Synthesis, structural aspects, antimicrobial activity and ion transport investigation of five new [1+1] condensed cycloheterophane peptides

Article abstract of DOI:10.1007/s10847-012-0282-x

Five novel [1+1] condensed cycloheterophane peptides were synthesized via reaction of pyridine-2,6-bis(2-aminothiophenoxymethyl) with several diacid chlorides: glutaryl dichloride, adipoyl dichloride, 2,2′-thiodiacetyl chloride, dithiodiglycoloyl chloride and 3,3′-thiodipropionoyl chloride combinations (L ₁ -L ₅ ). The compounds were characterized by elemental analyses, mass, FT-IR, ¹H, and ¹³C NMR spectral data. The antimicrobial activities of the compounds were evaluated using the disk diffusion method in dimethyl sulfoxide as well as the minimal inhibitory concentration dilution method, against several bacteria and yeast cultures. The results were compared with those of commercial antibiotic and antifungal agents. Structure activity relationships were also discussed. Permeability of compound L ₅ against Na⁺ and K⁺ were also investigated. Graphical abstract: Five novel cycoloheterophane amides were synthesized. Mass spectra reveal their [1+1] cyclic condensation. The compounds exert moderate microbial activity. Thia and aza groups seem to be a key element of microbial potency.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s10847-012-0282-x

J Incl Phenom Macrocycl Chem
DOI 10.1007/s10847-012-0282-x
ORIGINAL ARTICLE
Synthesis, structural aspects, antimicrobial activity and ion
transport investigation of five new [1+1] condensed
cycloheterophane peptides
¨
Omer Zaim Naz Mohammed Aghatabay
•
•
•
•
˘
Mustafa Ulvi Gu¨rbu¨z C¸ aglar Baydar
Bas¸aran Du¨lger
Received: 9 November 2012 / Accepted: 22 December 2012
Ó Springer Science+Business Media Dordrecht 2013
Abstract Five novel [1?1] condensed cycloheterophane
peptides were synthesized via reaction of pyridine-2,6-
bis(2-aminothiophenoxymethyl) with several diacid chlo-
rides: glutaryl dichloride, adipoyl dichloride, 2,20-thiodi-
acetyl chloride, dithiodiglycoloyl chloride and 3,30-
thiodipropionoyl chloride combinations (L1–L5). The
compounds were characterized by elemental analyses,
with a wide range of activities since their discovery [1, 2].
Cyclopeptides, systematically called cyclophane peptides,
having many similarities with crown ethers, are very com-
mon natural products and found in the marine environment,
as well as in higher plants. Marine cyanobacteria [3], green
algae [4], marine sponges [5, 6], tropical phoma [7] are
among the sources for naturally occurring cyclopeptides.
Hydrophilic cavity and hydrophobic outer shell of these
macrocycles enables them to carry cations from hydrophobic
non polar media to aqueous environment and vice versa.
Similar property has been observed in living organisms by
carrying cations in and out of the cells by passing the
membrane. Macrocyclic compounds containing both ether
and amide functionalities show high selectivity towards
earth alkaline metals [8] rather than alkali metals. The same
type of affinity towards metals such as Hg2?, Pb2?, Cd2?
makes them extremely useful in environmental chemistry
and biomedical research [9]. Another important aspect of
these macrocyclic compounds is that they can be used as
receptors for neutral molecules [10]. Hydrogen bonding in
the cavity establishes complexation between neutral mole-
cules and hetero macrocyclic compounds. Biological activ-
ity studies showed that large amount of macrocyclic peptides
exhibit antimicrobial, antiviral, immunosuppressive, anti-
tumor activities and inhibit fat accumulation and as a result,
they are also very important synthetic targets [11–16].
The syntheses of the cycloheterophane peptides by
cyclocondensation reactions are also well documented
[17–19]. Corporation of pyridine unit may add interesting
properties to cyclophane peptides due to their structural
versatility and suitability for synthetic modifications. One
possible application of these materials is in generating
supramolecular arrays, which embody additional functional
groups that are capable of molecular recognition, models
for intercalation, building blocks for organic catalysts and
1
mass, FT-IR, H, and 13C NMR spectral data. The anti-
microbial activities of the compounds were evaluated using
the disk diffusion method in dimethyl sulfoxide as well as
the minimal inhibitory concentration dilution method,
against several bacteria and yeast cultures. The results were
compared with those of commercial antibiotic and anti-
fungal agents. Structure activity relationships were also
discussed. Permeability of compound L5 against Na? and
K? were also investigated.
Keywords Antimicrobial ꢀ Cycloheterophane ꢀ
Diffusion ꢀ Ion transport ꢀ Permeability
Introduction
Crown ethers have evolved from simple macrocyclic ethers
to more complicated, multifunctional cyclic compounds
¨
O. Zaim (&) ꢀ C¸ . Baydar
Department of Chemistry, Trakya University, Edirne, Turkey
e-mail: omerzaim@trakya.edu.tr
N. M. Aghatabay ꢀ M. U. Gu
¨rbu¨z
Department of Chemistry, Fatih University, Bu¨yu¨kc¸ekmece,
Istanbul 34500, Turkey
B. Du¨lger
Department of Biology, Duzce University, Duzce, Turkey
123

J Incl Phenom Macrocycl Chem
metal complexation or interaction of biomolecules via
internally or externally available networks.
was carried out by using toluene and a Dean-Stark separator
to remove water. The diols were prepared from related esters
by reduction with NaBH4 and purified by using chloroform
and a soxhlet extractor. The synthetic procedure to obtain the
tosylates was adapted from that of [26] with some modifi-
cations. They were then reacted with 2-aminothiophenol to
generate diamine in a good yield 82 %. The full procedure
for the preparation of the compounds is given in the
‘‘Experimental’’ section. The spectroscopic data from the
Synthesis ofsimilarmixedaza-oxo-thiamacrocyclictetra-
peptides and their respective antimicrobial and cytotoxicity
studies have recently been reported [20–22]. In this paper we
focus on the synthesis, spectroscopic characterization, ion
transportation and pharmacological investigation of the five
new [1?1] condensed macrocycles, bearing nitrogen and
sulfur donor centers. IUPAC Phane Nomenclature System
has been used for naming the compounds (Fig. 1) [23, 24].
1
mass, FT-IR, H and 13C NMR spectroscopy together with
elemental analysis provide useful information about their
formation and structural characterization.
Chemistry
All the macrocyclic peptides were prepared through the main
route depicted in Scheme 1, based on the condensation of
diacid chlorides with diamines at low temperature in dry
dichloromethane. The synthetic procedure to obtain the
diacid chlorides was adapted from that of [25]. Esterification
Biological data
Standardized samples of penicillin G (blocking the for-
mation of bacterial cell walls, rendering bacteria unable to
multiply and spread); ampicillin (preventing the growth of
Fig. 1 1,9(1,2)-Dibenzena-2,8-
diaza-3,7-dioxo-10,14-dithia-
12(2,6)-pyridinacyclotetra
12
13
NH
11
1
decaphane(L1) H and 13C
NMR spectra in DMSO-d6
14
10
9
1
8
2
4
6
5
7
3
SCH2
COCH2
CH2
2.4
1.9
4.21
9.0
8.5
8.0
7.5
7.0
COCH2
CO
SCH2
CH2
41
38
168
152
136
120
104
88
72
56
40
24
ppm
123

J Incl Phenom Macrocycl Chem
Scheme 1 Reagents and
conditions: a benzene, EtOH,
H2SO4, Dean-Stark, 12 h, 87 %,
b THF, NaBH4, 12 h, 85 %,
c TsCl, KOH, CH2Cl2, 87 %,
d Na/EtOH, 60 °C, 3 h, 84 %,
e CH2Cl2, pyridine, 0 °C 5 h,
room temperature overnight,
&65 %
a
b
c
HO
OH
EtO
OEt
N
N
N
O
O
O
O
OH
OH
NH2
SH
d
2
N
O
O
N
S
S
X
OTs
OTs
Cl
Cl
NH2
H2N
e
L1 : X = CH2
L2 : X = CH2CH2
L3 : X = S
L4 : X = S S
L5 : X = CH2SCH2
bacteria mostly Gram-negative); cefotaxime (used against
most Gram-negative enteric bacteria); ofloxacin (inhibiting
DNA-gyrase, preventing the bacteria from reproducing);
tetracyclines (effects through the inhibition of protein
synthesis); nystatin (binding to sterols in the fungal cellular
membrane, causing leakage of the cellular contents);
ketoconazole (inhibiting the formation of the fungal cell
wall) and clotrimazole (causing the fungal cells leakage).
Bruker Ultrashield Plus 400 MHz instrument. Chemical
shifts (d) are expressed in units of parts per million relative to
TMS. The analytical data, Mass, FT-IR, NMR and physical
properties are summarized for each experiment.
Synthesis of pyridine-2,6-dicarbonyl dichloride
This was adapted from the procedure [25] using 2,6-pyridine
dicarboxylic acid (1.25 g, 7.5 mmol) and thionyl chloride
(12 mL, 16.5 mmol) in methylene chloride (25 mL) (1.31 g,
86 %). Selected IR bands (cm-1): 1,750 m(CO).
Experimental
General
Pyridine-2,6-dicarboxylic acid diethyl ester
All chemicals including isophthaloyl chloride and solvents
were reagent grade and were used as purchased without
further purification except dichloromethane (CaH2 under Ar)
and toluene (Na/benzophenone under Ar). Melting points
were determined using an Electro-thermal 9100 melting-
point apparatus. Analytical data wereobtainedwithaThermo
Finnigan Flash EA 1112 analyzer. Mass (ESI) analyses were
carried out in positive ion modes using a ZivacÒ Tandem
Gold Triple quadruple MS Spectrometer. FT-IR spectra were
recorded on a Bruker Alpha-P (mid 4,000–400 cm-1) as
solid and far IR spectra (700–180 cm-1) were recorded (as
polyethylene pellets) on a Jasco FT/IR-600 Plus Spectrom-
eter. Routine1H (400 MHz)and13C (100 MHz)spectrawere
recorded in DMSO-d6 or CDCl3 at ambient temperature on a
According to procedure [27] pyridine-2,6-dicarboxylic acid
(11.7 g, 70 mmol) and ethanol (100 mL), benzene (10 mL)
and concentrated sulfuric acid (3 mL) was refluxed using a
Dean-Stark receiver trap for 12 h. White crystalline solid
was obtained (13.6 g, 87 %). M.P. 41–42 °C. (Ref. [27]
45–46 °C). 1H NMR (CDCl3), dH ppm: 1.47 (t, 2CH3,
J = 7.25 Hz), 4.50 (q, 2CH2, J = 7.16 Hz), 8.01 (t, 2H,
J = 7.75 Hz), 8.29 (d, H, J = 7.92 Hz).
2,6-Bis(hydroxymethyl)pyridine
According to procedure [28], pyridine-2,6-dicarboxylic
acid diethyl ester (5.58 g, 25 mmol) and NaBH4 (4.16 g,
110 mmol) in THF (50 mL) were used and a white solid
123

J Incl Phenom Macrocycl Chem
was obtained (2.96 g, 85 %). M.P. 112–114 °C. (Ref. [28]
115–120 °C) ATR (solid, cm-1): 3,346 m(O–H),
3,099 m(C H), 1,593 m(C C). H NMR (CDCl3), dH ppm:
4.79 (s, 4H), 7.20–7.79 (m, 3H).
CH2Cl2 (50 mL) were reacted and white solid product was
obtained (320 mg, 72 %). M. P. 220 °C. Found (calculated)
C24H23N3O2S2: C, 63.98 (64.12); H, 5.21 (5.16); N, 9.21
(9.35); S, 14.06 (14.26). FT-IR (solid, cm-1): 3,298,
3,232 m(N–H), 3,060 m(C H), 1,692 m(C=O), 1,650 d(N–
H), 1,580 m(C C), 1,520 d(N–H), 746 d(C H). Far-IR
1
2,6-[(Tosyloxy)methyl]pyridine
1
(cm-1): 562, 491, 447, 432, 387, 334, 320, 214. H NMR
2,6-Bis(hydroxymethyl)pyridine (1.39 g, 10 mmol) in
CH2Cl2 (35 mL), KOH (40 % aq, 35 mL) and p-toluene-
sulfonyl chloride (3.81 g, 20 mmol) were reacted accord-
ing to procedure [26], affording white solid (3.89 g, 87 %).
M.P. 119–121 °C. (Ref. [26] 121–122 °C) ATR (solid,
cm-1): 1H NMR (CDCl3), dH ppm: 2.46 (s, 6H, CH3), 5.08
(s, 4H, CH2), 7.34–7.83 (m, 11H).
(DMSO-d6), dH ppm: 1.92 (quintet, CH2, J = 7.1 Hz), 2.43
(t, 2CH2, J = 7.0 Hz), 4.21 (s. 2CH2), 7–7.66 (m, 11H), 9.12
(s, 2NH). 13C NMR (DMSO-d6), dC ppm: 21.26 (CH2), 35.53
(2COCH2), 40.45 (2SCH2), 121.64, 124.42, 125.70, 127.41,
129.44, 131.46, 137.11, 137.54, 156.48, 170.73 (2CO).
1,10(1,2)-Dibenzena-2,9-diaza-3,8-dioxo-11,15-dithia-
13(2,6)-pyridinacyclopentadecaphane (L2)
2,6-Bis(2-aminophenylsulfanyl methyl) pyridine
3 mLPyridineinCH2Cl2 (50 mL), adipoylchloride(183 mg,
1 mmol) in CH2Cl2 (50 mL) and 2,6-bis(2-aminothiophen-
oxymethyl)pyridine (353 mg, 1 mmol) in CH2Cl2 (50 mL)
were reacted and white solid product was obtained (270 mg,
60 %). Dec. 210 °C. Found (calculated) C25H25N3O2S2: C,
64.45 (64.77); H, 5.28 (5.44); N, 8.97 (9.06); S, 13.76 (13.83).
MS (ESI) m/z calculated for C25H25N3O2S2, 463.61; found:
464.40 [M ? H] (75 %). FT-IR (solid, cm-1): 3,324, 3,255
m(N–H), 3,050 m(C H), 2,950 m(C–H), 1,689 m(C=O), 1,658
d(N–H), 1,576 m(C C), 1,506 d(N–H), 755 d(C H). Far-IR
(cm-1): 558, 537, 465, 449, 419, 371, 308, 282, 234, 189. 1H
NMR (CDCl3), dH ppm: 1.93 (t, 2CH2), 2.54 (t, 2COCH2),
4.04 (s, 2SCH2), 7.0–8.40 (m, 11H), 8.71 (s, 2NH). 13C NMR
(CDCl3), dC ppm: 29.11 (2CH2), 38.65 (2COCH2), 43.31
(2SCH2), 120.86, 122.26, 124.62, 130.76, 130.98, 136.16,
137.78, 140.12, 157.18, 169.67 (2C=O).
This was prepared according to the procedure [29]. Reac-
tion of 2-aminothiophenol (1.25 g, 10 mmol) in absolute
EtOH (8 mL) containing Na (0.23 g, 10 mmol) and 2,6-
[(tosyloxy)methyl]pyridine (2.23 g, 5 mmol) gave white
solid product (1.47 g, 84 %). M. P. 80–82 °C (Ref. [29]
80–82 °C). ATR (solid, cm-1): 3,410, 3,310 m(N–H);
1
1,605 d(N–C). H NMR (CDCl3), dH ppm: 4.00 (s, 4H,
CH2), 5.38 (s, 4H, NH), 6.40–7.58 (m, 11H). 13C NMR
(CDCl3), dC ppm: 41.48 (CH2), 115.09, 117.12, 118.42,
121.66, 130.37, 136.76, 136.82, 148.97, 158.01.
General procedure for [1?1] condensed cyclopeptides
synthesis
A 250 mL two-necked, round-bottomed flask fitted with two
dropping funnels was charged with dry CH2Cl2 (50 mL) and
dry pyridine (3 mL) at 0 °C. A solution of diacid chloride
(1 mmol) in dry methylene chloride (50 mL) and a solution
of diamine (1 mmol) in dry methylene chloride (50 mL)
were simultaneously added dropwise to a well stirred solu-
tion at the same rate under dry argon atmosphere for 5 h,
while maintaining the temperature at 0 °C. After the addition
was complete, the reaction mixture was left stirring at room
temperature overnight. The solvent was evaporated on a
rotary apparatus under vacuum and the residue was added
very slowly into 300 mL of ice-cold distilled water with
vigorous stirring. The white solid products were collected,
washed several times with cold water, than with cold acetone
and dried under vacuum.
1,9(1,2)-Dibenzena-2,8-diaza-3,7-dioxo-5,10,14-trithia-
12(2,6)-pyridinacyclotetradecaphane (L3)
Pyridine (3 mL) in CH2Cl2 (50 mL), 2,2-thio diacetyl
chloride (187 mg, 1 mmol) in CH2Cl2 (50 mL) and 2,6-
bis(2-aminothiophenoxymethyl)pyridine (353 mg, 1 mmol)
in CH2Cl2 (50 mL) were reacted and white solid product was
obtained (290 g, 62 %). Dec. 232 °C. Found (calculated)
C23H21N3O2S3: C, 58.93 (59.07); H, 4.61 (4.53); N, 8.75
(8.99); S, 20.38 (20.57). MS (ESI) m/z calculated for
C23H21N3O2S3, 467.63; found: 468.33 [M ? H] (81 %). FT-
IR (solid, cm-1): 3,274 m(N–H), 3,053 m(C H), 2,968 m(C–
H), 1,659 m(C=O), 1,576 m(C C), 1,505 d(N–H), 757
d(C H). Far–IR (cm-1): 570, 525, 497, 455, 405, 310, 277,
1,9(1,2)-Dibenzena-2,8-diaza-3,7-dioxo-10,14-dithia-
12(2,6)-pyridinacyclotetradecaphane (L1)
1
200. H NMR (CDCl3), dH ppm: 3.63 (s, 2CH2), 4.09 (s,
2CH2), 6.90–8.25 (m, 11H), 9.28 (s, 2NH). 13C NMR
(CDCl3), dC ppm: 43.93 (2CH2), 44.5 (2CH2), 120.28,
122.01, 122.88, 124.93, 130.77, 136.5, 137.68, 140.07,
157.01, 166.4 (2C=O).
3 mL Pyridine in CH2Cl2 (50 mL), glutaryl chloride
(168 mg, 1 mmol) in CH2Cl2 (50 mL) and 2,6-bis(2-ami-
nothiophenoxymethyl)pyridinde (352 mg, 1 mmol) in
123

J Incl Phenom Macrocycl Chem
1,10(1,2)-Dibenzena-2,9-diaza-3,8-dioxo-5,6,11,15-
tetrathia-13(2,6)-pyridinacyclopentadecaphane (L4)
Hanseniaspora guilliermondii DSM 3432) using both disc
diffusion and dilution methods.
Pyridine (3 mL) in CH2Cl2 (50 mL), 2,2-disulfanediyldiacetyl
chloride (219 mg, 1 mmol) in CH2Cl2 (50 mL) and 2,6-bis(2-
aminothiophenoxymethyl)pyridine (353 mg, 1 mmol) in
CH2Cl2 (50 mL) were reacted and white solid product was
obtained (290 mg, 58 %). Dec. 166 °C. Found (calculated)
C23H21N3O2S4: C, 55.17(55.28);H, 4.35 (4.24);N, 8.29 (8.41);
S, 25.49 (25.67). MS (ESI) m/z calculated for C23H21N3O2S4,
499.69; found: 500.33 [M ? H] (56 %). FT-IR (solid, cm-1):
3,257 m(N–H), 3,060 m(C H), 2,929 m(C–H), 1,664 m(C=O),
1,577 m(C C), 1,506 d(N–H), 751 d(C H). Far-IR (cm-1):
580, 531 m(S–S), 500, 465, 453, 389, 336, 298, 276, 258. 1H
NMR (CDCl3), dH ppm: 3.7 (s, 2SCH2CO), 4.08 (s, 2SCH2),
7.10–8.47 (m, 11H), 9.5 (s, 2NH).
Antimicrobial screening
Disc diffusion method
The antimicrobial activity of the compounds was evaluated
using a slightly modified disc diffusion method [30, 31].
The compounds were dissolved in DMSO (2 mg mL-1).
Sterilized antibiotic discs (6 mm, Schleicher & Schull No.
2668, Germany) were each impregnated with 20 lL of
solution. To ensure that the solvent had no effect on bac-
terial growth, a control test was performed with test med-
ium supplemented with DMSO in the same procedures
used in the experiments. All the bacteria were incubated at
30 0.1 °C for 24 h by inoculation into nutrient broth
(Difco) and the yeasts were incubated in malt extract broth
(Difco) for 48 h. An inoculum containing 106 bacterial
cells or 108 yeast cells/mL was spread on Mueller-Minton
agar (Oxoid) plates (1 mL inoculum/plate). The discs
injected with solutions were placed on the inoculated agar
by pressing slightly and incubated at 35 °C (24 h) for the
bacteria and at 25 °C (72 h) for the yeasts. On each plate
an appropriate reference antibiotic disc was applied
depending on the test microorganisms. In each case trip-
licate tests were performed and the average was taken as
the final reading.
1,11(1,2)-Dibenzena-2,10-diaza-3,9-dioxo-6,12,16-trithia-
14(2,6)-pyridinacyclohexadecaphane (L5)
Pyridine (3 mL) in CH2Cl2 (50 mL), 3,3-thiodipropanoyl
chloride (214 mg, 1 mmol) in CH2Cl2 (50 mL) and 2,6-
bis(2-aminothiophenoxymethyl)pyridine (353 mg, 1 mmol)
in CH2Cl2 (50 mL) were reacted and white solid product was
obtained (0.310 g, 63 %). Dec. 216 °C. Found (calculated)
C25H25N3O2S3: C, 60.72 (60.58); H, 5.19 (5.08); N, 8.37
(8.48); S, 19.36 (19.41). MS (ESI) m/z calculated for
C25H25N3O2S3, 495.68; found: 496.33 [M ? H] (100 %).
FT-IR (solid, cm-1): 3,358, 3,256 m(N–H), 3,057 m(C H),
2,954 m(C–H), 1,684 m(C=O), 1,658 d(N–H), 1,575
m(C C), 1,508 d(N–H), 751 d(C H). Far-IR (cm-1): 577,
Dilution method
1
552, 461, 454, 380, 302, 261, 190. H NMR (CDCl3), dH
Screening for antibacterial and antifungal activities was
carried out by preparing a broth micro dilution, following the
procedure outlined in Manual of Clinical Microbiology [32].
All the bacteria were incubated and activated into nutrient
broth at 30 °C for 24 h and the yeasts were incubated in malt
extract broth for 48 h. The compounds were dissolved in
DMSO (2 mg mL-1) and then diluted using caution adjusted
Mueller-Hinton broth (Oxoid). Two fold serial concentra-
tions of the compounds were employed to determine the
(MIC) ranging from 200 to 1.56 lg mL-1. Cultures were
grown at 37 °C (20 h) and the final inoculation was
approximately 106 cfu mL-1. Test cultures were incubated
at 37 °C (24 h). The lowest concentrations of antimicrobial
agents that result in complete inhibition of microorganisms
were represented as (MIC) lg mL-1. In each case triplicate
tests were performed and the results are expressed as means.
ppm: 2.71 (t, 2COCH2, J = 5.82 Hz), 3 (t, 2SCH2,
J = 5.80 Hz), 4.10 (s, 2SCH2), 7.0–8.41 (m, 11H), 8.73 (s,
2NH). 13C NMR (CDCl3), dC ppm: 29.92 (2SCH2), 38.47
(2COCH2), 43.31 (2SCH2), 121.41, 122.14, 123.55, 125.25,
130.49, 135.88, 137.60, 139.57, 156.97, 166.06 (2C=O).
Pharmacology
Microorganisms
The antimicrobial activities were evaluated against Gram-
positive (Staphylococcus aureus ATCC 6538, Bacillus
cereus ATCC 7064, Micrococcus luteus La 2971, Myco-
bacterium smegmatis CCM 2067, Listeria monocytogenes
ATCC 15313) and Gram-negative (Escherichia coli ATCC
11230, Proteus vulgaris ATCC 8427, Klebsiella pneumo-
niae UC57, Pseudomonas aeruginosa ATCC 27853) bac-
teria and yeast cultures (Candida albicans ATCC 10231,
Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra
DSM 70403, Debaryomyces hansenii DSM 70238 and
Ion transport
Macrocyclic peptide (L5) (60 mg, 0.12 mmol) was dis-
solved in dichloromethane (60 mL) and transferred in a
123

J Incl Phenom Macrocycl Chem
ion peaks [M ? H]? for each individual cyclic compound,
suggesting the monomeric nature of the compounds and
confirming the proposed formula. The mass spectra of
these compounds also show a series of peaks corresponding
to various fragments. Their intensity may give an idea of
the stability of the fragments.
NaCl
&
H2O
KCl
H2O
Vibrational spectra
In most cases secondary amides usually have their NH and
C=O groups trans to each other. cis-Configured secondary
amides are found practically only in forms of lactams,
where the trans-configuration is difficult because of the
high ring tension. However, the bands lie essentially in the
same regions as for the trans-configured derivatives. The
vibrational spectra for the cyclic compounds can be dis-
cussed in terms of two characteristic wave regions:
3,370–2,900 cm-1 corresponding to the mas(N–H), ms(N–
H), m(C H) aromatic, m(C–H) aliphatic characteristic
modes, and 1,750–1,500 cm-1 belonging to m(CO), d(N–
H), m(C C) vibration modes. The bands corresponding to
free aniline –NH2 m(N–H) (3,410 cm-1) and acid chloride
COCl m(CO) (1,750 cm-1) groups are not observed in the
IR spectra of the products, which suggests complete con-
densation of the reactions and possible formation of amide
linkages. The medium bands observed in the regions
3,370–3,320 and 3,280–3,230 cm-1 are assignable to
mas(N–H) and ms(N–H) amide vibration modes, respec-
tively. These modes of vibration are not dependent on the
backbone conformation but are very sensitive to the
strength of a hydrogen bonding, which may cause broad-
ening and as well as causing multiplicity in certain cases
[33]. The peptide bond formation could also be confirmed
due to the observance of two major bands in the frequen-
cies 1,690–1,650 (amide I) and 1,520–1,500 cm-1 (amide
II). The amide I band is the most intensive absorption in the
IR spectrum and it is almost fully governed by the
stretching vibration of the C=O mode with a very small
contribution of the C–N groups. Amide II is more complex
than amide I, it’s signal derives mainly from in-plane N–H
bending vibration with a certain contribution of m(C–N)
groups [33, 34]. Stretching vibration C=O amide is con-
siderably lower than corresponding acyl m(CO) vibration
frequencies. The characteristic m(CH) modes of aromatic
and aliphatic groups are observed in the wave region
3,100–3,000 and 2,980–2,920 cm-1 respectively (see
‘‘Experimental’’ section).
Cyclophane (L5)
in CH2Cl2 60 mL
Fig. 2 Apparatus used for ion transport study
glass vessel as shown in Fig. 2. One arm of the U tube is
filled with distilled water (6 mL) in which NaCl (1.17 g,
20 mmol) was dissolved and the other arm is filled with
triply distilled water. In another identical glass vessel
similar procedure was carried out using KCl (1.49 g,
20 mmol) instead of NaCl. The dichloromethane layer was
stirred gently for 72 h for each vessel. The arm that was
filled with triply distilled water showed the presence of K?
ion (500 mg) and Na? ion level was below the measuring
limit. Thus the experiment proved that K? ions were
transported by cyclophane peptide (L5) from one arm to the
other. This indicates that the size of K? ion (ionic radius:
˚
1.33 A) and the cavity size of the cyclophane amide (L5)
are complementary to each other, whereas the size of Na?
˚
ion (ionic radius: 0.95 A) does not match with cavity size.
Hence cyclophane amide (L5) could be used as a potential
ion filtering system for retaining the biologically important
K? ion and eliminating Na? ion or possible continuous
purification system for these ions. A blank experiment was
also performed in dichloromethane without cyclophane
amide and no such ion mobility was observed.
Results and discussions
Mass spectra
Magnetic resonance spectra
The mass spectra of the cyclic peptides give important
structural information about the nature of the condensation
(1?1), the ring size and molecular conformation of the
compounds. The mass spectra show a molecular positive
1
The H NMR spectra of the cyclic compounds do not give
any signal corresponding to aniline –NH2 at 5.38 ppm,
instead sharp bands appear in the regions 8.70–9.50 ppm
123

J Incl Phenom Macrocycl Chem
corresponding to amide (2CONH) proton atoms. As
expected no significant changes are observed for the
methylene proton chemical shifts compared with the
starting material diamine. They appear around 4 ppm as
sharp singlets (4H, 2SCH2) for each of the cyclic
compound.
hetero atoms capable of hydrogen bonding with biomole-
cules. Comparing the inhibition zone values, in most cases
the compounds baring sulfur atoms such as CSC (L3) and
CS2C (L4) containing units seems to show slightly higher
biological activity than the units such as C3 (L1), C4 (L2),
which has no sulfur atoms. As a result the inhibition
activity seems to be governed to a certain degree by the
percentage amount of nitrogen presence in these com-
pounds, because the (L4) macrocycle contains higher
numbers of nitrogen atoms compared with the other four
compounds. In a similar pattern, the least nitrogen con-
taining compound L1 in most cases shows the lowest
antibiotic activity. This pattern repeats itself for both
inhibition zone and MIC values (Tables 1, 2).
The peaks corresponding to acid chlorides COCl
m(C=O) (*173 ppm) groups are not observed in the 13C
NMR spectra of the cyclic products, instead the signals
which appeared for each product in the regions
166–171 ppm are attributed to a (CONH) group, suggest-
ing complete condensation of the reactions, which was also
predicted from IR spectra.
Microbial activity
Of all the tested compounds (L3) and (L4) showed
moderate activity against both the Gram-positive pathogen
B. cereus (L3 and L4 = 6.25 lg mL-1) and on Gram-
negative bacteria P. vulgaris both L3 and L4 showed strong
activity 6.25 lg mL-1 compared with Gentamycin
(6.25 lg mL-1 (Table 2). In the case of inhibition zone
values these two compounds also showed (in most cases)
slightly higher activity than the other compounds. Showing
moderate antibacterial activity on both Gram-positive and
Gram-negative bacteria, these compounds could be con-
sidered to possess a broad-spectrum potency.
The results concerning in vitro antimicrobial activities of
the macrocycles together with inhibition zone (mm) and
(MIC) values of compared antibiotic and antifungal
reagents are listed in Tables 1 and 2. The overall macro-
cycles can interact through van der Waals and hydrophobic
interactions, while the individual hetero atoms present in
the structure particularly nitrogen sulfur atoms, could
interact by strong hydrogen bonding. As far as hydrogen
bonding is concerned, there is an important electronegative
aspect regarding to nitrogen atoms. The nitrogen atom is
the most common atom involved as hydrogen bond
acceptor in biological systems. Moderate biological activ-
ity shown by the compounds may be related to their
hydrogen bonding capability due to the presence of several
The inhibition zone and (MIC) values indicate that
almost all the compounds exhibited a moderate antifungal
activity against most of the yeast cultures. Once more the
compound (L4) shows slightly higher activity (zone 20 mm
and MIC 6.25 lg mL-1) against K. fragilis in comparison
Table 1 In vitro antimicrobial activity (mm zone) of (L1–L5) and standard reagents
Microorganisms
L1
L2
L3
L4
L5
PE
AM
CT
OF
TE
NY
KE
CL
Staphylococcus aureus?
Bacillus cereus?
Micrococcus luteus?
Mycobacterium smegmatis?
Listeria monocytogenes?
Escherichia coli-
12
14
12
14
14
15
15
14
15
13
12
17
14
14
15
15
13
17
14
14
16
16
14
15
13
18
15
16
16
16
12
17
18
16
14
14
11
18
17
15
19
18
18
17
10
18
18
17
15
15
14
20
15
17
21
20
15
14
11
18
14
14
15
16
12
14
16
15
18
18
14
12
34
17
12
16
12
18
10
–
15
14
30
20
14
14
18
15
10
–
14
14
34
14
14
12
20
16
48
–
22
36
28
28
30
30
32
26
40
–
26
22
20
30
28
25
24
30
38
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Proteus vulgaris-
–
–
–
Klebsiella pneumoniae-
Pseudomonas aeruginose-
Kluyveromyces fragilis
Rhodotorula rubra
–
–
–
–
–
–
18
16
20
23
16
16
22
22
22
16
18
16
16
24
18
–
–
–
–
–
Candida albicans
–
–
–
–
–
Hanseniaspora guilliermo
Debaryomyces hansenii
–
–
–
–
–
–
–
–
–
–
In each case triplicate tests were performed and the average was taken as the final reading
? Gram positive, - Gram negative, PE penicillin G (10 units), AM ampicillin 10 lg, CT cefotaxime 30 lg, OF ofloxacin 5 lg, TE tetracycline
30 lg, NY nystatin 100 lg, KE ketaconazole 20 lg, CL clotrimazole 10 lg
123

J Incl Phenom Macrocycl Chem
Table 2 In vitro antimicrobial activity (MIC, lg mL-1) of (L1–L5) and standard reagents
Microorganisms/compounds
L1
L2
L3
L4
L5
GEN
NY
Staphylococcus aureus1
Bacillus cereus1
Micrococcus luteus?
Mycobacterium smegmatis?
Listeria monocytogenes?
Escherichia coli-
25
12.5
12.5
25
25
12.5
12.5
12.5
50
25
–
12.5
50
6.25
6.25
6.25
50
–
25
50
–
25
12.5
25
25
12.5
12.5
25
12.5
12.5
6.25
6.25
6.25
6.25
–
–
25
12.5
12.5
6.25
–
12.5
25
12.5
12.5
12.5
25
6.25
6.25
12.5
25
–
Proteus vulgaris-
6.25
–
Klebsiella pneumoniae-
Pseudomonas aeruginose-
Kluyveromyces fragilis
Rhodotorula rubra
12.5
50
12.5
50
12.5
25
6.25
12.5
50
–
–
12.5
50
12.5
25
6.25
25
6.25
3.125
3.125
3.125
12.5
12.5
12.5
12.5
12.5
12.5
12.5
–
Candida albicans
12.5
12.5
25
6.25
–
Hanseniaspora guilliermondii
Debaryomyces hansenii
12.5
12.5
6.25
6.25
3.125
3.125
6.25
6.25
–
–
In each case triplicate tests were performed and the average was taken as the final reading
? Gram positive, - Gram negative, GEN gentamycin, NY nystatin
with Nystatin (zone 18 mm and MIC 6.25 lg mL-1).
Nystatin acts by binding to ergosterol in the cell membrane
of susceptible species resulting in a change in membrane
permeability and the subsequent leakage of intracellular
components. Considering the presence of peptide bonds in
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Ion transport
The physical and chemical properties of Na? and K? ions
are quite similar. Both are closed shell, hard ions, they have
identical coordination geometries and display similar
exchange kinetics for bound water molecules. Despite
these similarities, K? ion channels, a class of transmem-
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Acknowledgments We would like to extend our gratitude to the Fatih
University Scientific Research Centre (Project-P50021004_G1452)
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¨
and the Trakya University (Grant # TUBAP-2010/174) for their
¨
financial support. Dr. Ahmet Ceyhan Goren and Gokhan Bilsel
(TUBITAK-UME), is acknowledged for the Mass analysis.
¨
˙
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