H NMR (500 (C-5’), 162.4 (C-3’), 168.0 (C-8). HRMS (ESI) m/z: Calcd. for
MHz, DMSO-d6): δ 5.63 (s, 2H, H-9), 7.10 (pseudo t, H-5), C19H13N3O5 [M - H]+ 362.07770 .Found: 362.7824.
7.29 (s, H-3), 7.29 (pseudo t, H-6), 7.40 (s, H-4’), 7.48 (d, J
(3-(4-methoxyphenyl)isoxazol-5-yl)methyl 1H-indole-
= 8.2 Hz, H-7), 7.68 (d, J = 8.0 Hz, H-4), 8.01 (d, J = 8.2 Hz, 2-carboxylate (7h)
H-3’’, H-5’’), 8.15 (d, J = 8.2 Hz, H-2’’, H-6’’), 12.00 (s, NH).
Yield 36%; white solid; mp: 146 - 149°C. 1H NMR (500
13C NMR (125 MHz, DMSO-d6): δ 57.1 (C-9), 103.3 (C-4’), MHz, DMSO-d6): δ 3.83 (s, 3H, OCH3), 5.56 (s, 2H, H-9),
109.4 (C-3), 113.1 (C-7), 113.3 (C-4’’), 118.8 (CN), 120.8 (C-5), 7.07 (d, J = 8.6 Hz, H-3’’, H-5’’), 7.10 (pseudo t, H-5), 7.19
122.6 (C-4), 125.5 (C-6), 126.5 (C-3a), 127.1 (C-2), 127.9 (C-2’’, (s, H-4’), 7.29 (s, H-3), 7.29 (pseudo t, H-6), 7.48 (d, J = 8.3
C-6’’), 133.0 (C-1’’), 133.6 (C-3’’, C-5’’), 138.2 (C-7a), 160.9 Hz, H-7), 7.68 (d, J = 8.0 Hz, H-4), 7.84 (d, J = 8.6 Hz, H-2’’,
(C-5’), 161.4 (C-3’), 169.0 (C-8). HRMS (ESI) m/z: Calcd. For H-6’’), 11.99 (s, NH). 13C NMR (125 MHz, DMSO-d6): δ 55.8
C20H12N3O3 [M - H]+ 342.08787. Found: 342.08763.
(OCH3), 57.0 (C-9), 102.7 (C-4’), 109.3 (C-3), 113.1 (C-7), 115.0

Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds
ꢀ145
(C-3’’, C-5’’), 120.8 (C-5), 121.6 (C-1’’), 122.6 (C-4), 125.5 (C-6), 7.15 (overlapped dd, J = 7.4, 7.7 Hz, H-5), 7.27 – 7.73 (m, 8H,
126.6 (C-3a), 127.1 (C-2), 128.4 (C-2’’, C-6’’), 138.1 (C-7a), 160.9 ArH). 13C NMR (75 MHz, DMSO-d6): δ = 14.6 (CH2CH3), 40.4
(C-5’), 161.1 (C-3’), 162.1 (C-4’’), 167.8 (C-8). HRMS (ESI) m/z: (NCH2C), 61.1 (OCH2CH3), 100.8 (C-4’), 111.6 (C-3), 111.7 (C-7),
Calcd. for C20H15N2O3 [M - H]+ 347.10373. Found: 347.10372.
121.7 (C-5), 123.1 (C-4), 124.2 (C-6), 126.1 (C-3a), 127.5 (C-1”),
Ethyl 1-((3-phenylisoxazol-5-yl)methyl)-1H-indole-2- 127.9 (C-2), 129.1 (C-2”, C-6”), 132.6 (C-3”, C-5”), 130.7 (C-4”),
carboxylate (8a)
139.5 (C-7a), 161.6 (C-5’), 161.6 (Ar-COO), 170.5 (C-3’). HRMS
1
Yield 60.5%; white solid; mp: 112 – 113°C. H NMR (ESI) m/z: Calcd. for C21H17BrN2O3Na + [M+Na]+ 447.03148.
(500 MHz, DMSO-d6): δ 1.33 (t, J = 7.10 Hz, CH2CH3), 4.34 Found: 447.03236.
(q, J = 7.10 Hz, OCH2CH3), 6.08 (s, NCH2C), 6.79 (s, H-4’),
Ethyl 1-((3-(4-cyanophenyl)isoxazol-5-yl)methyl)-1H-
6.89 (s, H-3), 7.19 – 7.81 (m, H-4, H-5, H-6, H-7, H-2”, H-3”, indole-2-carboxylate (8e)
13
1
H-4”, H-5”, H-6”). C NMR (125 MHz, DMSO-d6): δ = 14.6
Yield 50.3%; white solid; mp: 154 – 158°C. H NMR
(CH2CH3), 40.4 (NCH2C), 61.1 (OCH2CH3),100.8 (C-4’), 111.6 (300 MHz, DMSO-d6): δ 1.27 (t, J = 7.1 Hz, CH2CH3), 4.29 (q,
(C-7), 111.7 (C-3), 121.7 (C-5), 123.1 (C-4), 126.0 (C-6), 126.1 J = 7.1 Hz, OCH2CH3), 5.51 (s, NCH2C), 6.05 (s, H-4’), 6.89
(C-3a), 127.1 (C-2”, C-6”), 127.5 (C-2), 128.7 (C-4”), 129.5 (C-3”, (s, H-3), 7.16 (pseudo t, J = 7.8 Hz, H-5), 7.27 – 7.74 (m, H-4,
C-5”), 130.7 (C-1”), 139.4 (C-7a), 161.6 (C-5’), 162.3 (Ar-COO), H-6, H-7), 7.90 (d, J = 8.4 Hz, H-3”, H-5”), 7.98 (d, J = 8.4 Hz,
+
170.1 (C-3’). HRMS (ESI) m/z: Calcd. for C21H18N2O3 [M+H]+ H-2”, H-5”). 13C NMR (75 MHz, DMSO-d6): δ = 14.6 (CH2CH3),
347.13902. Found: 347.13903
40.6 (NCH2C), 61.2 (OCH2CH3), 101.2 (C-4’), 111.6 (C-3), 111.8
Ethyl 1-((3-(4-fluorophenyl)isoxazol-5-yl)methyl)-1H- (C-7), 113.2 (C-4”), 118.9 (C N), 121.7 (C-5), 126.1 (C-4), 127.0
indole-2-carboxylate (8b)
(C-6), 127.5 (C-3a), 128.0 (C-2”, C-6”), 133.0 (C-2), 133.5 (C-3”,
1
Yield 41.2%; white solid; mp: 109 – 110°C. H NMR C-5”), 139.0 (C-1”), 139. 5 (C-7a), 161.3 (C-5’), 161.6 (Ar-COO),
(500 MHz, DMSO-d6): δ 1.33 (t, J = 6.5 Hz, CH2CH3), 4.34 171.0 (C-3’). HRMS (ESI) m/z: Calcd. for C22H17N3O3 [M+H]+
+
(q, J = 6.5 Hz, OCH2CH3), 6.07 (s, NCH2C), 6.80 (s, H-4’), 372.13427. Found: 372.13426.
7.20 (overlapped dd, J = 7.1, 6.9, H-5), 7.31 (overlapped dd,
Ethyl 1-((3-(p-tolyl)isoxazol-5-yl)methyl)-1H-indole-2-
J = 8.1, 8.2 Hz, H-3”, H-5”), 7.41 (m, H-3, H-6), 7.76 (m, H-4, carboxylate (8f)
13
H-7), 7.87 (m, H-2”, H-6”). C NMR (125 MHz, DMSO-d6): δ
Yield 59.5%; white solid; mp: 80 – 81°C. 1H NMR (300
= 14.6 (CH2CH3), 40.4 (NCH2C), 61.1 (OCH2CH3), 100.8 (C-4’), MHz, DMSO-d6) : δ = 1.28 (t, J = 7.1 Hz, CH2CH3), 2.29 (s,
111.6 (C-3), 111.7 (C-7), 116.6 (2J C-F = 21.9 Hz, C-3”, C-5”), Ar-CH3), 4.29 (q, J = 7.1 Hz, OCH2CH3), 6.01 (s, NCH2C), 6.68
121.67 (C-5), 123.1 (C-4), 125.3 (4J C-F = 3.2 Hz, C-1”), 126.0 (s, H-4’), 7.13 (overlapped dd, J = 7.4, 7.3 Hz, H-5), 7.22 (d, J
(C-6), 126.1 (C-3a), 127.4 (C-2), 129.5 (3J C-F = 8.6 Hz, C-2”, =7.8 Hz, H-3”, H-5”), 7.30 – 7.43 (m, H-3, H-6), 7.64 (d, J = 7.8
C-6”), 139.5 (C-7a), 161.5 (Ar-COO), 161.6 (C-5’), 163.7 (1J C-F Hz, H-2”, H-6”), 7.70 (d, J = 7.8, H-4, H-7). 13C NMR (75 MHz,
= 247.8 Hz, C-4”), 170.3 (C-3’). HRMS (ESI) m/z: Calcd. for DMSO-d6) : δ = 14.8 (CH2CH3), 21.4 (Ar-CH3), 40.5 (NCH2C),
+
C21H17FN2O3 [M+H]+ 387.11154. Found: 387.11145.
61.1 (OCH2CH3), 100.7 (C-4’), 111.6 (C-3), 111.7 (C-7), 121.7 (
Ethyl 1-((3-(4-chlorophenyl)isoxazol-5-yl)methyl)-1H- C-5 ), 123.1 (C-4), 125.9 (C-2), 126.1 (C-6), 127.0 (C-2”, C-6”),
indole-2-carboxylate (8c)
127.5 (C-1”), 130.1 (C-3”, C-5”), 139.5 (C-7a), 140.5 (C-4”), 161.6
Yield 21.1%; white solid; mp: 117 – 119°C. 1H NMR (C-5’), 162.3 (Ar-COO), 169.9 (C-3’). HRMS (ESI) m/z: Calcd.
(300 MHz, DMSO-d6): δ 1.39 (t, J = 7.1 Hz, CH2CH3), 4.37 for C22H20N2O3 [M+H]+ 361.15467. Found: 361.15450.
+
(q, J = 7.1 Hz, OCH2CH3), 5.95 (s, NCH2C), 6.22 (s, H-4’), 7.19
Ethyl
1-((3-(4-nitrophenyl)isoxazol-5-yl)methyl)-1H-
(overlapped dd, J = 7.8, 7.2 Hz, H-5), 7.34 (d, J = 8.3 Hz, H-3”, indole-2-carboxylate (8g)
H-5”) ,7.39 (m, H-3, H-6), 7.49 (d, J = 8.5 Hz, H-4), 7.62 (d, J =
Yield 57.4%; green solid; mp: 111 – 114°C. 1H NMR (500
8.3 Hz, H-2”, H-6”), 7.69 (d, J = 7.8 Hz, H-7). 13C NMR (75 MHz, MHz, DMSO-d6): δ = 1.32 (t, J = 7.0 Hz, CH2CH3), 4.34 (q,
CDCl3): δ = 14.4 (CH2CH3), 40.2 (NCH2C), 60.9 (OCH2CH3), J = 7.0 Hz, OCH2CH3), 6.11 (s, NCH2C), 6.96 (s, H-4’), 7.21
100.3 (C-4’), 110.4 (C-3), 111.8 (C-7), 121.5 (C-5), 122.9 (C-4), (overlapped dd, J = 6.9, 7.7 Hz, H-5), 7.39 (m, H-3, H-6) ,7.77
125.9 (C-6), 126.2 (C-3a), 127.1 (C-1”), 127.3 (C-2), 128.1 (C-2”, (m, H-4, H-7), 8.11 (d, J = 7.4 Hz, H-2”, H-6”), 8.31 (d, J =
C-6”), 129.1 (C-3”, C-5”), 136.1 (C-4”), 139.2 (C-7a), 161.6 7.4 Hz, H-3”, H-5”). 13C NMR (125 MHz, DMSO-d6) : δ = 14.6
(C-5’), 162.0 (Ar-COO), 169.7 (C-3’). HRMS (ESI) m/z: Calcd. (CH2CH3) , 40.6 (NCH2C), 61.1 (OCH2CH3), 101.3 (C-4’), 111.5
for C21H17ClN2O3 [M+H]+ 381.10005. Found: 381.10003.
(C-3), 111.8 (C-7), 121.7 ( C-5 ), 123.1 (C-4), 124.7 (C-3”, C-5” ),
+
Ethyl 1-((3-(4-bromophenyl)isoxazol-5-yl)methyl)-1H- 126.1 (C-6), 127.4 (C-2), 128.4 (C-3a), 128.8 (C-2”, C-6”), 134.7
indole-2-carboxylate (8d)
(C-1”), 139.4 (C-7a), 148.8 (C-4”), 161.0 (C-5’), 161.6 (Ar-COO),
Yield 52.7%; white solid; mp: 108 – 109°C. H NMR 171.1 (C-3’). HRMS (ESI) m/z: Calcd. for C21H17N3O5+[M+H]+
(300 MHz, DMSO-d6): δ 1.27 (t, J = 7.1 Hz, CH2CH3), 4.29 392.12410. Found: 392.12397.
(q, J = 7.1 Hz, OCH2CH3), 6.02 (s, NCH2C), 6.77 (s, H-4’),
1

146ꢀ
Raed A. Al-Qawasmeh et al.
Ethyl 1-((3-(4-methoxyphenyl)isoxazol-5-yl)methyl)- dish. Sterile paper disks (6 mm in diameter; Becton,
1H-indole-2-carboxylate (8h) Dickinson & Co.) were placed on the agar plates and
1
Yield 31.8%; yellow solid; m.p: 144 – 146°C. H NMR impregnated with 30 uL solution of each compound.
(300 MHz, DMSO-d6): δ = 1.27 (t, J = 7.1 Hz, CH2CH3), 3.84 Plates were incubated for the recommended time
(s, Ar-OCH3), 4.29 (q, J = 7.1 Hz, OCH2CH3), 5.99 (s, NCH2C), periods (24–48 h and up to 7 days for Aspergillus) under
6.76 (s, H-4’), 7.07 – 7.38 (m, H-6, H-3’’, H-5”), 7.65 -7.74 (m, appropriate cultivation conditions. Antimicrobial activity
13
H-3, H-6), 7.82 (d, J = 2.1 Hz, H-2”, H-6”). C NMR (75 MHz, was determined if the tested compound produced an
DMSO-d6): δ = 14.6 (CH2CH3), 40.7 (NCH2C), 56.8 (OCH3), 61.2 inhibition zone around the impregnated disk. Disks
(OCH2CH3), 100.7 (C-4’), 111.5 (C-3), 111.7 (C-7), 113.7 (C-3”, impregnated with sterile DMSO served as negative
C-5”), 121.7 (C-5), 122.0 (C-4), 122.2 (C-1”), 123.1 (C-6), 126.1 controls and disks with standard antibiotics (vancomycin,
(C-2”, C-6”), 127.3 (C-3a), 127.5 (C-2), 139.4 (C-7a), 156.4 (C-5’), ciprofloxacin, and clarithromycin (Oxoid, UK)) served as
161.1 (Ar-COO), 161.6 (C-4”), 170.2 (C-3’). HRMS (ESI) m/z: positive controls. Measurements at each concentration
+
Calcd. for C22H20N2O4 [M+H]+ 377.14958. Found: 377.14969.
were performed in triplicates [36]. For MIC determination,
200 µL of diluted microbial suspension was added, (0.2-
500 µg/50 µL) of the synthesized compounds and standard
antibiotics (Cephtriaxone, Gentamycin and Levofloxacin)
in 20% H2O/DMSO were added to wells of ELISA plates,
and subsequently incubated at conditions described
2.2.7 Grwoth and Maintainance of Microorganisms and
Cancer Cell Lines
The synthesized compounds were tested against Gram- above. The effect of the tested compounds on the growth
positive strains namely; Methicillin-sensitive Staphylococcus of microorganisms was monitored by measuring the
aureus NCTC 10788 (MSSA), methicillin-resistant optical density at 600 nm using an ELISA reader. The
Staphylococcus aureus ATCC 33591 (MRSA), two standard MIC was defined as the lowest concentration of the tested
Gram-negative bacterial strains namely; Escherichia coli compounds allowing no visible growth. All measurements
NCTC 12923, Pseudomonas aeruginosa NCTC 12924 along of MIC values were repeated in triplicate.
with the two fungi Candida albicans (NCPF 3179) and
Antimicrobial interactions between the tested
Aspergillus brasiliences (NCPF 2275). The microorganisms compounds conventional antimicrobial agents like
were inoculated into Trypticase soy broth and Sabaroud cefzolidin and amoxicillin against MRSA were evaluated
dextrose broth for bacteria and fungi respectively and the by the standard checkerboard titration method [34].
pH of the medium was adjusted to 7.3 with sterile phosphate The bacterial suspensions, growth media, and culture
buffered saline. The cultures were subsequently incubated conditions were the same as those described for the
at 37°C for 24-48 hours except for Aspergillus which was MIC determination mentioned above. Experiments
incubated at 25°C for 5-7 days. The optical density (OD) of the were performed in triplicate. The fractional inhibitory
bacteria from mid-log phase of growth was measured at 600 concentrations (FICs) were calculated as follows:
nm and to obtain an optical density of 0.4 (corresponding to
1.5×108 colony forming units/mL).
MCF-7 (human breast adenocarcinoma), Hep2 (human
epiglottis cancer), and Hela (cervical cancer cells) cell
FIC = (MIC of drug A in combination/MIC of drug A alone)
+ (MIC of drug B in combination/MIC of drug B alone).
cultures were kindly provided by Jordan Company for The FIC indices were interpreted as follows: ≤0.5, synergy;
Antibody Production (MONOJO, Amman, Jordan). The cell 0.5–1, additive; 1–4.0, indifference; >4.0, antagonism [37].
lines were cultured in growth medium (RPMI-1640medium,
pH7.4), supplemented with 15% fetal bovine serum (FBS)
and antibiotics [(penicillin (100 units/mL) and streptomycin 2.2.9 Cell Cytotoxicity for Anticancer Activity Testing
(100 ug/mL)]. The cell lines were maintained at 37°C in a 5%
CO2 atmosphere with 95% humidity [33].
Microculture tetrazolium (MTT) assay was used to evaluate
cell vitality. Briefly, monolayers of each cell line were
trypsinized and the cells were seeded in 96-well plates at
the density of 5 x105 cell/well (100 µL/well) in a culture
medium and incubated for 24 h at 37oC, with 5% CO2 in
2.2.8 Antimicrobial Susceptibility Testing
The stock suspensions of microorganisms were prepared a humidified atmosphere. The medium was subsequently
to the standard McFarlands (0.5) and subsequently removed and fresh growth medium containing different
uniformly spread on a solid growth medium in a Petri concentrations of tested compounds (concentration range

Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds
ꢀ147
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of 5-500 µg/mL) were separately added. Control cells were
supplemented only with a medium and colchicine was
used as a positive control. 20 µL of 5 mg / mL MTT (pH
7.4) in 180 µL media was added per well and incubated
for another 4 h. The plates were then centrifuged and
supernatants were removed to all the addition of 100 µL of
1:1 ethanol: DMSO. The absorbance was then measured by
an ELISA reader Bio-Rad, USA) at a wavelength of 570 nm.
The values are presented as means of duplicate analyses.
The activity of the tested compounds on the proliferation
of cancer cells was expressed as the % of cytotoxicity [36].
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3 Conclusion
In summary, sixteen new hybrid compounds were
synthesized featuring the indole and the isoxazole
moieties. All new compounds were identified and tested
against three bacterial strains and three cancer cell lines.
It was found through this research work that these newly-
synthesized compounds did not show any promising
activities. Single X-ray crystal structure was determined for
a representative compound of these new hybrid systems.
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[17] Weidner-Wells MA, Henninger TC, Fraga-Spano SA, Boggs CM,
Matheis M, Ritchie DM, et al. Synthesis and structure-activity
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(IL-8) receptor antagonists. Bioorg Med Chem Lett. 2004
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Acknowledgments: This work was supported by grants
(1015-1014\19) from the Deanship of Scientific Research-
the University of Jordan, Amman 11942, Jordan.
Conflict of interest: Authors declare no conflict of interest.
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