Synthesis, characterization, and antimicrobial activity of some new phosphorus macrocyclic compounds containing pyrazole rings

Article abstract of DOI:10.3906/kim-1205-18

A simple synthetic method is reported for the preparation of some new phosphorus macrocycles in which the pyrazole rings are appended to a phosphorus atom. The methodology is based on the cyclocondensation reaction of bis(4-formylpyrazolyl) phosphine oxides (1a, 1b) with nitrogen nucleophiles that contain active terminal amino groups. The antimicrobial activities of the synthesized compounds were assayed. TUeBITAK.

Full text of DOI:10.3906/kim-1205-18

Turk J Chem
Turkish Journal of Chemistry
(2013) 37: 160 – 169
¨
http://journals.tubitak.gov.tr/chem/
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1205-18
Research Article
Synthesis, characterization, and antimicrobial activity of some new phosphorus
macrocyclic compounds containing pyrazole rings
Tarik El-Sayed ALI∗, Salah Abdel-Aziz ABDEL-GHAFFAR, Kamilia Mohamed EL-MAHDY,
Somaia Mohamed ABDEL-KARIM
Department of Chemistry, Faculty of Education, Ain Shams University,
Roxy, 11711 Cairo, Egypt
Received: 09.05.2012
•
Accepted: 04.12.2012
•
Published Online: 24.01.2013
•
Printed: 25.02.2013
Abstract: A simple synthetic method is reported for the preparation of some new phosphorus macrocycles in which
the pyrazole rings are appended to a phosphorus atom. The methodology is based on the cyclocondensation reaction of
bis(4-formylpyrazolyl) phosphine oxides (1a, 1b) with nitrogen nucleophiles that contain active terminal amino groups.
The antimicrobial activities of the synthesized compounds were assayed.
Key words: Phosphorus macrocycles, pyrazole, cyclocondensation, nitrogen nucleophiles
1. Introduction
The discovery of a family of macrocyclic compounds (cyclophanes) was a milestone in chemistry and opened
new frontiers in the synthesis of supramolecular host molecules. This group of compounds was recognized
as a source of receptors for many chemical species, such as metal cations and inorganic and organic anions,
as well as structurally variable organic molecules.1 Therefore, they are widely used for construction of metal
sequestering agents, selective sensors, mimetics of enzymes, and carriers for transport through membranes. 2−10
Phosphorus-containing macrocycles are interesting molecules with potential application in supramolecular and
synthetic organic chemistry.11 They were synthesized as phosphine oxides, phosphines, phosphonium salts,
phosphates, phosphonates, and phosphoranes.12 The importance of these molecules, as phosphorous analogs of
crown ethers, is their potential catalytic activity and ion-carrier properties. The synthesis of host molecules that
are capable of binding neutral organic molecules as guests is an area of rapidly expanding interest.13 Tucker et
al.,14 Fages et al.,15 Wallon et al.,16 Seward et al.,17 and Brelow et al.18 have made significant advances in
the field of host–guest complexation. Some of the past and present research has led to the construction of large
reorganized macrocyclic cavities bearing concave functionalities.19 They are also expected to function as good
‘hosts’ in the ‘host–guest chemistry’. This particular property enables them to carry the drug molecule to the
required site in the living system, thus suggesting a great future for them in the pharmaceutical industry. On
the other hand, the introduction of a pyrazole ring into molecular structures is one of the important aims of
the present work, which promises an approach leading to new biological properties.20,21 In view of these and
several other possible applications, the present work deals with the synthesis and characterization of some new
phosphorus macrocycles with nitrogen, oxygen, sulfur, and phosphorus as rich electronic centers. In addition,
their antimicrobial activities were also evaluated.
∗Correspondence: tarik elsayed1975@yahoo.com
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2. Experimental
The melting point was determined in an open capillary tube on a digital Stuart SMP-3 apparatus. Infrared
spectra were measured on a PerkinElmer 293 spectrophotometer (cm−1), using KBr disks. 1 H NMR spectra
were measured on a Gemini-200 spectrometer (200 MHz), using DMSO-d6 as a solvent and TMS (δ) as
the internal standard. 31 P NMR spectra were registered on a Varian Inova 202 MHz spectrometer at room
temperature using DMSO-d6 as a solvent, TMS as the internal standard, and 85% H3 PO4 as the external
reference. Mass spectra were recorded on a gas chromatographic GCMSqp 1000-EX Shimadzu instrument at
70 eV. Elemental microanalyses were performed at the microanalysis center of the National Research Center,
Giza. The results of elemental microanalyses were satisfactory and did not exceed 0.4% for carbon and hydrogen
and 0.42% for nitrogen. The purity of the synthesized compounds was checked by thin layer chromatography
(TLC). Bis(4-formylpyrazolyl)phosphine oxides (1a, 1b),22 carbohydrazide (2a),23 thiocarbohydrazide (2b),23
phosphonic dihydrazide (3a),24 and P-methoxyphosphonic dihydrazide (3b)24 were prepared according to the
reported methods in the literature.
2.1. General procedure for macrocycles 4a, 4b and 5a, 5b
A hot ethanolic solution (15 mL) of bis(4-formyl-3-phenyl-1H-pyrazol-1-yl) phosphine oxide (1a) (0.78 g,
2 mmol) was mixed with a hot aqueous solution of appropriate dihydrazide (2 mmol in 5 mL of water),
namely carbohydrazide (2a), thiocarbohydrazide (2b), phosphonic dihydrazide (3a), and P-methoxyphosphonic
dihydrazide (3b) in the presence of 2 drops of glacial acetic acid. The solution mixture was heated under reflux
for 4 h. The resulting solids were filtered off and crystallized from dimethylformamide to give the corresponding
macrocycles 4a, 4b and 5a, 5b, respectively, as yellow crystals with yields of 58%–71%.
13,33-Diphenyl-6,8-dihydro-5,6,8,9-tetraaza-2-phosphoryl-7-oxo-1,3-(1,4)-dipyrazola-cyc-
lodcaphane-4,9-diene (4a): Mp 211–212 ◦ C. IR (KBr, cm−1): 3300 (NH), 3029 (C–Harom), 2500 (P–H),
1690 (C=O), 1601 (C=Nexocyclic), 1539 (C=C), 1219 (P=O). 1 H NMR (DMSO-d6 , δ): 6.66 (d, 2H, J =
7.0 Hz, Ph–H), 7.18 (d, 1H, J = 8.0 Hz, Ph–H), 7.37–7.62 (m, 4H, Ph–H), 7.68 (d, 1H, JPH = 584 Hz, P–H),
8.00 (d, 3H, J = 8.6 Hz, Ph–H), 8.42 (brs, 2H, C5 –Hpyrazole), 9.86 (s, 2H, CH=Nexocyclic), 11.81 (br, 2H, NH
exchangeable with D2 O).m/z (relative intensity %): 444 (M+ , 25), 408 (50), 301 (50), 186 (50), 156 (50), 65
(100). Anal. Calcd. for C21 H17 N8 O2 P (444.38): C, 56.76; H, 3.86; N, 25.22. Found: C, 56.37; H, 3.53; N,
25.35%.
13,33-Diphenyl-6,8-dihydro-5,6,8,9-tetraaza-2-phosphoryl-7-thioxo-1,3-(1,4)-dipyrazo-lacyc-
lodecaphane-4,9-diene (4b): Mp 214–216 ◦ C. IR (KBr, cm−1): 3135 (NH), 3060 (C–Harom), 2500 (P–H),
1597 (C=Nexocyclic), 1540 (C=Nendocyclic), 1500 (C=C), 1218 (P=O), 1129 (C=S). 1 H NMR (DMSO-d6 ,
δ): 7.64 (d, 1H, JPH = 612 Hz, P–H), 6.51–7.96 (m, 10H, Ph–H), 8.49 (s, 2H, C5 –Hpyrazole), 9.80 (s, 2H,
CH=Nexocyclic), 11.72 (br, 2H, NH exchangeable with D2 O).m/z (relative intensity %): 462 (M+2, 0.02),
461 (M+1, 0.13), 460 (M+ , 0.45), 369 (2), 321 (1.4), 246 (7), 221 (18), 161 (12), 104 (47), 77 (100), 51 (16).
Anal. Calcd. for C21 H17 N8 OP (460.45): C, 54.78; H, 3.72; N, 24.34. Found: C, 54.42; H, 3.39; N, 24.47%.
13,33-Diphenyl-6,8-dihydro-5,6,8,9-tetraaza-2,7-diphosphoryl-1,3-(1,4)-dipyrazolacyc-lode-
caphane-4,9-diene (5a): Mp 242–244 ◦ C. IR (KBr, cm−1): 3427 (NH), 3056 (C–Harom), 2500 (P–H), 1615
(C=Nexocyclic), 1595 (C=Nendocyclic), 1533 (C=C), 1294, 1202 (2P=O). 1 H NMR (DMSO-d6 , δ): 6.52 (d,
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1H, J = 6.0 Hz, Ph–H), 7.16 (d, 1H,J =8.2 Hz, Ph–H), 7.45–7.98 (m, 8H, Ph–H), 7.39 (d, 1H, JPH = 512 Hz,
P–H), 8.06 (d, 1H, JPH = 406 Hz, P–H), 8.47 (s, 2H, C5 –Hpyrazole ), 9.80 (s, 2H, CH=Nexocyclic).31 P NMR
(DMSO-d6 , δ): 8.29 (pyrazole–P–pyrazole), 17.70 (NH–P–NH). m/z (relative intensity %): 462 (M–2, 0.2),
324 (100), 231 (18), 104 (16), 77 (70), 65 (24), 51 (24). Anal. Calcd. for C20 H18 N8 O2 P2 (464.36): C, 51.73;
H, 3.91; N, 24.13. Found: C, 51.45; H, 3.59; N, 23.72%.
13,33-Diphenyl-6,8-dihydro-5,6,8,9-tetraaza-2-phosphoryl-7-methoxyphosphoryl-1,3-(1,4)-
dipyrazolacyclodecaphan-4,9-diene (5b): Mp 232–234 ◦ C. IR (KBr, cm−1): 3429 (br, NH), 3055 (C–
Harom), 2992 (C–Haliph), 2353 (P–H), 1602 (C=Nexocyclic), 1534 (C=C), 1294, 1209 (2 P=O), 1053 (P–O–
C). 1 H NMR (DMSO-d6 , δ): 3.35 (s, 3H, OCH3), 5.24 (brs, 2H, NH exchangeable with D2 O), 6.52 (d, 1H,
J = 8.4 Hz, Ph–H), 7.02–7.20 (m, 3H, Ph–H), 7.40 (d, 1H, JPH = 513 Hz, P–H), 7.32–7.54 (m, 4H, Ph–H),
7.76 (d, 1H, J = 7.2 Hz, Ph–H), 8.02 (d, 1H, J = 7.2 Hz, Ph–H), 8.49 (s, 2H, C5 –Hpyrazole), 9.81 (s, 2H,
CH=Nexocyclic).m/z (relative intensity %): 494 (M+ , 3.3), 463 (3), 246 (41), 231 (15), 116 (12), 77 (100), 51
(32). Anal. Calcd. for C21 H20 N8 O3 P2 (494.38): C, 51.02; H, 4.08; N, 22.67. Found: C, 50.79; H, 3.69; N,
22.25%.
2.2. General procedure for macrocycles 8a, 8b and 9a, 9b
A hot ethanolic solution (10 mL) of bis(4-formyl-3-(4‘-biphenyl)-1H-pyrazol-1-yl) phosphine oxides (1b) (1.08
g, 2 mmol) was mixed with a hot ethanolic solution of appropriate diamine (2 mmol in 10 mL), namely
hydrazine hydrate (6a), 1,2-ethylenediamine (6b), 1,2-phenylenediamine (7a), and 1,4-phenylenediamine (7b)
in the presence of 2 drops of glacial acetic acid. The solution mixture was heated under reflux for 4 h. The
resulting solids were filtered off and crystallized from dimethylformamide to give the corresponding macrocycles
8a, 8b and 9a, 9b, respectively, as yellow crystals with yields of 48%–85%.
13,33,83,103-Tetris(4‘-biphenyl)-5,6,12,13-tetraaza-2,9-diphosphoryl-1,3,8,10(1,4)-tetrapy-
razolacyclotetradecaphane-4,6,11,13-tetraene (8a): Mp 232–235 ◦ C. IR (KBr, cm−1): 3057 (C–Harom),
2363 (P–H), 1614 (C=Nexocyclic), 1597 (C=Nendocyclic), 1533 (C=C), 1209 (P=O). 1 H NMR (DMSO-d6 ,
δ): 7.56 (d, 1H, JPH = 590 Hz, P–H), 7.67 (d, 1H, JPH = 612 Hz, P–H), 6.50–7.99 (m, 36H, Ar–H), 8.46
(s, 2H, C5 –Hpyrazole ), 8.65 (s, 2H, C5 –Hpyrazole), 9.78 (s, 4H, CH=Nexocyclic).m/z (relative intensity %):
1073 (M–4H, 0.1), 882 (0.2), 734 (0.3), 452 (0.5), 307 (3), 293 (11), 167 (24), 149 (100), 127 (13), 96 (11), 57
(35). Anal. Calcd. for C64 H46 N12 O2 P2 (1077.07): C, 71.37; H, 4.30; N, 15.61. Found: C, 71.63; H, 4.44; N,
15.23%.
13,33,103,123-Tetris(4‘-biphenyl)-5,8,14,17-tetraaza-2,11-diphosphoryl-1,3,10,12(1,4)-tet-
rapyrazolacyclooctadecaphane-4,8,13,17-tetraene (8b): Mp 218–220 ◦ C. IR (KBr, cm−1): 3053 (C–
H
arom), 2880, 2844 (C–Haliph), 2362 (P–H), 1633 (C=Nexocyclic), 1595 (C=Nendocyclic), 1539 (C=C), 1214
(P=O). 1 H NMR (DMSO-d6 , δ): 3.79 (t, 8H, 2 CH2 CH2), 7.59 (d, 1H, JPH = 619 Hz, P–H), 7.69 (d, 1H,
JPH = 633 Hz, P–H), 6.51–8.28 (m, 36H, Ar–H), 8.49 (s, 2H, C5 –Hpyrazole), 8.87 (s, 2H, C5 –Hpyrazole), 9.80
(s, 2H, CH=Nexocyclic), 9.99 (s, 2H, CH=Nexocyclic). Anal. Calcd. for C68 H54 N12 O2 P2 (1133.18): C, 72.07;
H, 4.80; N, 14.83. Found: C, 71.72; H, 4.59; N, 14.42%.
13,33,93,113-Tetris(4‘-biphenyl)-6,14(1,2)-dibenzene-5,7,13,15-tetraaza-2,10-diphosph-oryl-
1,3,9,11(1,4)-tetrapyrazolacyclohexadecaphane-4,7,12,15-tetraene (9a): Mp 299–300 ◦ C. IR (KBr,
cm−1): 3057 (C–Harom), 2361 (br, P–H), 1615 (C=Nexocyclic), 1595 (C=Nendocyclic), 1533 (C=C), 1209
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ALI et al./Turk J Chem
(P=O). 1 H NMR (DMSO-d6 , δ): 7.70 (d, 1H, JPH = 610 Hz, P–H), 7.79 (d, 1H,JPH = 631 Hz, P–H), 6.64–
8.01 (m, 44H, Ar–H), 8.63 (s, 4H, C5 –Hpyrazole ), 9.87 (s, 2H, CH=Nexocyclic), 10.05 (s, 2H, CH=Nexocyclic).
Anal. Calcd. for C76 H54 N12 O2 P2 (1229.268): C, 74.26; H, 4.43; N, 13.67. Found: C, 73.86; H, 4.12; N,
13.25%.
13,33,93,113-Tetris(4‘-biphenyl)-6,14(1,4)-dibenzene-5,7,13,15-tetraaza-2,10-diphosph-oryl-
1,3,9,11(1,4)-tetrapyrazolacyclohexadecaphane-4,7,12,15-tetraene (9b): Mp 313–315 ◦ C. IR (KBr,
cm−1): 3023 (C–Harom), 2363 (P–H), 1607 (C=Nexocyclic), 1537 (C=C), 1213 (P=O). 1 H NMR (DMSO-d6 ,
δ): 7.70 (d, 1H, JPH = 610 Hz, P–H), 7.79 (d, 1H,JPH = 630 Hz, P–H), 6.59–8.07 (m, 44H, Ar–H), 8.56 (s,
2H, C5 –Hpyrazole), 8.60 (s, 2H, C5 –Hpyrazole ), 9.86 (s, 2H, CH=Nexocyclic), 10.05 (s, 2H, CH=Nexocyclic).
31 P NMR (DMSO-d6 , δ): 8.38. m/z (relative intensity %): 1214 (M–16, 0.2), 643 (0.2), 501(0.4), 353 (30),
309 (100), 177 (10), 104 (16), 69 (53), 57 (48). Anal. Calcd. for C76 H54 N12 O2 P2 (1229.268): C, 74.26; H,
4.66; N, 13.64. Found: C, 73.87; H, 4.27; N, 13.29%.
3. Results and discussion
A series of some new phosphorus macrocycles containing pyrazole rings were achieved via reaction of bis(4-
formylpyrazolyl)phosphine oxides (1a, 1b) with some dihydrazides containing oxygen, sulfur, and/or phosphorus
atoms and also with aliphatic/aromatic diamines. The reactions of dicarbonyl compounds with diamines are
much more complicated and produce a wide spectrum of products that can be identified by mass spectrometry.25
The type [1+1] macrocycle is usually formed as the major product when any flexible diamine reacts with a
dicarbonyl compound.26 Thus, equimolar amounts (approximately <10−2 mol) of bis-{4-formyl-3-phenyl-1H-
pyrazol-1-yl}phosphine oxide (1a) and carbohydrazide (2a), thiocarbohydrazide (2b), phosphonic dihydrazide
(3a), and P-methoxyphosphonic dihydrazide (3b) were condensed in aqueous ethanol under reflux, leading to
the formation of [1+1] macrocycles 4a, 4b and 5a, 5b, respectively (Scheme 1).
When 2 amino groups are located in close proximity to each other (e.g., α,β- and α,γ-diamines), the
respective diamines can react with dicarbonyl compounds to produce [2+2] macrocycles.27,28 Thus, macrocycles
8a, 8b and 9a, 9b were obtained via reaction of bis-{4-formyl-3-(4‘-biphenyl)-1H-pyrazol-1-yl}phosphine oxide
(1b) with hydrazine hydrate (6a), 1,2-ethylenediamine (6b), 1,2-phenylenediamine (7a), and 1,4-phenylene-
diamine (7b), respectively, under the same previous conditions (Scheme 2).
3.1. The IR spectral study
The absorption bands corresponding to free aldehyde (–CHO) and primary amine (–NH2) groups were not
observed for the prepared macrocycles. This confirmed that there is a complete condensation of the terminal
amino groups of nitrogen nucleophiles with bis(4-formyl-pyrazolyl)phosphine oxides (1a, 1b). The recorded
absorption bands in the region of 3135–3429 cm−1 in the IR spectra of 4a, 4b and 5a, 5b were assignable to
NH stretching vibrations. In addition, the azomethine bonds were observed from the presence of strong bands
in the frequency region of 1597–1615 cm−1 for all the newly formed macrocycles. The pure characteristic ν
(C–H) modes of aromatic rings were also observed in the region of 3023–3060 cm−1 in all macrocycles, while ν
(C–H) modes of aliphatic groups in macrocycles 5b and 8b were displayed in the region of 2880–2992 cm−1
.
The vibrational frequencies of C=O and C=S functional groups in macrocycles 4a and 4b appeared at 1690
and 1129 cm−1 , respectively. Moreover, the P=O groups in all macrocycles were also recorded in the wave
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CHO
OHC
Ph
O
P
N
N
N
Ph
N
H
1a
(NH2NH)2P(O)R
(NH2NH)2C=X
2a,b
3a,b
X
O
R
7
8
9
P
HN
NH
N
HN
N
NH
N
6
5
N
10
4
H
H
5
1
2
N
N
N
N
N
N
4
N
P
3
1
P
O
N
3
O
2
5a,b
4a,b
Compd
3a, 5a
3b, 5b
R
Compd
X
H
O
2a, 4a
OCH3
S
2b, 4b
Scheme 1. Synthetic route for the preparation of the macrocycles 4a, 4b and 5a, 5b.
region of 1209–1294 cm−1
.
3.2. The 1 H NMR spectral study
The 1 H NMR spectra of the formed macrocycles in DMSO-d6 did not give any signal corresponding to free
aldehyde (–CHO) or primary amine (–NH2) protons; instead, they showed broad signals in the region of δ
11.72, 11.81, and 5.24 ppm corresponding to NH protons in macrocycles 4a, 4b, and 5b, respectively. The
signals of NH–P(O)H–NH moiety in the 1 H NMR spectrum of macrocycle 5a were displaced, presumably as
a result of rapid proton exchange in 2 types of tautomeric equilibrium. First, there is the tautomeric amide–
imide equilibrium (I, II, III) (Scheme 3). Second, the hydrophosphoryl unit in solutions easily undergoes the
tautomeric transition, providing it a unique combination of properties of pentavalent and trivalent phosphorus
atoms (phosphite–phosphonate I and IV) (Scheme 3).29−32 The protons of hydrophosphoryl units that attached
to pyrazole rings in all macrocycles appeared as doublets in the region of δ 7.39–8.06 ppm with coupling
constants in the range of 406–634 Hz. The high field protons for exocyclic CH=N and C5 –H pyrazole rings
were attributed to δ 9.78–10.05 and 8.46–8.87 ppm, respectively. It was interesting that the signals of protons of
exocyclic CH=N and C5 –H pyrazole rings were duplicated at different values of chemical shifts in macrocycles
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CHO
OHC
Ar
O
P
H
N
N
N
Ar
N
H2N
H2N
1b
NH2-X-NH2
6a,b
7a,b
Ar=
X
4
5
N
N
Ar
Ar
3
6
14
12
N
2
1
N
N
16
Ar
3
Ar
N
13
15
N
P
N
4
N
P
N
O
H
H
O
11
1
N
2
N
5
N
N1
10
N
2
N
H
P
O
O
P
H
N
9
N
3
Ar
N
Ar
N
N
N
X
7
Ar
5
Ar
N
8
N
4
8a,b
6
9a,b
Compd
6a, 8a
X
bond
Compd
Diamine
6b, 8b
CH2CH2
7a, 9a
7b, 9b
1,2-disubstituted
1,4-disubstituted
Scheme 2. Synthetic route for the preparation of the macrocycles 8a, 8b and 9a, 9b.
8a, 8b and 9a, 9b (see Experimental). This can be explained as the result of the presence of each 2 pyrazole
rings separated with the hydrophosphoryl unit at different planes. We failed to obtain suitable crystals to
conduct X-ray crystallography to confirm this suggestion. The chemical shifts of aromatic protons for all the
macrocycles appeared in the region of δ 6.50–8.28 ppm. In addition, the methoxy protons in macrocycle 5b
were observed as singlet at δ 3.35 ppm, while protons of the –CH2 CH2 – group in macrocycle 8b were displayed
as a triplet at δ 3.79 ppm.
3.3. The 31 P NMR spectral study
The 31 P NMR spectra of the selected macrocycles in DMSO-d6 were measured. Macrocycle 5a showed 2
doublets signals at δ 8.29 and 17.70 ppm with coupling constants 612 and 572 Hz, respectively, because of the
presence of 2 hydrophosphoryl units. The hydrophosphoryl unit of macrocycle 9b was also observed at δ 8.38
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OH
O
H
H
P
P
HN
N
N
HN
NH
N
N
N
H
H
N
N
N
N
N
N
N
N
P
P
O
O
Ph
Ph
Ph
Ph
5a (I)
5a (II)
OH
P
OH
P
H
HN
N
NH
N
NH
N
N
N
H
H
N
N
N
N
N
N
P
O
N
P
O
N
Ph
Ph
Ph
Ph
5a (IV)
5a (III)
Scheme 3. The tautomeric structures of compound 5a.
ppm with coupling constant 605 Hz.
3.4. The mass spectral study
The mass spectral study of macrocycles 4a, 4b and 5a, 5b confirmed that the cyclocondensation process
occurred at a 1:1 ratio. The mass spectral data of 4a and 4b showed the molecular ion peaks at m/z 444
(M+ , 25 %) and 460 (M+ , 0.45 %) with their base peaks at m/z 65 and 77, respectively. The mass spectral
data of 5a and 5b designated the molecular ion peaks at m/z 462 (M–2H, 0.2%) and 494 (M+ , 3.3%) with
their base peaks at m/z 324 and 77, respectively. On the other hand, the mass spectra of macrocycles 8a and
9b supported their formation at a 2:2 ratio, although they did not show the molecular ion peaks, indicating
the fragile nature of these macrocycles. Thus, the mass spectrum of 8a showed the highest value peak at
1073 (M–4H) with a base peak at m/z 149, whereas macrocycle 9b recorded the highest peak at m/z 1214
corresponding to its fragment ion peak after losing an oxygen atom, while its base peak was recorded at m/z
309.
3.5. Antimicrobial activity
All the newly synthesized compounds were evaluated in vitro for their antibacterial activities against Staphy-
lococcus aureus (ATCC 25923) and Streptococcus pyogenes (ATCC 19615) as representatives of gram-positive
bacteria and Pseudomonas fluorescens (S 97) and Pseudomonas phaseolicola (GSPB 2828) as examples of gram-
negative bacteria. They were also examined in vitro for their antifungal activities against Fusarium oxysporum
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and Aspergillus fumigatus fungal strains. The agar-diffusion technique was used for the determination of the
preliminary antibacterial and antifungal activities.33 The test was performed on medium potato dextrose agar
that contained an infusion of 200 g potatoes, 6 g dextrose, and 15 g agar.34 Uniformly sized filter paper disks
(3 disks per compound) were impregnated by an equal volume (10 μL) of compounds dissolved in dimethylfor-
mamide at concentrations of 1000 and 2000 μg/mL and were carefully placed on inoculated agar surface. After
incubation for 36 h at 27 ◦ C in the case of bacteria and 48 h at 24 ◦ C in the case of fungi, the antimicrobial
activities were determined by measuring the inhibition zones (Table 1). Cephalothin, chloramphenicol, and
cycloheximide were used as reference drugs (30 μg/mL) for gram-positive bacteria, gram-negative bacteria,
and fungi, respectively. The minimum inhibitory concentration (MIC, μg/mL) for some selected compounds
against all species of microbes was also determined (Table 2). The tube dilution technique35 was applied for
the determination of the MIC of the tested compounds against microbes. Dilution series were set up with 500,
250, 125, 62.5.. . . . . . . . 6.25 μg/mL of nutrient broth medium to each tube, 100 mL of standardized suspension
of the test microbes (107 cell/mL) was added, and tubes were incubated at 37 ◦ C for 24 h (Table 2).
Table 1. In vitro antimicrobial activities of the synthesized macrocycles 1a, 1b, 4a, 4b, 5a, b, 8a, b and 9a, b at
1000 and 2000 μg/mL by disk diffusion assay.
Zone of inhibition (mm)**
Compd.* Concentration
Gram-positive bacteria
Gram-negative bacteria
P. phaseolicola P. fluorescens
Fungi
no.
1a
(μg/mL)
1000
2000
1000
2000
1000
2000
1000
2000
1000
2000
1000
2000
1000
2000
1000
2000
1000
2000
1000
2000
30
S. aureus
S. pyogenes
F. oxysporum
A. fumigatus
8
10
8
–
7
5
8
6
–
–
–
–
–
–
–
–
–
–
10
–
8
–
1b
4a
4b
5a
5b
8a
8b
9a
9b
10
7
8
6
–
6
–
–
10
13
16
13
16
5
10
14
19
14
19
5
5
9
–
–
–
5
19
22
9
13
5
8
–
–
–
14
17
11
13
–
6
–
–
–
8
–
10
–
8
7
–
–
9
9
8
6
5
–
–
–
8
5
5
6
7
11
14
12
16
–
6
5
12
12
17
5
6
8
–
–
–
–
11
15
–
12
15
–
–
–
–
–
8
6
–
5
8
6
R1
R2
R3
28
NT
NT
30
NT
NT
NT
25
NT
NT
30
NT
NT
NT
28
NT
NT
31
30
30
*R1 = cephalothin, R2 = chloramphenicol, R3 = cycloheximide; used as reference drugs at concentration of 30 μg/mL.
**Lowly active: 6–12 mm; moderately active: 13–19 mm; highly active: 20–30 mm; –: no inhibition or inhibition less
than 5 mm; NT: not tested.
The obtained results were recorded for each tested compound as the average diameter of inhibition zones
of both the bacteria and fungi around the disks in millimeters at the concentrations of 1000 and 2000 μg/mL
(Table 1) and as MIC values (Table 2). The following points were revealed:
1) The results recorded in Table 1 revealed that most of the synthesized macrocycles possessed various
167

ALI et al./Turk J Chem
antimicrobial activities towards all the microorganisms.
Table 2. Minimum inhibitory concentration (MIC) of compounds 4b, 5a, and 9a.
Compd.
no.
4b
MIC*, μg/mL
S. aureus S. pyogenes P. phaseolicola P. fluorescens F. oxysporum A. fumigatus
500
500
250
6.25
–
250
250
500
6.25
–
>500
>500
>500
–
>500
>500
>500
–
250
500
500
–
>500
>500
>500
–
5a
9a
R1
R2
6.25
6.25
–
–
R3
–
–
–
–
12.50
6.25
*R1 = cephalothin, R2 = chloramphenicol, and R3 = cycloheximide; used as reference drugs.
2) Although compounds 4b, 5a, and 9a had the highest inhibition zones for antibacterial and antifungal
activities, they did not have strong MIC values.
3) All the synthesized macrocycles revealed better effects against gram-positive bacteria strains in com-
parison with the starting bis-(4-formylpyrazolyl)phosphine oxides (1a, 1b).
4) Most of the small macrocycles, 4a, 4b and 5a, 5b, revealed better effects than the large macrocycles,
8a, 8b and 9a, 9b.
5) Small macrocycles 4a and 5a showed more antibacterial activities than antifungal activities, while
macrocycles 4b and 5b revealed more antifungal activities than antibacterial activities.
6) Large macrocycles 8a, 8b and 9a, 9b displayed more antibacterial activities than antifungal activities.
7) The macrocycles that contained aromatic units, 9a and 9b, had more effects than the macrocycles
that contained aliphatic units, 8a and 8b.
8) Generally, the macrocycles showed lower to moderate activities. However, none of the tested com-
pounds were nearly equal to or more active than the reference drugs.
4. Conclusion
We achieved an efficient route for the preparation of previously unreported phosphorus macrocycles containing
pyrazole rings. The characterization of these macrocycles was discussed. The preliminary antimicrobial activities
of the tested compounds showed that they had lower to moderate activities. Although compounds 4b, 5a, and
9a had the highest recorded inhibition zones for antibacterial and antifungal activities, they did not have strong
MIC values. However, none of these tested compounds was equal to or better than the reference drugs in terms
of activity.
Acknowledgment
The authors are thankful to Dr Ibrahim Hassan, Faculty of Agriculture for Girls, Al-Azhar University, Nasr
City, Cairo, Egypt, for helping in evaluating antimicrobial activities.
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