The Development of Novel Antibiotics Using ... - Jacobs University

aimed at assessing molecular recognition ability of the novel macrocycles
in solution is under investigation.
Experimental Section
All the reagents used for the reactions were purchased from
Sigma-Aldrich, Applichem or Flucka (Germany) and were used as
obtained. Whenever possible the reactions were monitored by thin
layer chromatography (TLC). TLC was performed on Macherey-
Nagel aluminium-backed plates pre-coated with silica gel 60
(UV 254 ). Melting points were determined in open capillaries using
a Buechl B-545 melting point apparatus and are not corrected.
Infrared spectra were determined using a Vector-33 Bruker FT IR
spectrometer. The samples were measured directly as solids or oils;
ν max values were expressed in cm –1 and were given for the main
absorption bands. 1 H, 13 C and 2D NMR spectra were acquired on a
JEOL ECX-400 spectrometer operating at 400 MHz for 1 H NMR
and 100 MHz for 13 C NMR in DMSO-d 6 using a 5 mm probe. The
chemical shifts (δ) are reported in parts per million and were
referenced to the residual solvent peak. The following abbreviations
are used: s, singlet; m, multiplet; br, broad signal. Mass
spectra were recorded using HCTultra and ESI-TOF Bruker
Daltonics mass spectrometers and samples were dissolved in DMF,
acetonitrile and water using the positive or negative electrospray
ionisation modes. Calibration was carried out using a 0.1 M
solution of sodium formate in the enhanced quadratic mode prior to
each experimental run. The results of measurements were processed
using Compass 1.3 data analysis software for a Bruker
Daltonics time of flight mass spectrometer (micrOTOF). Preloaded
wang resin, N,N-diisopropylethylamine (DIEA), O-(benzotriazol-1-
yl)-hydroxybenzotriazole (HOBt), N,N,N',N'-tetramethyluranium
hexafluorophosphate (HBTU), 9-fluorenylmethoxycarbonyl)-
amino acid derivatives (1-Fmoc), DMF, N-methylpyrrolidone
(NMP), trifluoroacetic acid (TFA), 1,2-eth- anedithiol (EDT) and
other chemicals required for peptide synthesis were bought from
Iris Biotech GmbH (Marktredwitz, Germany). Peptides were
synthesized with standard solid phase peptide synthesis technique
and it was carried out on an automated peptide synthesizer (ABI-
433A, Applied Biosystems, Foster city, USA). Fmoc protected
preloaded resin was used to grow peptide chain on it. In detail,
deprotection of the N-terminal of resin bound amino acid was
performed by 20% piperidine in NMP and the C-terminal of other
protected amino acid was activated using HBTU/ HOBt/DIEA
(1:1:2) in DMF for coupling with the free N-terminal of the resin
bound amino acid. A mixture of 82.5% TFA, 5% phenol, 5%
water, 5% thioanisol and 2.5% EDT was used to separate the
peptide from resin as well as to remove the side chain protecting
groups. Subsequently, the peptide was isolated through
precipitation in cold (–20 ᴼC) diethyl ether and lyophilized by
Christ freeze dryers (Martin Christ Gefriertrocknungsanlagen
GmbH, Osterode am Harz, Germany). Peptides were purified by
high performance liquid chromatography (HPLC) and analyzed by
HRMS. Molecular modeling calculations were carried out with
HyperChem software (Release 8.0) at the MM+ levels in vacuo
and no influence of solvents was taken into account in the
calculations. Circular Dichroism measurements were performed
using Jasco-J-810 Spectropolarimeter in DMSO.
(4R,5R,25R,26R)-Tetra-(hydrazinecarboxamide) cyclophane 5
To a stirred solution of 4,4'-oxybis-(phenylisocyanate) 4a (500 mg,
1.98 mmol) in anhydrous THF (5 mL) were added (4R,5R)-1,3-
dioxolane-4,5-dicarbohydrazide 1 (396 mg, 2.08 mmol) and 4 mL
anhydrous THF. The mixture was stirred at room temperature for
24 h. The white precipitate was filtered off, washed successively
with water, MeOH and dried to yield 830 mg of the titled
compound (94%). M.p. > 260 ºC (decomp.). IR ṽ = 3283 (NH),
1676 (C=O) cm –1 . 1 H NMR (400 MHz, DMSO-d 6 ): δ = 10.07 (4H,
brs, NH), 8.71 (3H, brs, NH), 8.56 (1H, brs, NH), 8.11 (4H, brs,
NH), 7.39 (8H, d, J = 8.7, ArH), 6.85 (8H, d, J = 8.7, ArH), 5.15
(4H, brs, CH 2 ), 4.67 (4H, brs, CH) ppm. 13 C NMR (100 MHz,
DMSO-d 6 ): δ = 169.3 (CO), 155.6 (CO), 152.4 (C), 135.4 (C),
120.6 (CH), 119.1 (CH), 97.0 (CH 2 ), 77.2 (CH) ppm. HRMS
(ESI-TOF+, DMF/ACN): m/z calcd. for C 38 H 36 N 12 O 14 [M+H + ]
885.2544; found 885.2547.
(4R,5R,25R,26R)-Tetra- (hydrazinecarboxamide) cyclophane 6
To a stirred solution of bis-(4-isocyanatophenyl)-methane 4b (500
mg, 2 mmol) in THF (5 mL) were added (4R,5R)-1,3-dioxolane-
4,5-dicarbohydrazide 1 (399 mg, 2.1 mmol) and 4 mL THF. The
mixture was stirred at room temperature for 24 h. The white
precipitate was filtered off, washed successively with water,
diethyl ether and dried to yield 875 mg of the titled compound
(99%). M.p. > 260 ºC (decomp.). IR ṽ = 3274 (NH), 1683 (C=O)
cm –1 . 1 H NMR (400 MHz, DMSO-d 6 ): δ = 10.05 (4H, brs, NH),
8.64 (4H, brs, NH), 8.07 (4H, brs, NH), 7.31 (8H, d, J = 8.2, ArH),
7.03 (8H, d, J = 8.2, ArH), 5.14 (4H, brs, CH 2 ), 4.65 (4H, brs, CH),
3.74 (4H, brs, CH 2 ) ppm. 13 C NMR (100 MHz, DMSO-d 6 ): δ =
169.2 (CO), 155.5 (CO), 137.9 (C), 135.6 (C), 129.3 (CH), 119.2
(CH), 97.0 (CH 2 ), 77.2 (CH) ppm. HRMS (ESI-TOF+, DMF/
ACN): m/z calcd. for C 40 H 40 N 12 O 12 [M+Na + ] 903.2781; found
903.2799.
(4R,5R,25R,26R)-Tetra-(hydrazinecarboxamide) cyclophane 7
To a stirred solution of 4,4'-oxybis-(phenylisocyanate) 4a (500 mg,
1.98 mmol) in anhydrous THF (5 mL) were added (4R,5R)-2,2-
dimethyl-1,3-dioxolane-4,5-dicarbohydrazide 2 (454 mg, 2.08
mmol) and 4 mL anhydrous THF. The mixture was stirred at room
temperature for 24 h. The white precipitate was filtered off, washed
successively with water, MeOH and dried to yield 820 mg of the
titled compound (89%). M.p. > 240 ºC (decomp.). IR ṽ = 3298
(NH), 1670 (C=O) cm -1 . 1 H NMR (400 MHz, DMSO-d 6 ): δ =
10.02 (4H, brs, NH), 8.68 (4H, brs, NH), 8.15 (4H, brs, NH), 7.39
(8H, d, J = 8.7, ArH), 6.82 (8H, d, J = 8.7, ArH), 4.65 (4H, brs,
CH), 1.43 (12H, s, CH 3 ) ppm. 13 C NMR (100 MHz, DMSO-d 6 ): δ
= 169.4 (CO), 155.6 (CO), 152.4 (C), 135.4 (C), 120.6 (CH), 119.2
(CH), 113.1 (C), 77.2 (CH), 26.7 (CH 3 ) ppm. HRMS (ESI-TOF+,
DMF/ACN): m/z calcd. for C 42 H 44 N 12 O 14 [M+H + ] 941.3150; found
941.3173.
(4R,5R,25R,26R)-Tetra-(hydrazinecarboxamide) cyclophane 8
To a stirred solution of bis-(4-isocyanatophenyl)-methane 4b (500
mg, 2 mmol) in THF (5 mL) were added (4R,5R)-2,2-dimethyl-1,3-
dioxolane-4,5-dicarbohydrazide 2 (458 mg, 2.1 mmol) and 4 mL
anhydrous THF. The mixture was stirred at room temperature for
24 h. The white precipitate was filtered off, washed successively
with water, diethyl ether and dried to yield 850 mg of the titled
compound (90%). M.p. > 230 ºC (decomp.). IR ṽ = 3267 (NH),
1668 (C=O) cm -1 . 1 H NMR (400 MHz, DMSO-d 6 ): δ = 10.01 (4H,
brs, NH), 8.61 (4H, brs, NH), 8.11 (4H, brs, NH), 7.31 (8H, d, J =
8.2, ArH), 7.00 (8H, d, J = 7.7, ArH), 4.63 (4H, brs, CH), 3.72
(4H, brs, CH 2 ), 1.43 (12H, s, CH 3 ) ppm. 13 C NMR (100 MHz,
DMSO-d 6 ): δ = 169.3 (CO), 155.5 (CO), 137.9 (C), 135.6(C),
129.2 (CH), 119.2 (CH), 113.1(C), 77.2 (CH), 26.7 (CH 3 ) ppm,
signal for CH 2 overlapped with signals of DMSO-d 6 (For DEPT-
135 spectrum, see supporting information). HRMS (ESI-TOF+,
5