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

Results and Discussion
Dicarbohydrazides 1-3 were synthesised from commercially
available (+)-diethyl L-tartrate according to literature (Figure 1). 15-
16 Then they underwent successfully and selectively [2+2]-cyclocondensation
reactions with 4,4'-oxybis-(phenylisocyanate) 4a and
bis-(4-isocyanatophenyl)-methane 4b in anhydrous THF to afford
tetra-(hydrazinecarboxamide) cyclophanes 5-10 in excellent yields
(Figure 2). The [2+2]-cyclocondensation products 5-10 form in
almost quantitative yields and high purity at a stoichiometry of
1.1:1 of dihydrazides and diisocyanates. Structures of the novel
macrocycles were fully assigned and characterised by various
spectroscopic techniques such as 1 H NMR, 13 C NMR, FT IR, 2D
ROESY NMR, 2D HMBC, 2D HMQC, DEPT-135 and chirality
was confirmed by CD spectroscopy (Circular Dichroism). 1 H NMR
spectrum for (i.e., macrocycle 7) in DMSO-d 6 showed three broad
signals at δ 10.02, 8.68 and 8.15 ppm corresponding to 12 NH
protons (Figure 3).
FT IR spectrum showed a strong absorption band at ṽ 1670 cm –1
corresponding to the stretching vibration of the ṽ C=O. It showed
also weak absorption band at ṽ 3298 cm –1 for NH moieties. ESI-
TOF mass spectrum (Electrospray ionisation time of flight) showed
the expected pseudo-molecular ion peak at m/z 941.3173 [M+H + ].
High resolution ESI-TOF/MS data for the novel macrocycles are
shown in table 1. Tandem ESI-MS and proposed fragmentation
mechanisms of the novel macrocycles are shown with supporting
information.
Table 1. HRMS (ESI-TOF, +MS) data for macrocycles 5-10
Entry
Molecular
Formula
Calcd. m/z Meas. m/z Error
[ppm]
Yield
[%]
5 [a] C 38H 36N 12O 14 885.2544 885.2547 0.3 94
6 [b] C 40H 40N 12O 12
903.2781 903.2799 -2.0 99
7 [a] C 42H 44N 12O 14
941.3150 941.3173 2.4 89
8 [b] C 44H 48N 12O 12 959.3407 959.3439 -3.4 90
9 [b] C 48H 52N 12O 14
1043.3631 1043.3618 -1.2 97
10 [b] C 50H 56N 12O 12 1039.4033 1039.4066 -3.1 99
[a] Product ion appeared as [M+H + ], [b] Product ion appeared as [M+Na + ]
Molecular Modeling and Conformational Analysis
Figure. 1 [2+2]-cyclocondensation reaction of dicarbohydrazides 1-3 with
diisocyanates 4a and 4b in anhydrous THF.
Figure. 2 [2+2]-cyclocondensation products 5-10.
The novel tetra-(hydrazinecarboxamide) cyclophanes (i.e., 5)
assume a conformation in which the NH moieties are syn-anti
oriented (Figure 4, conformer 5a). 2D ROESY spectrum showed
through space interactions between H d and H e , while no interactions
were observed for H c and H d or H c and H e which suggested
a syn-anti conformer (Figure 5). Data from 2D ROESY NMR were
used for subsequent structure modeling calculations. Structure of
macrocycle 5 was optimised using HyperChem software (Release
8.0) at the MM+ level (Molecular mechanics). 19,20 The molecular
structure was optimised using the Polak-Ribiere algorithm till the
RMS (Root mean square) gradient was 0.01 Kcal.mol –1 . Molecular
modeling calculations for macrocycle 5 suggested conformer 5a as
the lowest-energy structure. Conformer 5b appeared to be the least
stable with the highest-energy due to the electrostatic repulsion
between the carbonyl groups C=O (6)-(9), C=O (21)-(24), C=O
(27)-(30) and C=O (3)-(42). Macrocycles 5-10 form under conformational
bias of the reacting dicarbohydrazide and diisocyanate
components. We envisage that the macrocycles form in a stepwise
reaction in which the final intermediates are conformationally
biased to form selectively [2+2]-cyclocondensation products rather
than formation of higher oligomers. We performed molecular
modeling simulations at the MM+ level to investigate the effect of
conformational bias on the final ring closure step. The closer the
nucleophilic hydrazide and the electrophilic isocyanate moieties
are in space, the more rapidly the ring closure will occur to form
the macrocycles. Molecular modeling suggested a distance of 3.59
Å between the reactive sites in intermediate B which permits facile
ring closure under conformational bias (Figure 6). Molecular
modeling also suggested formation of intramolecular hydrogen
bonds between NH…O=C (2)-(6) and NH…O=C (23)-(27) which
further stabilize and bias intermediate B towards formation of
macrocycle 5.
Investigation of Molecular Recognition by ESI-MS
Figure. 3 1 H NMR spectrum for macrocycle 7, 400 MHz, DMSO-d 6.
ESI-MS has been widely used as a soft ionisation tool for
studying weak non-covalent interactions in the gas phase. 21-25 The
2