Unexpected resistance to base-catalyzed hydrolysis of nitrogen pyramidal amides based on the 7-azabicyclic[2.2.1]heptane scaffold

Article abstract of DOI:10.3390/molecules23092363

Non-planar amides are usually transitional structures, that are involved in amide bond rotation and inversion of the nitrogen atom, but some ground-minimum non-planar amides have been reported. Non-planar amides are generally sensitive to water or other n

Full text of DOI:10.3390/molecules23092363

molecules
Article
Unexpected Resistance to Base-Catalyzed Hydrolysis
of Nitrogen Pyramidal Amides Based on the
7-Azabicyclic[2.2.1]heptane Scaffold
Diego Antonio Ocampo Gutiérrez de Velasco 1, Aoze Su 1, Luhan Zhai 1, Satowa Kinoshita 1,2
,
Yuko Otani 1 and Tomohiko Ohwada 1,
*
1
Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; daogv1@hotmail.com (D.A.O.G.d.V.);
aaronsusu@gmail.com (A.S.); zhailuhanzlh@gmail.com (L.Z.); s.kinoshita13@gmail.com (S.K.);
otani@mol.f.u-tokyo.ac.jp (Y.O.)
Department of Chemistry, St John’s College, University of Cambridge, St John’s Street,
Cambridge CB2 1TP, UK
2
*
Correspondence: ohwada@mol.f.u-tokyo.ac.jp; Tel.: +81-3-5841-4730
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Michal Szostak
Received: 25 August 2018; Accepted: 12 September 2018; Published: 15 September 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Non-planar amides are usually transitional structures, that are involved in amide bond
rotation and inversion of the nitrogen atom, but some ground-minimum non-planar amides
have been reported. Non-planar amides are generally sensitive to water or other nucleophiles,
so that the amide bond is readily cleaved. In this article, we examine the reactivity profile of
the base-catalyzed hydrolysis of 7-azabicyclo[2.2.1]heptane amides, which show pyramidalization
of the amide nitrogen atom, and we compare the kinetics of the base-catalyzed hydrolysis of
the benzamides of 7-azabicyclo[2.2.1]heptane and related monocyclic compounds. Unexpectedly,
non-planar amides based on the 7-azabicyclo[2.2.1]heptane scaffold were found to be resistant to
base-catalyzed hydrolysis. The calculated Gibbs free energies were consistent with this experimental
finding. The contribution of thermal corrections (entropy term, –T
term (
S‡) took a large negative value, indicating significant order in the transition structure,
which includes solvating water molecules.
∆
S‡) was large; the entropy
∆
Keywords: non planar amide; base-catalyed hydrolysis; water solvation; entropy
1. Introduction
In non-planar amides, distortion of the amide bond can arise from both twisting about the
C-N bond and pyramidalization at the nitrogen atom (Scheme 1) [
the amide bond are essentially mutually correlated, and the transition states of the amide rotation
involved bond twisting and nitrogen pyramidalization at the same time [ ] (see also Reference [ ]).
1,2]. These transformations of
3
4
The partial double-bond character of planar amides limits rotation about the C-N bond, and this feature
also contributes to stabilization, due to electron-delocalization. Decrease in sp2 nitrogen character,
with increase of sp3 character, tends to weaken the C-N bond and increase the electrophilicity of the
carbonyl carbon atom [1].
Molecules 2018, 23, 2363; doi:10.3390/molecules23092363
www.mdpi.com/journal/molecules

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Scheme 1. Amide transformation processes causing non-planarity: (1) N-C bond twisting (rotation)
and (2) nitrogen pyramidalization. These transformations are interconnected.
Non-planar amides are usually transitional structures that are involved in amide bond rotation
(Scheme 1 ( )) and inversion of the nitrogen atom (Scheme 1 ( )). However, even in ground-minimum
structures, amide distortion can be caused by several different factors, as illustrated in Figure 1A
lactam ring strain of the nitrogen atom at a bridgehead position [ ]; Figure 1B steric repulsion
between substituents at the carbonyl and nitrogen positions [ ]; Figure 1C angle strain at the
nitrogen position [10]; Figure 1D bulkiness of substituents at the nitrogen position [11]; Figure 1E
anomeric effect [12]; Figure 1F 1,3-allylic strain with respect to the pseudo C-N double bond [ 13].
1
2
5–8
9
4,
These compounds are examples of ground-minimum non-planar amides. One of the most significant
consequences of losing planarity of amides is an increase in lability: Reduction of the amide resonance
exposes the carbonyl functionality to nucleophilic attack and acyl transfer reaction. In particular,
hydrolysis by water under both acidic and basic conditions, and even under neutral conditions,
is greatly accelerated when planarity is disrupted [5–9].
Figure 1. Some examples of non-planar amides.
The torsional angle (
planar amides is 0.0◦. It is clear that the
values different from zero. Stable ground-state N,N-disubstituted tertiary amides, such as benzamide
derivatives (1a 1j) can also take non-zero values (Figure 2), as their calculated structures show
distortions from planarity [14 15]. Some of them (1c 1l and 1j) are activated for facile cleavage of the
amide C-N bond in the presence of various catalysts [16–19].
τ) (the mean twisting angle around the C-N bond, see Figure 2) for completely
τ
angle of ground-minimum non-planar amides can adopt
–
τ
,
,

Molecules 2018, 23, 2363
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a
Figure 2. Distortion angles (τ) of ground-state-stable benzoyl amides. B3LYP/6-31 + G(d) level of
b
theory (Reference [14]). X-ray data (Reference [15]). Calculated dihedral angle τ. τ = (ω1 + ω2)/2
(ω1 = ∠R-N-C-O and ω2 = ∠R-N-C-R’) [14].
Brown proposed a close relationship between nitrogen pyramidalization, C-N bond length and
kinetic reactivity to hydroxy anion attack [ ], based on a comparison of the hydrolysis kinetics of
7
analogous planar (molecule 1k in Figure 3) and non-planar amides (A3 in Figure 3). The base-catalyzed
◦
hydrolysis reaction at 25 C showed a striking activation by 7 orders of magnitude in passing from the
planar to the non-planar structure (see values in Figure 3).
Figure 3. Model molecules used by Brown in his comparative kinetics study.
Because of the lability of most non-planar amides in the presence of water [5–9], there have
been few applications of these scaffolds (Figure 1) in molecular design. On the other hand,
while 7-azabicyclo[2.2.1]heptane amides are highly suspicious of chemical stability, due to
nitrogen-pyramidalization [12
can be regarded as conformationally constrained
have been synthesized, and helical structures of homooligomers of
azabicyclo[2.2.1]heptane amide have been reported [20 21]. The helical structures were stable even in
the absence of intramolecular hydrogen bonds [22 23]. By introducing suitable bridgehead substituents,
,
13], 7-azabicyclo[2.2.1]heptane amides are of interest, beccause they
-proline mimics. Consequently, several derivatives
-proline mimics derived from
β
β
,
,
either all-cis amide or all-trans amide conformations were obtained. Conformational control favoring
the cis-isomer was achieved by introducing substituents at the C-4 bridgehead position (Figure 4(a)).
The cis-amide structure is heat-stable and the helical structure remains intact in a variety of solvents
(water, alcohol, halogenated solvents and cyclohexane) [24]. On the other hand, conformational control
favoring the trans-isomer was achieved by introducing substituents at the C-1 bridgehead position
(Figure 4(b)). The trans-amide structure also proved to be heat-stable and the helical structure remained
intact in both hydrophilic and hydrophobic solvents [25].

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Figure 4.
(a) Schematic representation of 7-azabicyclo[2.2.1]heptane amide cis-(S)-8mer. (b) Schematic
representation of the trans-(R)-8mer.
In the synthesis of oligomers of 7-azabicyclo[2.2.1]heptane amides (Figure 4) [24,25], acid-catalyzed
deprotection of the Boc group was compatible with the amide linkage (Figure 5). Therefore, we thought
that the bicyclic amide linkage might be stable under acidic conditions and convectional mild
reaction conditions. However, to our knowledge, neither qualitative nor quantitative data about
the base-catalyzed hydrolytic reactivity of this system have been reported.
Figure 5. Acidic deprotection procedure in the synthesis of the homooligomers.
Therefore, the aim of the present study is to establish the reactivity profile in the base-catalyzed
hydrolysis of this 7-azabicyclo[2.2.1]heptane amide system, which might serve as a model for the
enzymatic cleavage of peptide bonds. To this end, kinetic studies of the base-catalyzed hydrolysis of
the amide of 7-azabicyclo[2.2.1]heptane benzamides were conducted and the results were compared
with reported data for related monocyclic amide compounds. Theoretical calculations were also carried
out to aid in understanding the unexpectedly low reactivity.
2. Results and Discussion
In order to estimate the strength of amide bonding, we compared the reactivities of planar and
non-planar amides, specifically pyrrolidine amides (3a
(Figure 6). We also evaluated the effect of introducing substituents on the bridgehead position of the
bicycle (5a ). We utilized azetidine amides (2a ) (Figure 6) as reference compounds for non-planar
cyclic amides. In general, we found that the base-catalyzed hydrolysis of the bicyclic amides ( and
was rather slow. Among aromatic substituents, we focused on (H), (Cl) and (NO2) (Figure 6),
–e) and 7-azabicyclo[2.2.1]heptane amides (4a–e)
–
e
–e
4
5)
a
b
c
for which the reaction proceeds at acceptable speed, since the reactions in the cases of substituents
d
(Me) and e (MeO) are too slow to obtain kinetic data by means of NMR (see below).

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Figure 6. Model molecules used in this study. N-Benzoylazetidines (2a
(3a e) were examined as monocyclic amides. N-Benzoyl-7-azabicyclo[2.2.1]heptanes (4a
N-benzoyl-1-(methoxymethyl)-7-azabicyclo- [2.2.1]heptanes (5a ) were examined as bicyclic amides.
–
e) and N-benzoylpyrrolidines
–
–e
) and
–c
2.1. Synthesis
The monocyclic N-benzoylazetidines 2a
–e and N-benzoylpyrrolidines 3a–e were synthesized in
a straightforward manner by coupling the corresponding amines with different benzoyl chlorides
(Scheme 2). For the synthesis of the azetidine compounds, the chloride salt of the amine was used
as the starting material, with 3 equivalents of DIPEA (diispropylethylamine). For the synthesis of
pyrrolidine compounds, 1.2 equivalents of DIPEA sufficed. Compounds were obtained in good yields.
Scheme 2. Synthesis of monocyclic amides 2 and 3.
In addition to the monocyclic amides, we synthesized unsubstituted (4a
) bicyclic amides. N-Benzoyl-7-azabicyclo[2.2.1]heptanes were synthesized starting from
trans-4-aminocyclohexanol (Scheme 3) [ ]. The primary amine was substituted by benzyloxycarbonyl
–e) and substituted
(5a–e
4
chloride (ZCl), and the hydroxy group was changed to toluenesulfonate in order to facilitate bicycle
formation. Coupling of benzoyl chloride or para-substituted benzoyl chlorides gave the bicyclic
amides (4a–e).
Scheme 3. Synthesis of bridgehead-unsubstituted bicyclic amides 4.

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A different strategy was followed for the bridgehead-substituted bicyclic amides 5a–c
(Scheme 4). The hydroxy group was removed from the previously synthesized monomer scaffold
by Barton–McCombie deoxygenation using AIBN and tris(trimethylsilyl)silane (TTMSS) [26].
After that, the bridgehead ester functionality was first reduced to alcohol and then changed to ether.
After Boc-deprotection, the compounds were coupled with various para-substituted benzoyl chlorides
to afford the bridgehead-substituted bicyclic amides.
Scheme 4. Synthesis of bridgehead-substituted bicyclic amides 5.
2.2. Alkaline Hydrolysis of Planar Amide 3a
In order to assess the chemical reactivities of the non-planar 7-azabicyclo[2.2.1]heptane amides
4
and
5 in alkaline conditions, we first examined the kinetics of the planar amide N-benzoyl pyrrolidine
3a (X=H) in order to optimize the reaction conditions, because the hydrolysis of 3a is expected to be
the slowest among these compounds (2, 3, 4 and 5 in Figure 5).
2.2.1. Optimization of Reaction Conditions
Given that hydrolysis involves working with water as a solvent, it was necessary to confirm the
solubility of the reactants. In order to carry out the reactions, water-miscible co-solvents had to be
chosen. In addition, since alkaline conditions entail high concentrations of hydroxide, some solvents
(such as ketones or acetonitrile) are unsuitable. The list of possible co-solvents was narrowed down to
1,4-dioxane, methanol, THF and DMSO.
The first attempts at hydrolysis were conducted with 0.15 mmol (30 mg) of N-benzoylpyrrolidine
3a, 100
µL of deuterated methanol (as a co-solvent) and 400
µL of a solution of NaOD in D2O (40 w/w %).
The procedure was also done using 100
µL of deuterated 1,4-dioxane(1,4-dioxane-d8). The samples
were heated in a water bath at 37 ◦C and subjected to TLC. NMR spectra were recorded after 24 and
48 h. However, no hydrolysis product was detected by 1H-NMR, and no new product appeared on
TLC. Thus, the hydrolysis reaction did not proceed at 37 ◦C. Furthermore, the NaOD solution and the
1,4-dioxane solution separated into two phases.
Next, the concentration of the base was reduced to 0.4 M and that of the reactant to 0.1 M (i.e.,
a 4-fold excess of base over starting material). The total reaction volume was 1 mL (100 µL of co-solvent
and 900 µL of D2O).
Heating at 50 or 70 ◦C was applied, and the hydrolysis of 3a was monitored by TLC analysis.
◦
The reaction proceeded at 70 C. The starting material was no longer detectable after 48 h, and a single
spot corresponding to the hydrolysis product (benzoic acid) appeared. However, at 50 ◦C the starting
amide 3a was still detectable on the TLC plate after 48 h. In order to assess the working range for the
other compounds, a similar test was done at 70 ◦C for p-nitro derivative 3c (no spot of the starting
material was detected after 5 h) and p-methoxy derivative 3e (the starting material was still detected
after 65 h). Moreover, at the higher temperature, the 1,4-dioxane solution remained monophasic.
Therefore, it was decided to work at 70 ◦C.

Molecules 2018, 23, 2363
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In order to follow the progression of the reaction quantitatively, we recorded NMR spectra of
hydrolysis reaction mixtures of 3a
every two min at 70 ◦C. Rate constants were calculated from the
decrease of the integrals of the reactant (Figure 7).
–
e
◦
Figure 7. 1H-NMR monitoring of the hydrolysis of 3a, with NaOH in D2O and 1,4-dioxane-d8 at 70 C.
The intensity of amide peaks decreased (red box) over time, and product signals appeared (blue box).
From the 1H-NMR integration information we were able to determine the loss of amide over time.
Since working with excess deuteroxide anion guarantees pseudo-first-order kinetics, rate constants
were calculated using the following first-order equation:
d[Amide]
= −k[Amide].
(1)
dt
The integration of this rate equation gives the following equation:
ln[Amide] = −kt + ln[Amide]0
(2)
where [Amide] represents the molar concentration (M) of amide, k represents the reaction constant
(s−1), and
represents time ( ). A least-squares plot of the natural logarithm of amide concentration
t
s
versus time gave a straight line whose slope equals
the value of the y-intercept.
−
k. The initial amide concentration corresponds to
When either the aromatic protons or the pyrrolidine protons were used as a reference for signal
integration, all five pyrrolidine amides 3a showed good correlations between concentration and time,
and first-order reaction rate constants could be determined (Figure S1). The regression coefficients
R2 were high for all five compounds 3a
(Figure S1). The reaction showed Hammett-like behaviour,
–e
–e
that is the hydrolysis proceeded faster when an electron-withdrawing group was present at the para
position of the phenyl ring, and slower when an electron-donating group was present.

Molecules 2018, 23, 2363
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Based on these results, we next examined, the hydrolysis of the bicyclic compounds under
the same conditions. In order to hydrolyze compounds 4a and 4b it was necessary to increase the
proportion of 1,4-dioxane from 10% to 20%. The reaction proceeded smoothly, and the disappearance
of the reactants was successfully monitored by NMR. Unfortunately, the bicyclic compounds were not
sufficiently soluble under these conditions. Hence, we decided to increase the proportion of co-solvent.
The reaction volume was also scaled down from 1 mL to 500 µL. Solvent conditions were modified
on the basis of an examination of the hydrolysis of the p-NO2-substituted pyrrolidine benzamide 3c
(Table 1).
Table 1. Effect of variations of co-solvent proportions on the hydrolysis rate of 3c.
−1
1,4-Dioxane-d8/D2O (v/v%)
3c
NaOD
Temperature
−kobs (s
)
◦
−4
−5
−6
25/75
50/50
75/25
0.05 mmol
0.05 mmol
0.05 mmol
0.5 mmol
0.5 mmol
0.5 mmol
70 C
3.0 × 10
9.6 × 10
2.3 × 10
◦
70 C
◦
70 C
The sample containing 75% 1,4-dioxane did not form a homogenous solution even after heating
at 70 ◦C, possibly due to the high NaOD concentration in the water phase. On the other hand, the use
of 25% 1,4-dioxane resulted in a low R2 value (0.943). Despite these setbacks, it was seen that the
reaction proceeds faster in more polar solvent systems. This trend was also seen with other co-solvents
(Figure 8). The reaction time was shorter in DMSO-d6 than in methanol-d4, which in turn was shorter
than in 1,4-dioxane. The reaction was also carried out in THF-d8, but the compound was not sufficiently
soluble even at high temperature. The effect of solvent polarity on the hydrolysis rate can be explained
by the fact that the amide bond has a polar nature, and charges develop as the bond is broken.
Therefore, more polar solvent systems are better at stabilizing the developing charges in the transition
states and the products. Although DMSO is a good solvent, the presence of hydroxide anion can
produce the basic dimsyl anion (Na+ -CH2-SO-CH3) from DMSO. Therefore, we focused on 1,4-dioxane
and methanol, rather than DMSO.
Figure 8. Effect of polarity of the solvent system on the rate of alkaline hydrolysis. Higher polarity
of the solvent system accelerates the reaction. The rate of hydrolysis in methanol as a solvent was
arbitrarily set at unity (1.0).
2.2.2. Alkaline Hydrolysis of Amides in Two Solvents
Finally, the set of conditions, shown in Table 2, was selected for all compounds: 250
µL of
co-solvent (1,4-dioxane-d8 or methanol-d4), and 250 µL of water (total volume (500 µL)), and 10
equivalents of base with respect to the reactant amide. The reaction was carried out at 70 ◦C. These
conditions provided first-order kinetics with respect to amide concentration.
Table 2. Final conditions for alkaline hydrolysis of amide compounds.
Starting Amide
NaOD 40% wt.
D2O
Co-solvent
Temperature
◦
0.05 mmol
0.5 mmol
250 µL
250 µL
70 C

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2.2.3. Alkaline Hydrolysis in Dioxane
Several compounds were subjected to NaOD-catalyzed hydrolysis in 1,4-dioxane-d8-D2O (1:1)
under the conditions, shown in Table 3. The co-solvent was 1,4-dioxane-d8. The plots in Figure S2
are based on the raw data of selected hydrolysis experiments (Figure 9). Hydrolysis was repeated
three times for some of the compounds in order to assess the repeatability of the method. It was found
that the error when 1,4-dioxane-d8 was used as the co-solvent was
amine) were identified by mass spectrometry. NMR monitoring revealed signals corresponding to the
reactant and the hydrolysis products in all cases. The ring-opening product of azetidine amide was
±
13.8%. Products (carboxylate and
not detected. As a general trend, base-catalyzed hydrolysis of azetidine amides (
rapidly than that of pyrrolidine amides ( ), which in turn were hydrolyzed faster than unsubstituted
bicyclic amides ( ), while bridgehead-substituted bicycles ( ) were least reactive (for example, reaction
rate: 2a > 3a > 4a > 5a; 2b > 3b > 4b > 5b; 2c > 3c > 4c > 5c). The expected Hammett-like trend was
observed: The electron-withdrawing substituent NO2 ( ) on the phenyl moiety accelerated the reaction.
2) proceeded more
3
4
5
c
Table 3. Base-catalyzed hydrolysis rates ( –5x (x = a–e) in two solvent
kobs in M−1s−1) of amides 2x
−
systems.a Average values are shown where possible (see footnotes). Relative reaction rates (referenced
to 4) are shown in parentheses.
◦
1,4-Dioxane-D2O (1:1), NaOD, 70 C a
◦
Methanol-D2O (1:1), NaOD, 70 C b
x=
2x
3x
4x
5x
2x
3x
4x
5x
−5
−4
−4
−6
−5
−4
−6
−6
−5
−7
−6
−5
2.1 × 10
7.8 × 10
3.6 × 10
6.5 × 10
−4
−4
−5
−5
a (H)
ND
ND
1.1 × 10
2.9 × 10
(5.8)
(2.2)
(1)
(0.2)
−6
−5
1.1 ×
10−5(1)
7.0 ×
2.3 × 10
(82.1)
1.8 × 10
(6.4)
2.8 × 10
(1)
1.2 × 10
(0.4)
9.7 × 10
7.0 × 10
3.0 × 10
(0.3)
b (Cl)
c (NO2)
(88.2)
(6.4)
−3
−4
−5
−5
9.9 × 10
1.0 × 10
3.0 × 10
3.7 × 10
6.5 × 10
(92.9)
4.2 × 10
(6.0)
4.3 × 10
10−5(1)
4.1 ×
(33.0)
(3.3)
(1)
(1.2)
(0.6)
1.7 × 10
−6
−6
d (Me)
ND
ND
NA
NE
NE
NE
ND
ND
NE
NE
4.1 × 10
4.8 × 10
10−6(1)
NE
(4.2)
e (MeO)
1.9 × 10
a
Error estimation:
±
13.8% (in the 1,4-dioxane system). (
b
) Error estimation:
±
17.4% (in the methanol system).
ND = not determined; NA = not available (due to the solubility problem); NE = not executable (due to very
slow reaction).
9.7 x 10-4
2.3 x 10-4
(88)
(82)
7.0 x 10-5
1.8 x 10-5
(6)
(6)
2.8 x 10-6
(1)
1.1 x 10-5
(1)
3.0 x 10-6
(0.3)
1.2 x 10-6
(0.4)
Figure 9. Base-catalyzed hydrolysis rates of 2c, 3c, 4c and 5c in two solvent systems (1,4-dioxane
and methanol). Values of
(referenced to 4c).
−
kobs (s−1) are shown. Relative reaction rates are shown in parentheses

Molecules 2018, 23, 2363
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For example, N-(p-nitrobenzoyl-7-azabicyclo[2.2.1]heptane (4c) was hydrolyzed in 1,4-dioxane
and D2O at a slower rate than the analogous planar monocycle 2c (6 times faster than 3c) or the
non-planar monocycle 1c (82–88 times faster than 3c). Moreover, a bridgehead substituent (4c) further
slowed the hydrolysis rate (2.3–3.6 times slower than 3c) (Figure 9).
2.2.4. Alkaline Hydrolysis in Methanol
Several compounds were subjected to hydrolysis in methanol under the final conditions, shown
in Table 3 (methanol-d4-D2O 1:1). The co-solvent used in this case was methanol-d4. The following
plot shows the 1H-NMR spectral change corresponding to the slow consumption of 5b in methanol
(Figure 10). We could not detect intermediate formation of the methyl ester, which may be formed
by the attack of methoxide anion on the amide. Hydrolysis was repeated three times for some of
the compounds in order to assess the repeatability of the method. It was found that the error of
the method when using methanol-d4 as a co-solvent was
spectrometry. Signals corresponding to reactants and hydrolysis products were identified in all cases.
The ring-opening product of azetidine amide was not detected. Reactivity followed the same trend as in
±
17.4%. Products were identified by mass
1,4-dioxane (reaction rate: 2b
> 3b > 4b> 5b). The reactivity was higher in methanol than in 1,4-dioxane.
◦
Figure 10. Progress of the hydrolysis of 5b in methanol-d4/D2O (1:1) at 70 C, followed by 1H-NMR;
the intensity of amide peaks decreased, and new product signals appeared as the reaction progressed
(red box).
2.2.5. Comparison of Kinetic Data
As shown in Table 3, the same general trend was observed irrespective of the solvent system
employed. Azetidine amides (
2
) were the most reactive, followed by pyrrolidine amides (
3),
then unsubstituted bicyclic amides (
4
), and finally bridgehead-substituted bicycles ( ) (reaction rate:
5
2b > 3b > 4b> 5b). Phenyl substitution had the expected effects (according to the inductive effects) in
all series of amides.

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2.3. Computational Studies
2.3.1. Reaction Model
It was unexpected to find that non-planar amides based on the 7-azabicyclo[2.2.1]heptane scaffold
4) showed such poor susceptibility to base-catalyzed hydrolysis, even upon heating, as compared with
(
the corresponding monocyclic amides (
amides ( ) also decelerated base-catalyzed hydrolysis of the amide as compared with the unsubstituted
bicyclic derivative ( ). It has been established in previous studies on heavy atom isotope effects that
3). Bridgehead substitution of the 7-azabicyclo[2.2.1]heptane
5
4
formation of the tetrahedral intermediate is rate-determining in the base-catalyzed hydrolysis of
formamide (HCONH2) (Scheme 5) [27].
Scheme 5. General reaction path for the base-catalyzed hydrolysis of amides.
While there have been several ab initio and DFT calculation studies of the hydrolysis of planar
amides and non-planar amides [28–31], base-catalyzed hydrolysis of amides has been relatively
little studied until recently, Further, most studies have focused on rather simple amides, such as
formamide, N-methylacetamide, DMF, and DMA (dimethylacetamide) [32]. Here, we aimed to
rationalize the observed reactivity trends by computational studies of our more complex reactants and
transition structures.
It is known that that explicit water solvation is crucial for the calculation of amide hydrolysis.
Xiong and Zhan [32] showed that incorporation of five implicit water molecules is required, and there
are two kinds of hydrogen-bonding networks of water in the vicinity of the hydroxy ion (−OH) and
amide group in the presence of five explicit water molecules (Figure 11). These two patterns commonly
involve activation of the carbonyl group by hydrogen-bonding of two water molecules to the oxygen
atom (increasing its electrophilicity) and hydrogen bonding of three water molecules with the oxygen
atom of the hydroxy anion (decreasing its nucleophilicity and at the same time decreasing electronic
repulsions). There is a difference in the topology of the hydrogen-bonding networks. Type a is more
stable than Type b by approximately 1-2 kcal/mol, but finding a TS of Type a in our experimental amide
systems 2–5 was difficult, probably because the Type a hydrogen network is sensitive to the steric
interactions encountered in more complicated amide structures (see the detail in the Experimental
Section). Therefore, in the present work, we focused on the hydrogen network Type b, which seems
relevant to the present compounds.
Figure 11. Two hydrogen network patterns involving five H2O molecules in the attack of OH anion on
the amide carbonyl carbon atom [32].

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Geometry optimizations for the ground states of hydrated reactants (amide and hydroxide
anion) and the transition state for the nucleophilic addition of hydroxide anion to the amide carbonyl
group were performed in the presence of explicit water molecules at the B3LYP and M06-2X levels
of theory with a combination of two basis sets, 6-31+G(d) and 6-311++G(d, p). Bulk solvation
effects (self-consistent reaction field, SCRF) were also incorporated by means of IEFPCM (Polarizable
Continuum Model, PCM, using the integral equation formalism variant) and SMD methods in water.
Vibrational frequency calculations were performed at the same level of theory. The energies were
◦
corrected for the zero-point energies and Gibbs free energy at 25 C (298.15 K), obtained from frequency
calculations. Hereafter, we will focus on the calculation values based on M06-2X level with the SMD
solvent model. The calculations at the B3LYP level (see Tables S1–S4) were consistent with the trends
obtained in the M06-2X calculations.
2.3.2. Model with Five Explicit Water Molecules
Transition structures (TSs) for the hydroxide anion-catalyzed hydrolysis of the amides, including
five explicit water molecules were identified with M06-2X//6-31+G(d) (Figure 12). As previously
described by Xiong and Zhan [32], we calculated the activation Gibbs energies from the energy
difference between the TS structures and the summation of the energies of the two reagents, the amide
with two hydrogen-bonded water molecules (amide(H2O)2) and the hydroxide anion clustered with
−
three water molecules ( OH(H2O)3) (Scheme 6). We also estimated Gibbs free energies by single-point
calculation with M06-2X//6-311++G(d,p) on the basis of the M06-2X//6-31+G(d) optimized structures
(Table 4).
Scheme 6. Model of base-catalyzed hydrolysis reaction of amide.
The contribution of thermal corrections (entropy term, –T
term (
S‡) took a large negative value (Table 2), indicating the presence of significant order in the
∆
S‡) was significant. The entropy
∆
transition structure. Associated water molecules need to rearrange on the surface of the amide, and thus
would contribute to this large negative entropy term. A larger free-energy activation barrier was
seen for N-benzoyl-7-azabicyclo[2.2.1]heptane (4a) than for the monocyclic N-benzoylpyrrolidine (3a).
The pyramidal amide, the azetidine derivative 2a has the smallest activation energy. The order of the
magnitude of the Gibbs activation energies (4a > 3a, 5a > 2a, Table 4) is essentially consistent with the
experimental reactivity (Figure 8 and Table 3), with the exception of the bridgehead-substituted bicyclic
amide 5a, which is expected to have higher activation energy than 4a. However, we need to make
allowance for the simple harmonic oscillator approximation in the thermal energy correction, and also
we need to consider that this thermal energy correction is an approximation of the real entropy change
in the solvation process.
The enthalpy terms (
essentially consistent with the experimentally observed hydrolysis rates: 2a
∆
H‡) were underestimated (Table 4), but the order of their magnitudes is also
3a, 4a, 5a. This trend is
>
consistent with the trajectory in the TS structures (Figure 12): The shorter the distance between the
amide carbonyl carbon atom and hydroxide oxygen atom (i.e., later the TS), the larger the enthalpy
term (∆H‡).

Molecules 2018, 23, 2363
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Table 4. Calculated free energy barrier for the formation of the transition state at 25 ◦C (298.15 K) at
the M06-2X/6-31+G(d) and M06-2X/6-311++G(d, p) levels, considering five water molecules. Solvent
effect is SMD (solvent = water) a.
∆H‡
kcal/mol
−T∆S‡
∆S‡
cal/(mol·K)
∆G‡
25 ◦
kcal/mol
C
Compound
kcal/mol
M06-2X/6-31+G(d) SMD=water b
2a
3a
4a
5a
2.51
5.03
6.80
4.66
+17.38
+17.03
+16.57
+17.95
−58.29
−57.12
−55.58
−60.20
19.89
22.06
23.37
22.61
M06-2X/6-311++G(d,p) SMD=water c
2a
3a
4a
5a
4.26
7.13
8.80
7.50
+16.16
+16.03
+15.76
+15.44
−54.20
−53.76
−52.86
−51.79
20.42
23.16
24.56
22.94
a
Calculation and experimental Gibbs free energy of hydroxide anion-catalyzed hydrolysis of formamide was
b
c
21–22 kcal/mol [32]. Full optimizations. Single-point calculations.
Other reaction models, a model with four explicit water molecules and a model with implicit
water molecules, were also examined and the order of the energy demand is consistent with that of the
present five-water model (the results are described in Supporting Information).
Non-planar amides based on the 7-azabicyclo[2.2.1]heptane scaffold were found to be rather
inert to base-catalyzed hydrolysis. The calculated Gibbs free energies are also consistent with the
experimental results.
H 2O
-
O H
-
O H
1.925Å
1.976Å
azetidine
pyrrolidine
3a-T S
2a-T S
-
-
O H
1.928Å
O H
1.961Å
bicyclic
bicyclic
5a-T S
4a-T S
Figure 12. M06-2X/6-31+G(d)-optimized transition structures of hydroxide anion addition to the
amide. Distance between the amide carbonyl carbon atom and hydroxide oxygen atom was shown.

Molecules 2018, 23, 2363
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A close scrutiny of the trajectory in the TS (Figure 12) revealed the optimal trajectory for
the hydroxide anion attack on the carbonyl group accompanied with hydrating water molecules.
The positions of the hydroxy anion were similar in the respective TS structures. In the bicyclic amides
(Figure 13, left), the water molecules in the bridgehead-substituted 5a were placed a little further
from the amide substrate as compared with those in bridgehead-unsubstituted 4a. A comparison of
azetidine 2a and bridgehead-unsubstituted 4a also indicated that some of the hydrated waters were
placed far from the substrate in the case of 4a (Figure 13, right). Therefore, these water molecules
cannot get close to the substrate, due to greater steric congestion in 4a, as compared with the azetidine
(2a). Hydrogen bonding of water molecules stabilized the developing negative charge during the
attack of hydroxide anion on the amide carbonyl carbon atom, while the entropy cost compensates for
the stabilization. Inefficient hydration is one of the possible reasons that could explain the increase of
the activation energy of the bicyclic amides 4 and 5.
Figure 13. Superimposed TS structures of 4a and 5a (left) and 2a and 4a (right), showing the disrupted
water network in bulky 5a (left, magenta) and 4a (right, blue).
3. Materials and Methods
3.1. General Procedures
All analyzed compounds were synthesized from commercially available reagents. All compounds
were purified before use by column chromatography on silica gel (spherical, neutral silica gel 60 N
1
(100–210
µ
m), Kanto). Characterization was done by multiple techniques. H- (400 MHz) and 13C-
(100 MHz) NMR spectra were recorded in a 400 MHz Bruker Avance 400 NMR spectrometer at 25 ◦C.
Chemical shifts ( ) are shown in ppm, and coupling constants are given in hertz. Spectral data was
δ
obtained using NMR data processing software Brucker TOP-Spin. The NMR probe temperature was
calibrated by the temperature-dependent chemical shift difference in ppm between OH proton and
CH2 proton of ehtyleneglycol [33].
ESI-TOF mass spectra were recorded in a Bruker Daltonics, micrO-TOF-05. Elemental analyses
were done by an independent group in this department and were given within a
Melting points were measured with a Yanaco Micro Melting Point Apparatus and are uncorrected.
±
0.4% error range.

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3.2. Synthesis of Amides
All the amide compounds except 5a
–5c have been synthesized previously [12] and stock samples
were used for the present work. Some compounds among 2a–2e, 3a–3e, and 4a–4e were resynthesized,
as described below, including the new compounds 5a–5c.
Synthesis of N-Benzoylazetidines
N-Benzoylazetidine (2a). Azetidine chloride (100 mg, 1.1 mmol) was dissolved in dry CH2Cl2
(5 mL) and the solution was cooled to 0 ◦C. DIPEA (0.7 mL, 2.9 mmol) was added, and the mixture
was stirred for 10 min. Then, benzoyl chloride (186 µl, 1.6 mmol) was added slowly to the solution,
and stirring was continued for 30 min. The ice bath was removed and the reaction mixture was
allowed to cool to r.t., then quenched by pouring it into water. The aqueous and organic layers were
separated, and the aqueous phase was extracted with dichloromethane (3
organic phase was washed with 0.5 M HCl, 0.5 M aq. NaHCO3 and brine, dried over sodium sulfate,
filtered, and evaporated under reduced pressure to afford a yellowish oil. The crude product was
×
10 mL). The combined
purified by open column chromatography (ethyl acetate/DCM, 1:1) to afford 2a as a transparent oil
1
(130.8 mg, 0.81 mmol, 76%). H-NMR (400 MHz, CDCl3),
δ
(ppm): 7.616–7.592 (m, 2H), 7.444–7.353
(ppm): 170.39, 133.36,
(m, 3H), 4.296–4.191 (m, 4H), 2.352–2.274 (m, 2H). 13C-NMR (100 MHz, CDCl3),
130.93, 128.40, 127.91, 53.46, 49.01. HRMS (ESI-TOF): [M + Na]+: Calcd. for C10H11NNaO+: 185.0766.
δ
Found: 185.0782.
N-(p-Chlorobenzoyl)azetidine (2b). Azetidine chloride (100 mg, 1.1 mmol) was dissolved in dry
CH2Cl2 (5 mL) and the solution was cooled to 0 ◦C. DIPEA (0.7 mL, 2.9 mmol) was added, and the
mixture was stirred for 10 min. 4-Chlorobenzoyl chloride (205 µL, 1.6 mmol) was added slowly,
and stirring was continued for 30 min. The ice bath was removed and the mixture was allowed to
warm to r.t., then quenched by pouring it into water. The aqueous and organic layers were separated,
and the aqueous phase was extracted with dichloromethane (3
phase was washed with 0.5 M HCl, 0.5 M aq. NaHCO3 and brine, dried over sodium sulfate, filtered,
and evaporated under reduced pressure to afford a pale oil. The crude product was purified by
×
10 mL). The combined organic
open column chromatography (ethyl acetate/DCM, 1:2) to provide 2b as colorless crystals (171.9 mg,
1
0.88 mmol, 82%). H-NMR (400 MHz, CDCl3),
δ
(ppm): 7.587–7.554 (m, 2H), 7.391–7.358 (m, 2H),
(ppm): 169.18, 137.08, 131.76,
4.309–4.199 (m, 4H), 2.386–2.309 (m, 2H). 13C-NMR (100 MHz, CDCl3),
δ
129.39, 128.70, 53.51, 49.09. HRMS (ESI-TOF): [M + Na]+ Calcd. for C10H10ClNNaO+: 218.0343. Found:
218.0360. Anal. Calcd. for C10H10ClNO: C, 61.39; H, 5.15; N, 7.16. Found: C, 61.02; H, 5.47; N, 6.96.
N-(p-Nitrobenzoyl)azetidine (2c). Azetidine◦chloride (100 mg, 1.1 mmol) was dissolved in dry
CH2Cl2 (5 mL) and the solution was cooled to 0 C. DIPEA (0.7 mL, 2.9 mmol) was added, and stirring
was continued for 10 min. Then, 4-nitrobenzoyl chloride (186 µL, 1.6 mmol) was added slowly,
and stirring was continued for 30 min. The ice bath was removed and the mixture was allowed to
warm to r.t., then quenched by pouring it into water. The aqueous and organic layers were separated,
and the aqueous phase was extracted with dichloromethane (3
phase was washed with 0.5 M HCl, 0.5 M aq. NaHCO3 and brine, dried over sodium sulfate, filtered,
and evaporated under reduced pressure to give a yellow oil. The crude product was purified by
×
10 mL). The combined organic
open column chromatography (ethyl acetate/DCM, 1:2) to afford 2c as a yellow solid (172.6 mg,
1
0.84 mmol, 78%). H-NMR (400 MHz, CDCl3),
δ
(ppm): 8.268–8.235 (m, 2H), 7.793–7.760 (m, 2H),
(ppm): 168.00, 139.22, 131.19,
4.311–4.231 (m, 4H), 2.425–2.347 (m, 2H). 13C-NMR (100 MHz, CDCl3),
129.01, 123.76, 53.41, 49.32. HRMS (ESI-TOF): [M + Na]+ Calcd. for C10H10N2NaO3+: 229.0585.
δ
Found: 229.0585.
Synthesis of N-Benzoylpyrrolidines
N-Benzoylpyrrolidine (3a). Pyrrolidine (1 mL, 12.2 mmol) was dissolved in dry CH2Cl2 (5 mL) and
the solution was cooled to 0 ◦C. DIPEA (3.4 mL, 14.5 mmol) was added, and the mixture was stirred

Molecules 2018, 23, 2363
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for 10 min. Then, benzoyl chloride (1.7 mL, 14.75 mmol) was added slowly, and stirring was continued
for 30 min. The ice bath was removed, and the mixture was allowed to warm to r.t., then quenched by
pouring it into water. The aqueous and organic layers were separated, and the aqueous phase was
extracted with dichloromethane (3
×
10 mL). The combined organic phase was washed with 0.5 M
HCl, 0.5 M aqeouse solution of NaHCO3 and brine, dried over sodium sulfate, filtered, and evaporated
under reduced pressure to give a yellow liquid. The crude product was purified by open column
chromatography (ethyl acetate) to afford 3a as a transparent yellow liquid (1.5530, 8.86 mmol, 73%).
1H-NMR (400 MHz, CDCl3),
δ (ppm): 7.504–7.356 (m, 5H), 3.615 (t, J = 6.8 Hz, 2H), 3.381 (t, J = 6.6 Hz,
2H), 1.929–1.804 (m, 4H). 13C-NMR (100 MHz, CDCl3), δ (ppm): 169.35, 137.10, 129.51, 128.00, 126.86,
49.33, 45.93, 26.17, 24.21. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd. for C11H13NNaO+: 198.0890. Found:
198.0872. Anal. Calcd. for C11H13NO: C, 75.40; H, 7.48; N, 7.99. Found: C, 75.21; H, 7.63; N, 7.97.
N-(p-Chlorobenzoyl)pyrrolidine (3b). Pyrrolidine (0.5 mL, 6.1 mmol) was dissolved in dry CH2Cl2
◦
(3 mL) and the solution was cooled to 0 C. DIPEA (1.7 mL, 7.7 mmol) was added, and the mixture was
stirred for 10 min. Then, 4-chlorobenzoyl chloride (1.6164 mL, 7.4 mmol) dissolved in CH2Cl2 (2 mL)
was added slowly, and stirring was continued for 1 h. The ice bath was removed, and the mixture was
allowed to warm to r.t., then quenched by pouring it into water. The aqueous and organic layers were
separated, and the aqueous phase was extracted with dichloromethane (3
×
10 mL). The combined
organic phase was washed with 0.5 M HCl, 0.5 M aq. NaHCO3 and brine, dried over sodium sulfate,
filtered, and evaporated under reduced pressure to afford a transparent oil. The crude product was
purified by open column chromatography (hexane/ethyl acetate) to provide 3b as a white amorphous
◦
1
solid (444 mg, 2.1 mmol, 35%). m.p.: 62–64 C. H-NMR (400 MHz, CDCl3),
δ (ppm): 7.410 (dd;
J = 4.3 Hz, 1.8 Hz; 2H), 7. 307 (dd; J = 8.8 Hz, 2 Hz; 2H), 3.569 (t, J = 6.6 Hz, 2H), 3.349 (t, J = 6.6 Hz, 2H),
1.930–1.786 (m, 4H). 13C-NMR (100 MHz, CDCl3),
δ (ppm): 168.48, 135.73, 135.50, 128.63, 128.44, 49.55,
46.25, 26.36, 24.36. HRMS (ESI-TOF): [M + Na]+ Calcd. for C11H12ClNNaO+: 232.0500. Found 232.0517.
Anal. Calcd. for C11H12ClNO: C, 63.01; H, 5.77; N, 6.68. Found: C, 62.95; H, 5.78; N, 6.51.
N-(p-Nitrobenzoyl)pyrrolidine (3c). Pyrrolidine (0.5 mL, 6.1 mmol) was dissolved in dry CH2Cl2
(5 mL) at 0 ◦C. DIPEA (1.7 mL, 7.7 mmol) was added to the solution, and the mixture was stirred for
10 min. Then, 4-nitrobenzoyl chloride (1.1301 g, 7.4 mmol) dissolved in CH2Cl2 (10 mL) was added
slowly, and stirring was continued for 1 h at 0 ◦C. The ice bath was removed, and the mixture was
allowed to warm to r.t., then quenched by pouring it into water. The aqueous and organic layers were
separated, and the aqueous phase was extracted with dichloromethane (3 × 30 mL). The combined
organic phase was washed with brine, dried over sodium sulfate, filtered, and evaporated under
reduced pressure to give a a yellow liquid. The crude product was purified by open column
chromatography (ethyl acetate/acetone 1:1) to afford 3c as a slightly yellow solid (821.6 mg, 3.7 mmol,
◦
61.3%). m.p.: 78–80 C. 1H-NMR (400 MHz, CDCl3),
δ (ppm): 8.293–8.207 (m, 2H), 7.698–7.665 (m, 2H),
3.676 (t, J = 7.0 Hz, 2H), 3.382 (t, J = 6.6 Hz, 2H), 2.039–1.887 (m, 4H). 13C-NMR (100 MHz, CDCl3),
δ
(ppm): 167.61, 143.19, 128.29, 123.84, 49.61, 46.56, 26.52, 24.50. HRMS (ESI-TOF): [M + Na]+ Calcd. for
C11H12N2NaO3+: 244.0774. Found: 244.0742. Anal. Calcd. for C11H12N2O3: C, 59.99; H, 5.49; N, 12.72.
Found: C, 59.82; H, 5.45; N, 12.65.
N-(p-Toluoyl)pyrrolidine (3d)◦. Pyrrolidine (0.5 mL, 6.1 mmol) was dissolved in dry CH2Cl2 (5 mL)
and the solution was cooled to 0 C. DIPEA (1.7 mL, 7.7 mmol) was added, and the mixture was stirred
for 10 min. Then, a solution of p-tolyl chloride (975
and the mixture was stirred for 1 h at 0 C. The ice bath was removed, and the mixture was allowed to
warm to r.t., then quenched by pouring it into water. The aqueous and organic layers were separated,
µ
L, 7.4 mmol) in CH2Cl2 (10 mL) was slowly added,
◦
and the aqueous phase was extracted with dichloromethane (3
×
30 mL). The combined organic
phase was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced
pressure to provide a yellow liquid. The crude product was purified by open column chromatography
(hexane/ethyl acetate 4:1) to afford 3d as a white solid (654.4 mg, 3.5 mmol, 56.8%). M.p.: 75–76 ◦C.
1H-NMR (400 MHz, CDCl3),
δ
(ppm): 7.438–7.408 (m, 2H), 7.202–7.179 (m, 2H), 3.639 (t, J = 7.0 Hz,
2H), 3.439 (t, J = 6.6 Hz, 2H), 2.372 (s, 3H), 1.986–1.829 (m, 4H). 13C-NMR (100 MHz, CDCl3),
δ
(ppm):

Molecules 2018, 23, 2363
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169.93, 139.98, 134.52, 128.93, 127.35, 49.75, 46.30, 26.55, 24.59, 21.50 HRMS (ESI-TOF) m/z: [M + Na]+
Calcd. for C12H15NNaO+: 212.1046. Found 212.1036. Anal. Calcd. for C12H15NO: C, 76.16; H, 7.99;
N, 7.40. Found: C, 75.80; H, 8.09; N, 7.33.
N-(p-Anisoyl)pyrrolidine (3e). Pyrrolidine (0.5 mL, 6.1 mmol) was dissolved in dry CH2Cl2 (5 mL)
◦
and the solution was cooled to 0 C. DIPEA (1.7 mL, 7.7 mmol) was added, and the mixture was
stirred for 10 min. Then, a solution of 4-nitrobenzoyl chloride (1◦.2573 g, 7.4 mmol) in CH2Cl2
(10 mL) was slowly added, and stirring was continued for 1 h at 0 C. The ice bath was removed,
and the mixture was allowed to warm to r.t., then quenched by pouring it into water. The aqueous
and organic layers were separated, and the aqueous phase was extracted with dichloromethane
(
3 × 30 mL). The combined organic phase was washed with brine, dried over sodium sulfate, filtered,
and evaporated under reduced pressure to provide a yellow liquid. The crude product was purified by
open column chromatography (n-hexane/ethyl acetate 1:1) to afford 3e as a pale-brown solid (808.3 mg,
◦
3.9 mmol, 65%). m.p.: 73.5–75.5 C. 1H-NMR (400 MHz, CDCl3),
δ
(ppm): 7.515 (ddd, J = 9.2, 2.4, 2 Hz,
4H), 3.832 (s, 3H), 3.634 (t, J = 6.4 Hz, 2H), 3.476 (t, J = 6.2 Hz, 2H), 1.966–1.851 (m, 4H). 13C-NMR
(100 MHz, CDCl3),
δ (ppm): 169.56, 160.93, 129.63, 129.29, 113.55, 55.45, 49.90, 46.44, 26.64, 24.59.
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd. for C12H15NNaO2+: 228.0995. Found 228.0993. Anal. Calcd.
for C12H15NO2: C, 70.22; H, 7.39; N, 6.82. Found: C, 70.01; H, 7.48; N, 6.76.
Synthesis of N-Benzoylazetidines
N-Benzoyl-7-azabicyclo[2.2.1]heptane (4a). Trans-4-aminocyclohexyl p-toluensulfonate hydrobromide
(1 g, 7.5 mmol) was dissolved in ethanol (110 mL) and water (30 mL), and then NaOH 1 M (25 mL)
was added. The mixture was stirred at room temperature for 20 h, and then quenched by adding HCl
4 M in 1,4-dioxane (5 mL). Stirring was continued for 20 min at r.t., and the mixture was evaporated
under reduced pressure. NaOH 10% (10 mL) was added to the residue, and free amide was extracted
with ether (3
×
30 mL). This solution was evaporated, and the residue was redissolved in dry CH2Cl2
(30 mL). DIPEA (2 mL, 9.3 mmol) was added to the resulting solution, and the mixture was stirred
for 10 min at 0 C. Then, benzoyl chloride (1.1 mL, 9.1 mmol) was slowly added. Stirring was
◦
continued for 30 min. The ice bath was removed, and the mixture was allowed to warm to r.t. Stirring
was continued for 4 h, and then the mixture was quenched by pouring it into water. The aqueous
and organic layers were separated, and the aqueous phase was extracted with dichloromethane
(
3 × 50 mL). The combined organic phase was washed with brine, dried over sodium sulfate, filtered,
and evaporated under reduced pressure to give a yellow oil. The crude product was purified by
open column chromatography (CH2Cl2/ethyl acetate 9:1) to afford 4a as a white solid (52.0 mg, 35%).
1H-NMR (400 MHz, CDCl3),
δ (ppm): 7.813–7.696 (m, 2H), 7.506–7.288 (m, 3H) 4.613–4.460 (m, 1H),
4.121–3.900 (m, 1H), 2.239–1.240 (m, 8H). ESI-HRMS (m/z): Calculated for C13H15NNaO+ [M + Na]+:
225.1079. Found: 225.1081.
N-(p-Toluoyl)-7-azabicyclo[2.2.1]heptane (4d). Trans-4-aminocyclohexyl p-toluensulfonate hydrobromide
(1 g, 7.5 mmol) was dissolved in ethanol (110 mL) and water (30 mL), and then NaOH 1 M (25 mL)
was added. The solution was stirred at room temperature for 20 h, and then quenched by adding
concentrated HCl 4 M (5 mL). Stirring was continued for 20 min at r.t., and then the solution was
evaporated under reduced pressure. NaOH 10% (10 mL) was added to the residue. Free amide
was extracted with ether (3
×
30 mL), then HCl in 1,4-dioxane (1 mL) was added, and the mixture
was evaporated. The residue was redissolved in dry CH2Cl2 (30 mL). DIPEA (2 mL, 9.3 mmol) was
added to the resulting solution, and the mixture was stirred for 10 min at 0 ◦C. Then, p-tolyl benzoyl
chloride (1.6 mL, 9.1 mmol) was slowly added. Stirring was continued for 30 min. The ice bath was
removed, and the mixture was allowed to warm to r.t., and further stirred for 4 h, then quenched
by pouring it into water. The aqueous and organic layers were separated, and the aqueous phase
was extracted with dichloromethane (3
brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give a yellow
×
30 mL). The combined organic phase was washed with
oil. The crude product was purified twice by open column chromatography (n-hexane/ethyl acetate

Molecules 2018, 23, 2363
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(1:1) and dichloromethane/ethyl acetate (9:1)) to afford 4d as a yellow solid. Recrystallization afforded
◦
transparent crystals (238.6 mg, 11%). M.p.: 105–107 C. 1H-NMR (400 MHz, CDCl3),
δ (ppm): 7.449 (d,
J = 8 Hz, 2H), 7.188 (d, J = 8Hz, 2H), 4.720 (br. s, 1H), 4.148 (br. s, 1H), 2.377 (s, 3H), 1.899–1.810 (s, 4H),
1.508–1.466 (m, 4H). 13C-NMR (100 MHz, CDCl3),
δ (ppm): 169.03, 140.72, 133.50, 128.96, 127.97, 77.36,
30.60, 28.87, 21.56. ESI-HRMS: Calculated for C14H17NNaO+ [M + Na]+: 238.1202. Found: 238.1217.
Anal. Calcd. for C14H17NO2: C, 78.10; H, 7.96; N, 6.51. Found: C, 77.76; H, 7.90; N, 6.58.
Synthesis of N-Benzoyl-1-(Methoxymethyl)-7-azabicyclo[2.2.1]heptanes
N-Benzoyl-1-(Methoxymethyl)-7-azabicyclo[2.2.1]heptane (5a). Boc-protected 1-(methoxymethyl)-7-
azabicyclo[2.2.1]heptane (114.6 mg, 0.47 mmol) was dissolved in a 1:1 mixture of dry CH2Cl2 (1 mL)
and TFA (1 mL), and the solution was stirred at room temperature for one h. The solvent was removed
in vacuo, and the residue was dissolved in CH2Cl2. The resulting solution was evaporated again (a total
of three times). Finally, the residue was dissolved in dry CH2Cl2 (5 mL) and DIPEA (130
was added to the solution. The mixture was stirred for 10 min at 0 C, then benzoyl anhydride
µ
l, 0.61 mmol)
◦
(170 µL, 0.9 mmol) was slowly added. Stirring was continued for 30 min. The ice bath was removed,
and the mixture was allowed to warm to r.t., then quenched by pouring it into water. The aqueous
and organic layers were separated, and the aqueous phase was extracted with dichloromethane
(
3 × 10 mL). The combined organic phases were washed with brine, dried over sodium sulfate, filtered,
and evaporated under reduced pressure to give a yellow liquid. The crude product was purified by
open column chromatography (CH2Cl2/ethyl acetate 9:1) to afford 5a as a white solid (520.0 mg, 35%).
◦
M.p.: 53–55 C. 1H-NMR (400 MHz, CDCl3),
δ (ppm): 7.524–7.512 (m, 2H), 7.437–7.342 (m, 3H), 4.225 (s,
2H), 4.106 (t, J = 5 Hz, 1H), 3.425 (s, 3H), 2.013–1.932 (m, 2H), 1.867–1.771 (m, 2H), 1.728–1.667 (m,
2H), 1.487–1.425 (m, 2H). HRMS (ESI-TOF): [M + Na]+ Calcd. for C15H19NNaO2+ [M + Na]+: 268.1308.
Found: 268.1312.
N-(p-Chlorobenzoyl)-(1-(methoxymethyl)-7-azabicyclo[2.2.1]heptanes (5b). To a solution of the
corresponding Boc-protected 1-(methoxymethyl)-7- azabicyclo[2.2.1]heptane (41.2 mg) in dry CH2Cl2
(2 mL) was added 3 mL of TFA. The solution was stirred at room temperature for 2 h The organic
solvent was evaporated, and the residue was dissolved in 10 mL of CH2Cl2. Triethylamine (93.6
µ
L)
◦
was added at 0 C, followed by p-chlorobenzoyl chloride (58.8 mg). The reaction mixture was stirred for
2 h at 0 ◦C, and then poured into saturated aqueous NaHCO3. The whole was extracted with CH2Cl2
(30 mL
×
3). The combined organic layer was dried over Na2SO4, and the solvent was evaporated.
1
Column chromatography (n-hexane:ethyl acetate = 4:1) of the residue gave 5b (47.8 mg, 77%). H-NMR
(400 MHz, CDCl3),
δ (ppm): 7.489 (2H, d, J = 8.4 Hz), 7.356 (2H, d J = 8.0 Hz), 4.198 (2H, s), 4.087 (1H,
m), 3.423 (3H, s), 2.076–1.571 (6H, m), 1.568–1.369 (2H, m). 13C-NMR (100 MHz, CDCl3),
δ (ppm):
169.11, 136.37, 135.45, 129.20, 128.43, 73.91, 68.03, 61.37, 59.34, 32.65, 29.59. HRMS (ESI-TOF): [M + Na]+
Calcd. for C15H18ClNNaO2 [M + Na]+: 302.0918. Found: 302.0924. Anal. Calcd. for C15H18ClNO2: C,
+
64.40; H, 6.49; N, 5.01. Found: C, 64.27; H, 6.52; N, 4.92.
N-(p-Nitrobenzoyl)-(1-(Methoxymethyl)-7-azabicyclo[2.2.1]heptanes (5c). Boc-protected 1-(methoxymethyl)-
7-azabicyclo[2.2.1]heptane (119.3 mg, 0.49 mmol) was dissolved in a 1:1 mixture of dry CH2Cl2 (1 mL)
and TFA (1 mL), and the solution was stirred at room temperature for one h. The solvent was removed
in vacuo, then the residue was dissolved in CH2Cl2 and this solution was evaporated again (a total of
three times). Finally, the residue was dissolved in dry CH2Cl2 (5 mL), DIPEA (132 µL, 0.63 mmol) was
added to it, and the mixture was stirred for 10 min at 0 ◦C. Then, p-nitrobenzoyl chloride (91.70 mg,
0.6 mmol) was slowly added, and stirring was continued for 30 min. The ice bath was removed,
and the mixture was allowed to warm to r.t. Stirring was continued for 5.5 h, and then the mixture was
quenched by pouring it into water. The aqueous and organic layers were separated, and the aqueous
phase was extracted with dichloromethane (3
brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give a a yellow
×
10 mL). The combined organic phase was washed with
liquid. The crude residue was purified by open column chromatography (n-hexane/ethyl acetate 1:1)
to afford 5c as a white solid (63.9 mg, 45%). m.p.: 140–142 ◦C. 1H-NMR (400 MHz, CDCl3),
δ (ppm):

Molecules 2018, 23, 2363
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8.256 (dd, J = 2 Hz, 6.8 Hz, 2H), 7.685 (dd, J = 2 Hz, 6.8 Hz, 2H), 4.178 (s, 2H), 4.010 (t, J = 4.4 Hz,
1H), 3.419 (s, 3H), 2.003–1.961 (m, 2H), 1.873–1.783 (m, 2H), 1.750–1.689 (m, 2H), 1.549–1.487 (m, 2H).
13C-NMR (100 MHz, CDCl3),
δ
(ppm): 167.52, 148.90, 143.12, 128.68, 123.74, 73.64, 68.66, 61.24, 59.46,
32.79, 29.71. HRMS (ESI-TOF): [M + Na]+ Calcd. for C15H18N2NaO4 [M + Na]+: 313.1159. Found:
+
313.1169. Anal. Calcd. for C15H18N2O4: C, 62.06; H, 6.25; N, 9.65. Found: C, 61.92; H, 6.28; N, 9.63.
3.3. Kinetic Studies
1
Kinetic data were collected by recording the H-NMR (400 MHz) spectra at 2- min intervals.
◦
Data were obtained at 70.0 C under a N2 flow of 400 L/h. Deuterated solvents were used for all
measurements: Deuterium oxide, 99.9% D from Wako; methanol-d4, 99.8% D from Kanto Chemical
Co. Inc.; 1,4-dioxane-d8, 99% D from Cambridge Isotope Laboratories, Inc.; and sodium deuteroxide,
40% wt. % in D2O, 99.5% D from Sigma-Aldrich, Co. Parameters were set using the reaction solution at
room temperature. Reactions were initiated by raising the temperature of the NMR machine. Spectral
data were then recorded for at least one half-life. Hydrolysis experiments were repeated three times.
1
Hydrolysis products were identified by comparison of the H-NMR data with those of authentic
samples, or by MS-ESI. Pseudo-first-order rate constants (kobs) were evaluated by a linear least-squares
fitting of a plot of the logarithm of reactant concentration versus time.
3.4. Computational Studies
Computational studies were carried out using the Gaussian 09 software package [34]. Geometry
optimizations for the model molecules were performed at the M06-2X/6-31+G(d) level with the bulk
solvent model SMD (solvent = water) together with explicit water molecules. Vibrational frequency
calculations were performed at the same level of theory. Optimized geometries were verified by
frequency calculations as minima (zero imaginary frequencies) or transition structures (TS, a single
imaginary frequency) (the coordinates, frequency and thermodynamic values are shown in Supporting
Information). Intrinsic reaction coordinate (IRC) computations of the transition structures verified the
reactants and products in the case of simple amide (formamide and acetamide (data not shown)). In the
cases of more realistic substrates (2a–5a), particularly in combination of the solvent model, the IRC
calculations were unsuccessful. In all the transition states the validity of transition state structures
was confirmed by inspecting the direction of vibration of the negative frequency, which matched the
trajectory of the nucleophilic attack of the hydroxy anion onto the carbonyl carbon atom. Geometry
minimization of the transition structures lead to the hydroxide-addition adducts, which also supported
the validity of the transition state structures, which represented the trajectory of the nucleophilic attack
of the hydroxy anion. We also estimated Gibbs free energies by single-point calculations (frequency
calculations) with M06-2X//6-311++G(d,p) for the M06-2X//6-31+G(d)-optimized structures. In these
single-point frequency calculations, a single negative frequency was found respectively, which also
corresponded to the trajectory of the nucleophilic attack of the hydroxy anion onto the carbonyl carbon
atom. The energies were corrected for zero-point energies and Gibbs free energy at 25 ◦C (298.15 K),
obtained from frequency calculations. Calculations at the B3LYP level showed similar trends to the
M06-2X calculations.
Identification of the transition states structures of the attack of the hydroxide anion to the carbonyl
carbon atom of the amide group was carried out in the following multiple procedures: The transition
state structures of the attack of the hydroxide anion to the carbonyl carbon atom of the amide were
first identified in the absence of the explicit water molecules (data not shown). Addition of explicit
water molecules in the arrangement similar to Types a and b (Figure 11) [32], followed by geometry
optimization (OPT = TS). Or we reproduced the TSs of the hydrolysis of formamide and acetamide
in the presence of four and five water molecules, respectively, which were consistent with the two
hydrogen network models (Types a and b, Figure 11), reported in the previous literature [32]. Then the
amide functionality was morphed into the realistic amide substrates (2a
–5a). The initial minimum
conformation of the neutral amide substrates (2a 5a) were obtained by conformational search in
–

Molecules 2018, 23, 2363
20 of 22
Marcomodel software (Schrödinger, LLC, New York, NY, USA), followed by optimization with the
DFT methods. The Type a arrangement of waters (Figure 11) did not converge to the optimization or
transformed to the Type b arrangement of water molecules. Therefore, we discussed the activation
energies on the basis of the Type b hydrogen network model, while the Type
model might be the energy minimum (see the main text).
a hydrogen network
The change of Gibbs free energy for the reaction was calculated on the basis of the reaction model,
shown in Scheme 6. The results of the present calculations (Table 4) were reasonable and reliable,
because the present calculations gave the activation energies of similar magnitude to the cases of simple
amide: The calculated activation energies of the present amides 2a–5a were in similar magnitude to
the calculated and experimental Gibbs free energy change of hydroxide anion-catalyzed hydrolysis of
formamide, 21–22 kcal/mol [32], (see also Table 4, footnote a).
4. Conclusions
Herein we measured and compared the kinetics of base-catalyzed hydrolysis of non-planar
N-benzoyl-7-azabicyclo[2.2.1] heptane amides (
4
) and related compounds (
2
,
3
and
5
) under
◦
pseudo-first-order conditions at 70 C. Excess sodium deuteroxide was used as the base in two different
solvent systems (methanol or 1,4-dioxane in a 1:1 D2O solution). Reaction progress was monitored
by 1H NMR spectroscopy. Unexpectedly we found that 7-azabicyclo[2.2.1]heptane amides (
4) were
resistant to base-catalyzed hydrolysis. As a general trend, independently of whether methanol or
1,4-dioxane was used as a co-solvent, it was found that the reactivity of nitrogen-pyramidal azetidine
amides (2) was greater than that of the planar pyrrolidine amides (3), followed by unsubstituted
bicyclic amides (
4
), while bridgehead-substituted bicycles ( ) were the least reactive (reaction rate:
5
2a > 3a > 4a > 5a, etc.). Phenyl substituents showed the expected electronic trends. We also executed
DFT calculations of the rate-determining process, addition of the hydroxide anion to the amide carbonyl
group, and found that the experimental kinetic data is consistent with the magnitude of the calculated
Gibbs free energies of activation. Our results confirm the stability of the 7-azabicyclo[2.2.1]heptane
◦
amides at least at 37 C. This is important, because it implies that the 7-azabicyclo[2.2.1]heptane amide
scaffold is available for practical molecular design, e.g., as an amino acid surrogate.
Supplementary Materials: The following are available online, Supporting Information: Raw kinetic data
(Figures S1 and S2), additional calculation data (Tables S1–S5), calculation coordinates, NMR spectra
(Figures S3–S5).
Author Contributions: D.A.O.G.d.V.: synthesis of compounds and kinetics measurement, data analysis;
A.S.: synthesis of compounds; L.Z.: synthesis of compounds; S.K.: synthesis of compounds; Y.O.: plotting,
synthesis, data analysis, funding, writing manuscript; T.O.: plotting, data analysis, calculations, funding,
and writing manuscript.
Funding: This work was supported by JSPS KAKENHI Grant Numbers JP 26104508 and 16K08157 (Y.O.) and
26293002 and 18H02552 (T.O.).
Acknowledgments: The computations were performed at the Research Center for Computational Science,
Okazaki, Japan. D.A.O.G.d.V. and A.S. were supported by Japanese Government (MEXT) scholarship.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).