Dual demeanour of norcantharidin derived dicarboxamides in acidic media: An insight

Article abstract of DOI:10.1016/j.tet.2019.07.016

The hydrolytic stability of norcantharidine derived conformationally constrained diamides in acidic media is solely governed by the type of amide. Tertiary diamides underwent smooth acid catalyzed hydrolysis due to anchimeric assistance whereas other diamides were stable. This was corroborated from conformational proximity of the amide groups for anchimeric assistance based on single crystal X-ray structure analysis. Theoretical calculations on the diamide structures also predict the similar proximity of the diamides in those conformations. Thus norcantharidine based diamides could probably serve as promising systems for delayed release of certain types of drugs which possess secondary amine component as exemplified by the release of m-chlorophenylpiperazine at a pH range of 1–2.

Full text of DOI:10.1016/j.tet.2019.07.016

Accepted Manuscript
Dual demeanour of norcantharidin derived dicarboxamides in acidic media: An insight
Chandrakant S. Gholap, Rekha Singh, Mukesh Kumar, Dilip K. Maity, Sunil K. Ghosh
PII:
S0040-4020(19)30758-6
https://doi.org/10.1016/j.tet.2019.07.016
TET 30458
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 27 May 2019
Revised Date: 4 July 2019
Accepted Date: 9 July 2019
Please cite this article as: Gholap CS, Singh R, Kumar M, Maity DK, Ghosh SK, Dual demeanour of
norcantharidin derived dicarboxamides in acidic media: An insight, Tetrahedron (2019), doi: https://
doi.org/10.1016/j.tet.2019.07.016.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Dual Demeanour of Norcantharidin Derived Dicarboxamides in Acidic
Media: An Insight
Chandrakant S. Gholap,a Rekha Singh,a Mukesh Kumarb,c Dilip K. Maityc and Sunil K. Ghosh*a,c
aBio-Organic Division; bRadiation Biology and Health Sciences Division; Bhabha Atomic
Research Centre, Trombay, Mumbai 400085, India
cHomi Bhabha National Institute, Training School Complex, Anushaktinagar,
Mumbai 400094, India
Email: ghsunil@barc.gov.in; Tel: +91-22-25595742; fax: +91-22-25505151
Key words: Amide hydrolysis, neighbouring group participation, theoretical calculation, X-ray
crystal structure, conformationally constrained diamide, slow drug release
Abstract
The hydrolytic stability of norcantharidine derived conformationally constrained diamides in
acidic media is solely governed by the type of amide. Tertiary diamides underwent smooth acid
catalyzed hydrolysis due to anchimeric assistance whereas other diamides were stable. This was
corroborated from conformational proximity of the amide groups for anchimeric assistance based
on single crystal X-ray structure analysis. Theoretical calculations on the diamide structures also
predict the similar proximity of the diamides in those conformations. Thus norcantharidine based
diamides could probably serve as promising systems for delayed release of certain types of drugs
1

ACCEPTED MANUSCRIPT
which possess secondary amine component as exemplified by the release of m-
chlorophenylpiperazine at a pH range of 1-2.
Introduction
The amide linkage is a characteristic feature in proteins. Proteinaceous biomolecules like
enzymes, hormones etc. are involved in vital functions like catalysis of reactions, transport of
molecules and transmission of messages from cell to cell. Amide hydrolysis is often used as a
model for the cleavage of peptide bonds.1,2 Proteolysis catalyzed by proteases regulates several
physiological, biochemical and cellular processes. Amide linkage also features in several drugs
as well as in a plethora of natural products having a wide range of bioactivities.3 Owing to this,
the amide hydrolysis has been a subject of study by many research groups. Usually amide
linkage is chemically robust4-6 which is due to the resonance between the nitrogen lone pair and
the pi-CO bond. Due to the delocalization of the lone pair, amides show a rigid planar
conformation with characteristic shortening of C-N bond and elongation of C=O bond.7 Its non-
enzymatic hydrolysis is not an easy task and has to be performed under harsh acidic or basic
conditions. Amide activation to perform various synthetic transformations has also been
achieved.8 Lewis acids have been designed to facilitate the alcoholysis and hydrolysis of
amides.9-13 Specific alkyl group on amide structure such as N-tropolonyl amides are sensitive to
acid catalyzed hydrolysis due to the ease of formation of tropolonium cation.14 But there are
instances when this hydrolysis becomes facile due to structural features of the amides and
embedded functional groups. Non-planar deformations activate the amide bond towards the
hydrolysis. Twisted amides have been shown to hydrolyze several orders of magnitude faster
than their corresponding planar analogs.15-22 Amide bond cleavage gets accelerated due to 1,3-
diaxial interaction with a carboxylic acid in Kemps' acid amides.23,24 Anchimeric assistance also
2

ACCEPTED MANUSCRIPT
known as neighbouring group participation (NGP) by certain functional groups accelerates the
amide hydrolysis. The acid catalyzed amide hydrolysis is facilitated by NGP of an alkoxy,25
a
hydroxyl,26,27 carboxylic acid28-38 or an amide39-42 group (Fig. 1). It is a well-known fact that
under physiological conditions proteolytic enzymes hydrolyze an amide bond by NGP via a
tetrahedral intermediate.
Hydroxy
Participation
Carboxylic acid
Participation
Amide
Participation
Ref 26
O
Ref 29
O
Ref 39
O
C6H11
NH2
N
NH2
C6H11
O
O
O
C6H11
HO
N
NH2
NH2
OH
C6H11
NH
O
O
C6H5
Rate enhancement
40 times
105 times
104 times
Fig. 1: Rate enhancement of amide hydrolysis due to NGP
Hydroxyl group in γ or δ position of butyramide, valeramide26 and butyranilide27 increase
the rate constant of acidic hydrolysis. Participation of a carboxylic acid group in the hydrolysis
has been reported in conformationally mobile dipeptides,27 succinanilic acids31 and aliphatic
amide in a cyclohexane system37 as well as in rigid systems like phthalamic acid derivatives,29,31
N-o-carboxybenzoylimidazole,36 N-(o-carboxybenzoyl)-L-leucine,38 2-acylamidobenzamides,40
and maleamic acids.32-35 Amide functionality also aids in the hydrolysis of another proximal
amide group as exemplified in o-benzamido-N,N-dicyclohexylbenzamide39 wherein the
hydrolysis occurs at a more bulky amide group. The participation of an amide group is also
found in the hydrolysis of N-acylimidazole derivatives of N-acetylphenylalanine, N-
3

ACCEPTED MANUSCRIPT
acetylvaline41 and carbonate diesters.42 More recently, ammonolysis of a secondary amide
assisted by unsubstituted vicinal amide43 has been reported.
The diamide systems studied so far include either conformationally flexible systems or
structurally rigid molecules. None of the previous reports dealt with the hydrolysis of amides in
conformationally constrained bridged bicyclic systems with juxtaposed cis-dicarboxamides or
their closest analogs. Besides, the systems studied never addressed the sensitivity of hydrolysis
to the structure of the amine component, i.e. the type of amide. To address these issues, we
herein present a systematic study of norcantharidine derived conformationally constrained
bridged 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxamides (Fig. 2) towards acid catalyzed
hydrolysis.
Previous studies on NGP
assisted diamide hydrolysis
Ref 39
Ref 40
Ref 41
O
O
R
O
C6H11
R
N
N
N
NH
C6H11
HN
NH2
O
NH
O
O
C6H5
Present work
O
O
O
NR2
NR2
2
Fig. 2: Hydrolysis studies of diamides
Results and Discussion
During the development of CHON based diamides for nuclear reprocessing,44 we synthesized
conformationally constrained 7-oxabicyclo[2.2.1]heptane2,3-dicarboxamides45 (Fig. 2) from 7-
oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid and long-chain dialkylamines. We experienced a
4

ACCEPTED MANUSCRIPT
rapid hydrolytic degradation of N,N,N′,N′-tetrakis(2-ethylhexyl)-7-oxabicyclo[2.2.1]heptane-2,3-
dicarboxamide in acidic media. This strange observation prompted us to systematically
investigate the hydrolytic behaviour of a series of other 7-oxabicyclo[2.2.1]heptane-2,3-
dicarboxamides vis-a-vis their conformational preferences.
In order to study the structure-reactivity relationship of the hydrolysis of diamides,
dicarboxamides 2a-e featuring different amine components were synthesized from readily
available starting materials as shown in Scheme 1. The reaction of various amines with oxa-
bridged tricyclic anhydride 1 in the presence of DMAP and diisopropylcarbodiimde (DIPC)
afforded these diamides in good yields. The anhydride 1, in turn, was prepared by the
hydrogenation of the Diels-Alder adduct of furan and maleic anhydride. Amines with shorter
alkyl groups were chosen with the anticipation of obtaining good solids and eventually good
single crystals. Although the chemical reactivity of both long as well as short carbon chain
diamides 2, would be comparable.
5

ACCEPTED MANUSCRIPT
O
O
O
Diisobutylamine,
DIPC, DMAP,
N
N
CH2Cl2, 0 °C to r.t.
2a, 82%
O
O
O
O
H
Isobutylamine,
DIPC, DMAP
N
N
H
2b, 85%
CH2Cl2, 0 °C to r.t.
1.
Dii
DM
sob
uty
lam
ine
O
,
AP
, C
H
2Cl2
N
N
H
O
O
O
2. Isobutylamine,
H
O
DIPC, 0 °C to r.t.
O
2c, 90%
H
1
O
N
NH2
O
O
1. Diisobutylamine,
DMAP, CH2Cl2
2. Liq. NH3, DIPC
-34 °C to r.t.
O
2d, 90%
O
1.
Isob
uty
lam
H C
ine
,
H
O
N
DM
AP
, C
l2
2
NH2
2. Liq. NH3, DIPC
-34 °C to r.t.
2e, 95%
O
O
O
N
N
mCPP, DIPC,
DMAP, CH2Cl2
N
Cl
N
0 °C to r.t.
Cl
2f, 98%
Scheme 1: Synthesis of bridged 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxamides.
The hydrolysis studies were performed by stirring these diamides in 0.1 M HCl in
EtOH/H2O (30/70) and was monitored by TLC. Under these conditions, diamide 2a completely
hydrolyzed within 0.5 h to produce diacid 3 and diisobutylamine whereas diamides 2b-e did not
undergo any hydrolysis (Scheme 2).
6

ACCEPTED MANUSCRIPT
Scheme 2: Hydrolysis of 2a
We were bit surprised to see this unusual hydrolytic behavior reflecting contrasting
stability of these diamides 2a-e in acidic media. The reactivity vs. structural variations in
intramolecular systems has been interpreted by several research groups on the basis of orbital
steering46 (angular dependence), proximity,47 stereopopulation control48 and spatiotemporal
hypothesis.49 According to Menger’s spatiotemporal hypothesis,49 the type of reaction i.e.
intermolecular or intramolecular is governed by the distance between the two reactive centers.
When the distance between the two reactive centers exceeds 3 Å, intermolecular reaction is
preferred whereas distances below this value allow an intramolecular participation. Further,
Karaman50 expanded and analyzed this study, and combined the hypothesis of all the previous
researchers. He postulated that strain energy essentially is a function of distance and angle; both
the distance and angle of attack are the governing factors in rate enhancement. In addition to this,
the steric dependence on the rate of hydrolytic reactions has also been studied. In case of o-
benzamido-N,N-dicyclohexylbenzamide, the hydrolysis occurs at more sterically hindered
carbonyl group30 while an enhancement in rate with every increase in substitution going from
succinanilic acid to tetramethylsuccinanilic acid was observed.39
7

ACCEPTED MANUSCRIPT
The stepwise hydrolysis of diamide 2a to acid-amide and then to diacid 3 may not be
happening as we were unable to trace the formation of monoamide intermediate while
monitoring by TLC. The plausible mechanism for the hydrolysis of diamide 2a has been
delineated in Scheme 3. The first step is the protonation of one of the amide carbonyl group. Due
to the close proximity, the oxygen of the second carbonyl then attacks the protonated carbonyl
group. Subsequent to this in presence of water tetrahedral intermediate “A” is formed. The
formation of tetrahedral intermediate51 was first reported by Bender in the hydrolysis of esters.
This intermediate then gets protonated which results in loss of amines and formation of
anhydride 1 which eventually in presence of acid and water gets hydrolyzed to the diacid 3.
H
O
O
O
O
O
O
NR1R2
NR1R2
O
H3O+
NR1R2
NR1R2
H2O
H
O
HO
O
NR1R2
NHR1R2
H3O+
O
O
H
NR1R2
NHR1R2
HO
O
A
- 2NHR1R2
O
O
H3O+
O
O
O
OH
OH
O
1
3
O
Scheme 3: Plausible mechanism for the hydrolysis of diamide 2a
It was expected that the 3-dimensional structures of these diamides would let us know the
proximity of the two carbonyls and their orientation in space shedding light on the reasons
behind the quick hydrolysis of diamide 2a and stability of other diamides 2b-e. Thus diffraction
8

ACCEPTED MANUSCRIPT
quality crystals for these diamides 2a-d52 were grown and X-ray crystallographic study was
performed.53 The crystal structures revealed that in all these molecules the two amide
substituents are unsymmetrically arranged. The crystallographic asymmetric unit of 2a has four
molecules, whereas 2d has two molecules and crystallized as a dihydrate. The asymmetric unit of
2b and 2c has only one molecule each. In 2a, the carbonyl oxygen atom of one of the amides lies
in close proximity of the carbonyl carbon atom of another amide group. The distance between
these carbon and oxygen atoms in 2a ranges from 2.604-2.676 Å in the four molecules of the
asymmetric unit,54 whereas in case of 2b and 2c the corresponding distance (minimum of the two
pairs) is 3.058 Å and 3.525 Å, respectively. These distances in 2d are 3.674 Å and 3.692 Å for
the two molecules in the asymmetric unit respectively. In the crystal structure of 2d, a water
molecule bridges the two diamides in the asymmetric unit. It makes a hydrogen bond with the
oxygen atoms of the amide from two different diamide molecules directing the crystal packing.
Thus the distance between the carbonyl oxygen atom of one of the amide groups and the
carbonyl carbon atom of another amide group is relatively small in case of 2a in comparison to
2b-d and is below the critical distance of 3 Å for intramolecular participation (Fig. 3).
9

ACCEPTED MANUSCRIPT
Fig. 3: Single crystal X-ray structures of amides 2a-d. For simplicity, only one of the
molecules present in the asymmetric unit of 2a and 2d are shown.54
Thus, as the steric crowding in the molecule increases it brings the carbonyl oxygen of
one of the amides closer to the carbonyl carbon of another amide, and in case of 2a, the value
was less than 3 Å. This is in accordance with the Menger’s postulation49 on the critical distance
of less than 3 Å (diameter of water) to achieve rate acceleration. Also, it has been proposed
earlier that the Bürgi–Dunitz angle, 55 O---C(=O) angle, for a successful nucleophilic attack on a
carbonyl must be in the range of 105 ± 5°. In the case of 2a,54 this angle has been found to be
106.86° (Fig. 4), which lies in this optimum range. In the case of 2b the angle is 88.4° which is
10

ACCEPTED MANUSCRIPT
much less than the optimum for nucleophilic attack. In the case of 2c and 2d,54 the angles are
found to be 135.55° and 138.53° respectively, which are larger than the optimum value.
Fig. 4: Bürgi–Dunitz angle for amide 2a
Another interesting observation was the angle between the planes of two carbonyls. It
was found to be about 50° for amides 2b-d whereas in case of amide 2a it was nearly 64.0°
(Table 1).54 Due to steric hindrance of the bulky substituents, the diamide 2a could attain an
appropriate conformation, proximity and the angle of attack leading to neighbouring group
participation and facilitating rapid hydrolysis in acidic media, whereas other diamides were
stable. The twist angle that represents the dihedral angle between C=O and N-R is close to zero
for planar amide bond. As the twist angle increases, the amide becomes susceptible for
hydrolysis or other nucleophilic attacks. The twist angle in amide-1 for diamides 2b-2d is lower
compared to that of the amide 2a which is also reflected in the slightly shorter C=O and slightly
longer C-N bond in case of 2a compared to 2b-d (Table 1). These values, although not very
significant, facilitates NGP in 2a during acid catalysed hdrolysis. In all cases, the distortion
angles were found to be <10° which also suggests that individual amide groups are not reactive
enough to directly react with water during hydrolysis but are getting hydrolyzed by NGP of C=O
11

ACCEPTED MANUSCRIPT
followed by water attack. Although, the conformations adopted by diamides 2a-d in the solid
state might not be the minimum energy conformations (vide infra), they show such
conformations may facilitate NGP induced hydrolytic instability.
Table 1: Structural details of diamides 2a-d from X-ray crystallography54
Diamide
Distance (Å)
between carbonyl
“O” of amide-1 &
carbonyl “C” of
amide-2
O---C(=O)
angle
Amide C=O & C-N bond length
Angle
(Å) [cis-O=C-N-R
between
C=O planes
of amide-1
and amide-2
(Bürgi–
dihedral angle]
Dunitz angle)
2a
2b
2c
2d
2.645 ± 0.029 &
3.851 ± 0.041
106.86
88.40
1.224 & 1.366 [-8.68] (amide-1)
1.247 & 1.348 [0.10] (amide-2)
63.92
50.57
51.50
49.02
3.058 & 3.536
3.525 & 3.726
1.268 & 1.315 [2.47] (amide-1)
1.261 & 1.331 [4.61] (amide-2)
135.55
138.53
1.231 & 1.349 [0.09] (amide-1)
1.219 & 1.341 [1.56] (amide-2)
3.692 ± 0.009 &
3.795 ± 0.0055
1.232 & 1.342 [-0.42] (amide-1)
1.235 & 1.330 [2.06] (amide-2)
12

ACCEPTED MANUSCRIPT
Theoretical calculations were performed using the GAUSSIAN 16 program56 to examine
if the conformations of amides 2a-d obtained from X-ray crystal structures are the most stable
ones and also to predict energy of different possible conformations of these systems. Input
structures of different conformations for geometry optimizations were generated considering all
possible orientations of the groups attached to the bicyclic bridged moiety. The electronic
structure calculations based on first principle have been carried out on systems 2a-d applying
dispersion corrected DFT functional, ωB97X-D in conjunction with 6-311++G(d,p) basis
functions. These calculations are carried out in water and ethanol solvents following a
macroscopic solvent model SMD, which is known to capture solvent effect reasonably well. In
this implicit solvation model, the solute electron density is allowed to interact with the solvent,
which is represented as a dielectric continuum. Conformation analyses have been performed on
all the four bridged 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxamides, 2a-d keeping bridged
oxabicyclic scaffold intact. It is observed that in all the four systems, the conformer that has two
carbonyl groups in anti position is the most stable one in contrary to the X-ray crystal structure
obtained for 2a and 2b. In case of 2a, the most stable conformer (see Fig 5, 2a1) is calculated to
be 3.7 kcal/mol lower in energy than the next stable conformer (see Fig 5, 2a2) calculated which
is similar to the X-ray crystal structure. In case of 2b, the most stable conformer (see Fig 5, 2b1)
is calculated to be 2.6 kcal/mol lower in energy than the next stable conformer (see Fig 5, 2b2)
calculated which is again similar to the X-ray crystal structure. For, 2c and 2d systems, the most
stable structures are similar to the X-ray crystal structure. It may be noted from Fig. 5 that for
2a2, the inter-atomic distance between carbonyl oxygen on one amide and carbonyl carbon of
other amide is suitable for NGP as the calculated shortest distance is 2.7 Å. In case of 2b2, the
said distance is 2.96 Å and it is expected to be suitable for NGP as per Menger postulate.49
13

ACCEPTED MANUSCRIPT
However, no reaction took place in case of 2b2 suggesting the distance of 2.96 Å is not close
enough to accelerate NGP induced hydrolysis. The ꢀE in the figure shows the relative energy
with respect to the most stable conformer of 2a and 2b i.e. 2a1 and 2b1, respectively. The
calculated ꢀE for 2a2 and 2b2 indicates the strain energy of the two systems with respect to the
most stable conformers. Similar calculations on diamide 2c showed that the syn carbonyls
conformer is 2.4 kcal/mol higher in energy than that of the anti i.e. the stable conformer while in
compound 2d, the stable anti carbonyls conformer is 1.8 kcal/mol lower in energy than that of
the syn carbonyls conformer. Also, the minimum distance between the two amides in syn
carbonyls conformer of 2c and 2d was calculated to be 2.92 Å and 2.97 Å, respectively which
are not favorable for NGP. Single crystal X-ray crystallography suggests the presence of a water
molecule with the diamide in case of structure 2d. However, present calculations in presence and
absence of an explicit solvent water molecule do not indicate any extra stability due to H-
bonding interaction with the solvent water molecule.
14

ACCEPTED MANUSCRIPT
Fig. 5: Calculated conformers of amides 2a and 2b with syn and anti C=O groups of two amides
We perceived that the exceptional property of the tertiary diamides of type 2a could
probably be employed for delayed release57 of certain drugs. To exemplify this, anhydride 1 was
reacted with m-chlorophenylpiperazine (mCPP, an antidepressant metabolite of Trazodone) in
the presence of DMAP, DIPC and Et3N to obtain the diamide 2f (Scheme 1). This was then
subjected to acidic hydrolysis in 0.1 M and 0.01 M HCl in EtOH/H2O (9:1) whereby mCPP got
completely released in a span of about 0.5 h and 13 h, respectively.
15

ACCEPTED MANUSCRIPT
Conclusions
In conclusion, the structural and functional requirements for acid catalyzed hydrolytic stability of
conformationally constrained bridged 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxamides were
derived in combination with hydrolytic studies, X-ray structures and theoretical calculations.
The hydrolysis of tertiary-tertiary diamides was attributed to the NGP of the amide groups which
are in close proximity and correct orientation. Except for tertiary-tertiary diamides, rest all types
were stable under acidic hydrolytic conditions. Owing to this dual property, these diamides 2
hold promises for multiple uses including pH dependent sensor, metal extraction from acidic
waste and slow release of certain drugs.
Experimental Section
General Methods. All reactions were performed in oven-dried (120 °C) or flame-dried glass
apparatus under dry N2 or argon atmosphere. Diisobutylamine and isobutylamine were obtained
from Aldrich and distilled over CaH2 prior to use. DIPC was freshly distilled before use. Other
chemicals were used as obtained from Aldrich. TLC was carried out using Merck silica gel 60
1
F254 plates. Column chromatography was performed on silica gel (230–400 mesh). H NMR
spectra were recorded on 200, 500 and 600 MHz spectrometers and 13C{1H} NMR spectra were
recorded with 50 and 125 MHz spectrometers using CDCl3 as the solvent. The spectra were
referenced to residual chloroform (δ 7.25 ppm, 1H; 77.00 ppm, 13C). IR spectra were recorded on
the FT-IR spectrometer using NaCl discs, and wavenumbers are given in cm −1. Melting points
(mp) were uncorrected. Elemental analyses (C, H, N) were carried out by Elementar, vario
MICRO CHNS instrument. HR-MS spectra were obtained using a high-resolution ESI-TOF
mass spectrometer.
16

ACCEPTED MANUSCRIPT
Computational method. The electronic structure calculations are carried out applying density
functional theory (DFT) based method. As the systems are expected to have non-bonding
interactions, a DFT functional including empirical dispersion correction, namely, ωB97X-D is
considered for present calculations. This DFT functional is known to perform well for such
systems. A large set of atomic basis function, 6-311++G(d,p) is adopted for all calculations to
account non-bonding interactions accurately. All calculations are carried out in water as well as
in ethanol medium applying SMD macroscopic solvent model. Input geometries are considered
based on X-ray structures as well as all structures possible from different orientations of groups
attached to the rigid bicyclic bridged moiety. Geometry optimizations for all the input guess
structures are calculated following Newton-Raphson algorithm. Hessian calculations are carried
out for all the predicted equilibrium structures and it is observed that these are true minima.
These calculations are performed using GAUSSIAN 16 Revision A.03 suit of ab initio program.
X-ray crystallographic method. Single crystal X-ray diffraction data were collected on Agilent
Supernova system equipped with a microfocus Cu-source (λ = 1.5418 Ǻ) and a Titan CCD
detector. The crystals were separated, coated with paraffin oil and mounted on a loop for X-ray
diffraction data collection at specified temperature. The data reduction and analysis were carried
out with CrysAlisPro software suit. Analytical absorption correction using a multifaceted crystal
model based on expressions derived by Clark & Reid58 and as implemented in the CrysAlisPro
software suit was carried out for both the crystals. The structures were solved by direct method
using Shelxs and refined using Shelxl softwares59 using Olex2 interface.60 All the non-hydrogen
atoms were refined anisotropically and hydrogens were generated at their idealized positions and
refined isotropically according to riding model.
17

ACCEPTED MANUSCRIPT
Synthetic procedure and analytical data of 2a-f
N2,N2,N3,N3-tetraisobutyl-7-oxa-bicyclo[2.2.1]heptane-2,3-dicarboxamide (2a). A solution of
diisobutylamine (1.4 mL, 8 mmol) in dry CH2Cl2 (2 mL) was added to a stirred solution of
anhydride 1 (672 mg, 4 mmol) and DMAP (98 mg, 0.8 mmol) in dry CH2Cl2 (10 mL) at room
temperature. After 0.5 h, the reaction mixture was cooled on ice-water bath and DIPC (0.6 mL, 4
mmol) was added drop-wise into it. The reaction mixture was stirred overnight and allowed to
attain room temperature. The reaction mixture was diluted with 1/1 ethyl acetate-petroleum ether
and washed with a dilute citric acid solution, several times with water to remove DMAP and
diisopropylurea. The organic extract was concentrated under reduced pressure and the residue
was purified by column chromatography to give 2a45 (1.35 g, 82%) as a white solid. The product
was crystallized from ethyl acetate/petroleum ether to obtain suitable crystals for single-crystal
1
X-ray analysis. mp 144-146 °C; IR (film) νmax = 2959, 2872, 1633, 1454, 1152 cm-1; H NMR
(500 MHz, CDCl3) δ 4.89 (s, 2 H), 3.14 (dd, J = 13.5, 8.0 Hz, 2 H), 3.07 (dd, J = 13.5,7.0 Hz, 2
H), 3.00-2.91 (m, 6 H), 2.12 (nonet, J = 6.5 Hz, 2 H), 1.94 (nonet, J = 6.5 Hz, 2 H), 1.86-1.84
(m, 2 H), 1.56-1.53 (m, 2 H), 0.92 (d, J = 6.5 Hz,12 H), 0.88 (d, J = 6.5 Hz, 6 H), 0.85 (d, J = 6.5
Hz, 6 H) ppm; 13C{1H} NMR (125 MHz, CDCl3) δ 170.0 (2C), 79.6 (2C), 55.7, 54.0, 51.5 (2C),
29.7 (2C), 27.8 (2C), 26.4 (2C), 20.7 (2C), 20.6 (4C), 20.4 (2C), 20.1 (2C) ppm; Anal. Calcd for
C24H44N2O3: C, 70.5; H, 10.85; N, 6.9. Found: C, 70.2; H, 10.6; N, 7.2.
N2,N3-diisobutyl-7-oxa-bicyclo[2.2.1]heptane-2,3-dicarboxamide (2b).
A
solution of
isobutylamine (6 mmol) in dry CH2Cl2 (1 mL) was added to a solution of anhydride 1 (504 mg, 3
mmol) and DMAP (73 mg, 0.6 mmol) in dry CH2Cl2 (9 mL) at room temperature. The reaction
mixture was stirred and cooled on ice water bath. DIPC (0.7 mL, 4.5 mmol) was added drop-
wise to stirred reaction mixture. The reaction mixture was allowed to attain room temperature
18

ACCEPTED MANUSCRIPT
and stirred overnight. The reaction mixture was diluted with 1/1 ethyl acetate-petroleum ether
and washed with dilute citric acid solution, several times with water to remove DMAP and
diisopropylurea. The organic extract was concentrated under reduced pressure and the residue
was purified by column chromatography to give 2b (760 mg, 85%) as a white solid. The product
was crystallized from ethyl acetate/petroleum ether to obtain suitable crystals for single-crystal
1
X-ray analysis. mp 224-225 °C; IR (film) νmax = 3332, 3293, 2955, 1655, 1649, 1215 cm-1; H
NMR (500 MHz, CDCl3) δ 6.08 (s, broad, 2 H), 4.94 (bd, J = 2 Hz, 2 H), 3.01-2.92 (m, 4 H),
2.92 (s, 2 H), 1.86-1.78 (m, 2 H), 1.70 (nonet, J = 6.5 Hz, 2 H), 1.55-1.48 (m, 2 H), 0.89 (d, J =
6.5 Hz, 12 H) ppm.13C{1H} NMR (125 MHz, CDCl3) δ 171.0 (2C), 78.5 (2C), 54.7 (2C), 47.0
(2C), 28.8 (2C), 28.3 (2C), 20.1 (4C) ppm; Anal. Calcd for C16H28N2O3: C, 64.8; H, 9.5; N, 9.45.
Found: C, 64.9; H, 9.3; N, 9.8.
N2,N2,N3-triisobutyl-7-oxa-bicyclo[2.2.1]heptane-2,3-dicarboxamide (2c).
A
solution of
diisobutylamine (4 mmol) in dry CH2Cl2 (1 mL) was added to a solution of anhydride 1 (672 mg,
4 mmol) DMAP (49 mg, 0.4 mmol) in dry CH2Cl2 (10 mL) at room temperature. After overnight
stirring, the reaction mixture was cooled on an ice-water bath and a solution of isobutylamine (4
mmol) and DIPC (0.6 mL, 4 mmol) in dry CH2Cl2 (1 mL) was added to reaction mixture. After
stirring for 5 h, the reaction mixture was diluted with 1/1 ethyl acetate-petroleum ether and
washed with dilute citric acid solution, several times with water to remove DMAP and
diisopropylurea. The organic extract was concentrated under reduced pressure and the residue
was purified by column chromatography to give 2c (1.26 g, 90%) as a white solid. The product
was crystallized from ethyl acetate/petroleum ether to obtain suitable crystals for single-crystal
1
X-ray analysis. mp 122-124 °C; IR (film) νmax = 3383, 2962, 2931, 1656, 1633, 1215 cm-1; H
NMR (500 MHz,CDCl3) δ 6.61 (bd, J = 4.0 Hz, 1 H), 5.06 (d, J = 4.5 Hz, 1 H), 4.64 (d, J = 5.0
19

ACCEPTED MANUSCRIPT
Hz,1 H), 3.64 (dd, J =13.5, 6.0 Hz, 1 H), 3.31 (dd, J = 14.5, 8 Hz, 1 H), 3.29 (t, J = 7.5 Hz, 1 H),
3.02 & 3.00 (ABq, J = 10.0 Hz, 2 H), 2.91 (dd, J = 15.5, 6.0 Hz, 1 H), 2.57-2.50 (m, 1 H), 2.48
(dd, J = 13.5, 8.5 Hz, 1 H), 1.96-1.81 (m, 3 H), 1.77 (tt, J = 11.5, 4 Hz, 1 H), 1.71 (nonet, J = 6.5
Hz, 1 H), 1.55 (td, J = 11.5, 3.5 Hz, 1 H), 1.49 (dq, J = 11.0, 3 Hz, 1 H), 0.95 (d, J = 6.5 Hz, 3
H), 0.91-0.85 (m, 12 H), 0.82 (d, J = 7.0 Hz, 3 H) ppm; 13C{1H} NMR (125 MHz, CDCl3) δ
170.7, 170.4, 79.8, 78.3, 56.9, 55.7, 54.0, 50.8, 46.4, 29.5, 28.5, 28.4, 27.5, 26.5, 20.7, 20.5, 20.4,
20.1, 20.0, 19.9; Anal. Calcd for C20H36N2O3: C, 68.1; H, 10.3; N, 7.95. Found: C, 68.1; H, 10.5;
N, 7.7.
N2,N2-diisobutyl-7-oxa-bicyclo[2.2.1]heptane-2,3-dicarboxamide (2d).
A
solution of
diisobutylamine (0.5 mL, 3 mmol) in dry CH2Cl2 (5 mL) was added to a solution of anhydride 1
(500 mg, 3 mmol) and DMAP (80 mg, 0.6 mmol) in dry CH2Cl2 (5 mL) at room temperature.
After stirring the reaction mixture for about 1 h, about 20 mL of liquid ammonia was collected
into the reaction mixture followed by addition of DIPC (0.465 mL, 3 mmol). The reaction
mixture was then allowed to attain room temperature gradually over 4 h. The reaction mixture
was diluted with chloroform, washed with dilute citric acid solution and several times with water
to remove DMAP and diisopropylurea. The organic layer was concentrated under reduced
pressure. The residue was purified by column chromatography to give 2d (795 mg, 90%) as a
white solid. The product was crystallized from ethyl acetate/petroleum ether to obtain suitable
crystals for single-crystal X-ray analysis. mp 130-131.7 °C; IR (film) νmax = 3441, 3501, 2957,
2872, 1676, 1631, 1554, 1466, 1431, 1387, 1236, 1148, 996 cm-1; 1H NMR (600 MHz, CDCl3) δ
6.51 (s, 1 H), 5.10 (d, J = 4.8 Hz, 1 H), 5.03 (s, 1 H), 4.70 (d, J = 5.4 Hz, 1 H), 3.73 (dd, J =
13.2, 6.0 Hz, 1 H), 3.27 (dd, J = 15.0, 9.6 Hz, 1 H), 3.03 (d, J = 9.6 Hz, 1 H), 2.97 (d, J = 9.6 Hz,
1 H), 2.94 (dd, J = 15.6, 6.0 Hz, 1 H), 2.46 (dd, J = 13.2, 7.8 Hz, 1 H), 2.00-1.88 (m, 2 H), 1.86
20

ACCEPTED MANUSCRIPT
(ddd, J = 12.0, 4.8, 3.6 Hz, 1 H), 1.80 (tt, J = 12.6, 4.8 Hz, 1 H), 1.56 (td, J = 12.0, 4.2 Hz, 1 H),
1.49 (td, J = 10.2, 3.6 Hz, 1 H), 0.95 (d, J = 6.6 Hz, 3 H), 0.89 (d, J = 6.6 Hz, 3 H), 0.87 (d, J =
13
7.2 Hz, 3 H), 0.83 (d, J = 6.6 Hz, 3 H) ppm; C{1H} NMR (50 MHz, CDCl3) δ 173.1, 170.2,
79.1, 78.0, 56.4, 55.3, 53.4, 50.1, 29.1, 28.4, 27.1, 26.1, 20.3, 20.2, 20.1, 19.5 ppm; HRMS (ESI-
TOF) m/z [M + Na]+C16H28N2NaO3 calcd for 319.1992, found 319.1992.
N2-isobutyl-7-oxa-bicyclo[2.2.1]heptane-2,3-dicarboxamide (2e). A solution of isobutylamine
(0.3 mL, 3 mmol) in dry CH2Cl2 (5 mL) was added to a stirred solution of anhydride 1 (500 mg,
3 mmol) and DMAP (80 mg, 0.6 mmol) in dry CH2Cl2 (5 mL) at room temperature. After stirring
the reaction mixture for about 1 h, about 20 mL of liquid ammonia was collected into the
reaction mixture followed by addition of DIPC (0.465 mL, 3 mmol). The reaction mixture was
then allowed to attain room temperature gradually over 4 h. The reaction mixture was diluted
with chloroform, washed with citric acid solution and several times with water to remove DMAP
and diisopropylurea. The organic layer was concentrated under reduced pressure. The residue
was purified by column chromatography to give 2e (680 mg, 94%) as a white solid. mp 222.3-
223.2 °C; IR (film) νmax = 3289, 3336, 2956, 2870, 1650, 1554, 1463, 1385, 1242, 1168, 936,
816 cm-1; 1H NMR (200 MHz, CDCl3) δ 6.29 (s, 1 H), 6.14 (s, 1 H), 5.32 (s, 1 H), 4.97 (br t, J =
2.6 Hz, 1 H), 4.89 (br t, J = 2.8 Hz, 1 H), 3.02 (dd, J = 6.2, 3.0 Hz, 1 H), 2.98 (dd, J = 6.2, 2.8
Hz, 1 H), 2.95 (s, 2 H), 1.90-1.60 (m, 3 H), 1.60-1.45 (m, 2 H), 0.90 (s, 3 H), 0.87 (s, 3 H) ppm;
13C{1H} NMR (125 MHz, CDCl3) δ 172.8, 171.3, 78.8, 78.1, 54.8, 54.3, 47.1, 28.9, 28.8, 28.3,
20.1, 20.1 ppm; HRMS (ESI-TOF) m/z [M + Na]+C12H20N2NaO3 calcd for 263.1366, found
263.1364.
7-Oxabicyclo[2.2.1]heptane-2,3-diyl)bis((4-(3-chlorophenyl)piperazin-1-yl)methanone
(2f).
Triethylamine (0.84 mL, 6 mmol) was added to a suspension of anhydride 1 (504 mg, 3 mmol),
21

ACCEPTED MANUSCRIPT
m-chlorophenylpiperazine (1.4 g, 6 mmol) and DMAP (73 mg, 0.6 mmol) in dry CH2Cl2 (10 mL)
at room temperature. The reaction mixture was cooled on ice bath and DIPC (0.5 mL, 3 mmol)
was added drop-wise to it. The reaction mixture was stirred overnight and allowed to attain room
temperature. The reaction mixture was diluted with 1/1 ethyl acetate-petroleum ether and washed
with dilute citric acid solution, several times with water to remove DMAP and diisopropylurea.
The organic extract was concentrated under reduced pressure and the residue was purified by
column chromatography to give 2f (1.57 g, 98%) as a white solid. mp 195.5-196.5 °C; IR (film)
1
νmax = 2827, 1649, 1593, 1485, 1432, 1230, 1103, 990 cm-1; H NMR (500 MHz, CDCl3) δ
7.16 (t, J = 8 Hz, 2 H), 6.84 (br s, 4 H), 6.75 (d, J = 8 Hz, 2 H), 4.93 (s, 2 H), 3.90-3.80 (br m, 2
H), 3.70-3.55 (br m, 4 H), 3.55-3.45 (m, 2 H), 3.30-3.00 (m, 10 H), 1.87 (d, J = 8 Hz, 2 H), 1.59
(d, J = 7.5 Hz, 2 H) ppm; 13C{1H} NMR (125 MHz, CDCl3): δ 169.2, 151.9, 135.0, 130.2,
120.0, 116.2, 114.3, 78.8, 51.8, 48.9, 48.7, 45.2, 41.8, 29.5 ppm; Anal. Calcd for C28H32Cl2N4O3:
C, 61.9; H, 5.9; N, 10.3. Found: C, 62.1; H, 5.55; N, 10.1.
Acknowledgements
Mr Chandrakant S. Gholap is thankful to the Department of Atomic Energy (DAE), Govt. of
India for a research fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org........
References
1. R. S. Brown, A. J. Bennet, H. Slebocka-Tilk, Acc. Chem. Res. 25 (1992) 482-488;
22

ACCEPTED MANUSCRIPT
2. R. S. Brown, The Amide Linkage: Selected Structural Aspects in Chemistry, Biochemistry
and Materials Science; eds. Greenberg, A.; Breneman, C. M.; Liebman, J. F. John Wiley
Sons: New York, 2000;
3. V. Kumar, V. Bhatt, N. Kumar, Studies in Natural Products Chemistry, Vol. 56, Chapt.
9, Elsevier, 2018;
4. D. Kahne, W. C. Still, J. Am. Chem. Soc. 110 (1988) 7529-7534;
5. R. M. Smith, D. E. Hansen, J. Am. Chem. Soc. 120 (1998) 8910-8913;
6. A. Radzicka, R. Wolfenden, J. Am. Chem. Soc. 118 (1996) 6105-6109;
7. A. J. Bennet, V. Somayaji, R. S. Brown, B. D. Santarsiero, J. Am. Chem. Soc. 113 (1991)
7563-7571;
8. D. Kaiser, A. Bauer, M. Lemmerer, N. Maulide, Chem. Soc. Rev. 47 (2018) 7899-7925;
9. S. A. Ruider, N. Maulide, Angew. Chem. 54 (2015) 13856-13858;
10. Y. Nishii, T. Hirai, S. Fernandez, P. Knochel, K. Mashima, Eur. J. Org. Chem. 0 (2017)
5010-5014;
11. M. Yashiro, T. Takarada, S. Miyama, M. Komiyama, J. Chem. Soc. Chem. Commun. 0
(1994) 1757-1758;
12. Y. Kita, Y. Nishii, T. Higuchi, K. Mashima, Angew. Chem. Int. Ed. 51 (2012) 5723-
5726;
13. N. M. Milović, J. D. Badjić, N. M. Kostić, J. Am. Chem. Soc. 126 (2004) 696-697;
14. C. Balchandra, N. K. Sharma, Org. Lett. 17 (2015) 3948-3951;
23

ACCEPTED MANUSCRIPT
15. J. Clayden, W. J. Moran, Angew. Chem. Int. Ed. 45 (2006) 7118-7120;
16. M. Szostak, J. Aubé, Org. Biomol. Chem. 9 (2011) 27-35;
17. G. M. Blackburn, J. D. Plackett, J. Chem. Soc., Perkin Trans 2. 0 (1972) 1366-1371;
18. Q. P. Wang, A. J. Bennet, R. S. Brown, B. D. Santarsiero, J. Am. Chem. Soc. 113 (1991)
5757-5765;
19. A. J. Kirby, I. V. Komarov, P. D. Wothers, Angew. Chem. Int. Ed. 37 (1998) 785-786;
20. A. J. Kirby, I. V. Komarov, N. Feeder, J. Am. Chem. Soc. 120 (1998) 7101-7102;
21. A. J. Kirby, I. V. Komarov, N. Feeder, J. Chem. Soc., Perkin Trans 2. 0 (2001) 522-529;
22. K. Tani, B. M. Stoltz, Nature. 441 (2006) 731-734;
23. J. J. Gerschler, K. A. Wier, D. E. Hansen, J. Org. Chem. 72 (2007) 654-657;
24. M. L. Dougan, J. L. Chin, K. Solt, D. E. Hansen, Bioorg. Med. Chem. Lett. 14 (2004)
4153-4156;
25. M. N. Levine, R. T. Raines, Chem. Sci. 3 (2012) 2412-2420;
26. T. Yamana, A. Tsuji, Y. Mizukami, Chem. Pharm. Bull. 20 (1972) 1217–1229;
27. B. A. Cunningham, G. L. Schmir, J. Am. Chem. Soc. 89 (1967) 917–922;
28. S. J. Leach, H. Lindley, Trans. Faraday Soc. 49 (1953) 921–925;
29. M. L. Bender, Y. L. Chow, F. Chloupek, J. Am. Chem. Soc. 80 (1958) 5380–5384;
30. T. Higuchi, L. Eberson, A. K. Herd, J. Am. Chem. Soc. 88 (1966) 3805–3808;
31. J. Brown, S. C. K. Su, J. A. Shafer, J. Am. Chem. Soc. 88 (1966) 4468–4474;
24

ACCEPTED MANUSCRIPT
32. A. J. Kirby, P. W. Lancaster, J. Chem. Soc., Perkin Trans. 2. 0 (1972) 1206–1214;
33. R. Kluger, J. Chin, W. W. Choy, J. Am. Chem. Soc. 101 (1979) 6976–6980;
34. R. Kluger, J. Chin, J. Am. Chem. Soc. 104 (1982) 2891–2897;
35. R. Kluger, J. C. Hunt, J. Am. Chem. Soc. 111 (1989) 5921–5925;
36. J. H. Smith J. Am. Chem. Soc. 98 (1976) 3598–3601;
37. F. M. Menger, M. Ladika, J. Am. Chem. Soc. 110 (1988) 6794–6796;
38. A. B. Onofrio, J. C. Gesser, A. C. Joussef, F. Nome, J. Chem. Soc., Perkin Trans. 2. 0
(2001) 1863–1868;
39. T. Cohen, J. Lipowitz, J. Am. Chem. Soc. 86 (1964) 5611–5616;
40. A. Tsuji, T. Yamana, Y. Mizukami, Chem. Pharm. Bull. 22 (1974) 623–627;
41. R. L. Kogan, T. H. Fife J. Org. Chem. 49 (1984) 5229–5232;
42. S. W. King, R. Natarajan, R. Bembi, T. H. Fife, J. Am. Chem. Soc. 114 (1992) 10715–
10721;
43. A. Arcelli, G. Porzi, S. Sandri, Tetrahedron. 52 (1996) 4141–4148;
44. S. A. Ansari, P. Pathak, P. K. Mohapatra, V. K. Manchanda, Chem. Rev. 112 (2012)
1751–1772;
45. S. Sharma, S. Panja, A. Bhattachariya, P. S. Dhami, P. M. Gandhi, S. K. Ghosh, Dalton
Trans. 44 (2015) 12771–12779;
46. A. Dafforn, Jr. D. E. Koshland, Proc. Nat. Acad. Sci. USA. 68 (1971) 2463–2467;
47. T. C. Bruice, Annu. Rev. Biochem. 45 (1976) 331–373;
25

ACCEPTED MANUSCRIPT
48. P. S. Hillery, L. A. Cohen, J. Org. Chem. 48 (1983) 3465–3471;
49. F. M. Menger, Pure Appl. Chem. 77 (2005) 1873–1886;
50. R. Karaman, Tetrahedron Lett. 49 (2008) 5998–6002;
51. H. Hirohara, M. L. Bender, R. S. Stark, Proc. Nat. Acad. Sci. USA. 71 (1974) 1643–
1647;
52. Crystallographic data for diamide derivatives 2a-d have been deposited with the
Cambridge Crystallographic Data Center as supplementary publication nos. CCDC
1888170 (2a), 1888171 (2b), 1888168 (2c) and 1888169 (2d).
53. The X-ray crystal structure revealed that in the asymmetric unit, four 2a molecules are
present and two 2d molecules are present; For details, see SI.
54. The relevant data for other structures in the asymmetric unit of 2a and 2d are provided in
the SI.
55. H. B. Bürgi, J. D. Dunitz, J. M. Lehn, G. Wipff, Tetrahedron. 30 (1974) 1563–1572;
56. M. J. Frisch, et al. Gaussian, Inc., Wallingford CT, 2016. Gaussian 16 Revision A.03;
Gaussian Inc: Wallingford, CT, 2016;
57. R. Karaman, Chem Biol Drug Des. 82 (2013) 643-668;
58. R. C. Clark and J. S. Reid, Acta Cryst. A51 (1995) 887-889.
59. G. M. Sheldrick, Acta Cryst. A64 (2008) 112-122;
60. O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, H. Puschmann, J.
Appl. Cryst. 42 (2009) 339-341;
26

ACCEPTED MANUSCRIPT
Highlights
Norcantharidine derived tertiary diamides hydrolysed easily under acid catalysis
Their primary, secondary and mixed diamides do not hydrolyse easily
Hydrolysis of tertiary diamides occurred due to NGP of proximal amide carbonyls
Hydrolysis of tertiary diamides has been supported by X-ray structure & calculations
These tertiary diamides could serve for delayed release of certain types of drugs