Synthesis and preliminary biological evaluations of (+)-isocampholenic acid-derived amides

Article abstract of DOI:10.1007/s11030-016-9668-9

The synthesis of two novel (+)-isocampholenic acid-derived amines has been realized starting from commercially available (1S)-(+)-10-camphorsulfonic acid. The novel amines as well as (+)-isocampholenic acid have been used as building blocks in the constru

Full text of DOI:10.1007/s11030-016-9668-9

Mol Divers
DOI 10.1007/s11030-016-9668-9
ORIGINAL ARTICLE
Synthesis and preliminary biological evaluations of
(+)-isocampholenic acid-derived amides
Uroš Grošelj1 · Amalija Golobicˇ1 · Damijan Knez2 · Martina Hrast2 ·
Stanislav Gobec2 · Sebastijan Ricˇko1 · Jurij Svete1
Received: 24 January 2016 / Accepted: 12 March 2016
© Springer International Publishing Switzerland 2016
Abstract The synthesis of two novel (+)-isocampholenic
acid-derived amines has been realized starting from com-
mercially available (1S)-(+)-10-camphorsulfonic acid. The
novel amines as well as (+)-isocampholenic acid have been
used as building blocks in the construction of a library of
amides using various aliphatic, aromatic, and amino acid-
derived coupling partners using BPC and CDI as activating
agents. Amide derivatives have been assayed against sev-
eral enzymes that hold potential for the development of
new drugs to battle bacterial infections and Alzheimer’s dis-
ease. Compounds 20c and 20e showed promising selective
sub-micromolar inhibition of human butyrylcholinesterase
to consider camphor and its commercially available deriv-
atives as starting materials for the preparation of easily
available building blocks such as acids and amines. These can
be conveniently used for the construction of a diverse library
of compounds by means of simple amide bond formation.
Camphor-based compounds have been reported for several
biological actions such as antibacterial [1,2] and antiviral
[3] activity. In addition, camphor is available in both enan-
tiomeric forms. Using either form as a starting material,
enantiomerically pure compounds can be synthesized. Out
of the broad plethora of chemical transformations associated
with camphor [4,5], we decided to investigate the applica-
tion of camphor-derived acids 1 and 2 and their amines 3–5
(Fig. 1) for the construction of a library of amides. Addi-
tionally, the presence of the double bond enables a multitude
of further chemical transformations. A SciFinder search [6]
(as of January 2016) revealed 31 amides derived from 1–5
building blocks. Moreover, only one urea and no sulfon-
amides derived from amines 3–5 were reported. In 1956,
Kagawa studied the influence of alkaline substances on 10-
bromocamphor leading to Grob fragmentation products, i.e.
derivatives of acids 1 and 2 as well as many other basic sys-
tematic transformations and interconversions [7,8]. On the
other hand, Money and co-workers applied Grob fragmenta-
tion reactions of suitably functionalized 10-bromocamphor
derivatives for the preparation of various natural products
and their analogues [4,5,9]. (+)-Isocampholenic acid (1) is
conveniently prepared from 10-bromo- or 10-iodocamphor
[10] by treatment with KOH via Grob [11] fragmentation
[12]. (+)-α-Campholenic acid (2) [13], on the other hand,
can easily be prepared via Retro-Prins fragmentation of (S)-
(+)-camphorsulfonic acid [14] by KOH fusion at 200–220◦C
for 5 min [15]. A literature search for amines 3–5 derived
from acids 1 and 2 revealed only the preparation of amine
3 via the Curtius rearrangement of acid 2 using DPPA [15].
(hBChE)(IC50 values 0.80
0.05 and 0.25
0.02 μM,
respectively).
Keywords 10-Iodocamphor · Grob fragmentation ·
Curtius rearrangement · Camphor-derived amines ·
Biological evaluation · Butyrylcholinesterase inhibitor
Introduction
The ever present need for new diverse scaffolds used for the
development of biologically active compounds prompted us
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-016-9668-9) contains supplementary
material, which is available to authorized users.
B
Uroš Grošelj
uros.groselj@fkkt.uni-lj.si
1
Faculty of Chemistry and Chemical Technology, University
of Ljubljana, Vecˇna pot 113, 1000 Ljubljana, Slovenia
2
Faculty of Pharmacy, University of Ljubljana, Aškercˇeva 7,
1000 Ljubljana, Slovenia
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Fig. 1 Building blocks 1–5
suitable for amide library
construction
Surprisingly, amines 4 and 5 have not been prepared so far
(Fig. 1).
because the concomitant deprotection (by acidolysis or cat-
alytic hydrogenation) would compromise the exocyclic C=C
bond. Treatment of methyl carbamate 10 with KOH in aque-
ous methanol furnished the second building block, amine 5,
in 72% yield (Scheme 1).
Herein we report on the preparation of amines 4 and 5
from (+)-isocampholenic acid (1) and a library of 35 amides
derived from acid 1 and amines 4 and 5 (Fig. 1). All the
reported amides have been assayed against several enzymes,
where several amide derivatives displayed promising selec-
tive inhibition of human butyrylcholinesterase (hBChE), an
emerging target in Alzheimer’s disease therapy [16,17].
Acetic acid derivative 1 was the first building block
used for the preparation of a library of amides. Following
our previous successful applications of BPC as an acti-
vating agent for acids, i.e. for the formation of activated
pentafluorophenyl esters for the peptide bond formation
[18–20], 1 was treated with BPC in the presence of Et3N
in anhydrous MeCN. The in situ formed reactive ester 11
was coupled with a variety of aliphatic amines 12a–k,
thus furnishing the corresponding amides 13a–k in 59–
91% isolated yields. The selected method of activation of
acid 1 was not suitable for the formation of an amide
bond with less nucleophilic (hetero)aromatic amines. An
alternative way to obtain the amides 13 via the Grob
fragmentation of 10-iodocamphor (7) with amines was
explored as well. However, treatment of 7 with primary
aliphatic amines in DMSO at elevated temperatures did
not give carboxamides 13, but rather mixtures of prod-
ucts. Only the reaction of 7 with n-butylamine (12l) fur-
nished the Grob fragmentation product 13l in 11% yield
(Scheme 2).
Next, amines 4 and 5 were used as the building blocks for
the preparation of the corresponding amide libraries 16a–
j and 17a–n, respectively. Carboxylic acids 1 and 14a–n
were first activated using 1,1ꢀ-carbonyldiimidazole (CDI),
a known robust activating agent for peptide bond forma-
tions and β-keto ester synthesis [21,22]. In addition, CDI
and its by-product imidazole did not interfere with the chro-
matographic isolation of rather non-polar amides of type 16
and 17, which was not the case when BPC was applied.
Thus, treatment of acids 1 and 14 with CDI in anhydrous
MeCN gave in situ corresponding reactive acyl imidazoles
15, followed by amide bond formation upon the addition
of amine 4 or 5, furnishing amides 16a–j and 17a–n in
61–98 and 61–99% yields, respectively. Reaction of 16i
Results and discussion
Thepreparationofaminebuildingblocks4and5commenced
from commercially available (1S)-(+)-10-camphorsulfonic
acid (6). Thus, following a slightly modified literature pro-
cedure [10], treatment of 6 with PPh3/I2 in toluene under
thermal conditions afforded 10-iodocamphor (7) in 62%
yield and 82% purity; the O=PPh3 side-product (18% yield)
did not interfere with the upcoming transformation. Next, 10-
iodocamphor (7) was subjected to the base-induced (KOH)
Grob fragmentation, yielding the desired acid 1 [12] in 76%
yield. Amine 4 was prepared in two steps from the acid
1. First, in situ activation of 1 with bis(pentafluorophenyl)
carbonate (BPC) followed by the addition of excess aque-
ous ammonia gave primary amide 8 [7] in 97% yield.
Kagawa reported the preparation of amide 8 by the treat-
ment of 10-bromocamphor with ammonia in MeOH in an
autoclave at elevated temperature [7]. Next, LiAlH4 reduc-
tion of 8 furnished the desired building block 4 in 42%
yield. Diphenylphosphoryl azide (DPPA)-mediated Curtius
rearrangement of acid 1 was used for the preparation of amine
5. Thus, reaction of 1 with DPPA in the presence of Et3N in
anhydrous toluene yielded in situ isocyanate A. Addition of
aqueous NaOH to isocyanate A failed to give amine5, instead
affording urea 9 in 14% yield. On the other hand, addition
of anhydrous MeOH to in situ made isocyanate A furnished
methyl carbamate 10, which was isolated in 71% yield. The
formation of benzyl or tert-butyl carbamate was not an option
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Scheme 1 Synthesis of amine
building blocks 4 and 5
Scheme 2 Formation and yields
of amides 13a–l
with meta-chloroperoxybenzoic acid (MCPBA) furnished an
inseparable mixture of epoxides 18 and 18ꢀ in a 76:24 ratio
and in 67% yield (Scheme 3).
After the initial screening in several assay systems,
selected amides of type 16 and 17 with the most promising
hBChE inhibitory potencies (Table 1) have been selectively
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Scheme 3 Formation and yields of amides 16a–j and 17a–n and epoxidation of amide 16i
N-Boc, O-Boc, or N-Cbz deprotected in order to evaluate
the inhibitory activities of their more polar counterparts. For
the removal of the Cbz protecting group, standard Pd-C cat-
alytic hydrogenolysis in MeOH was applied. The exocyclic
C=C bond of compounds 16 and 17 was reduced as well,
which resulted in the formation of inseparable mixtures of
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Table 1 Initial screening of amides of type 13a, 16, and 17
Compound
hBChE
mAChE
Compound
hBChE
mAChE
IC50 SEM (μM) or
%RA at 10 μM SEM
RA at 10 μM SEM
IC50 SEM (μM) or
%RA at 10 μM SEM
RA at 10 μM SEM
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
17a
17b
a
5.43 0.35
2.49 0.22
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
17c
17d
17e
17f
1.94 0.12
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
17g
17h
17i
2.61 0.24
n.a.b
55.1 1.2 %
n.a.b
2.81 0.35
9.11 1.58
n.a.b
5.95 0.68
66.4 9.9 %
n.a.b
62.6 9.4 %
50.5 2.4 %
60.4 1.9 %
n.a.b
17j
17k
17l
n.a.b
n.a.b
n.a.b
n.a.b
8.67 1.94
n.a.b
n.a.b
3.27 0.46
n.a.b
17m
17n
n.a.b
52.8 7.9 %
Compounds 13a–k were not active on hBChE and mAChE (residual activities—RA at 10 μ M compound concentration > 70 %)
n.a. not active (RA at 10 μM > 70 %). Tacrine was used as a positive control (IC50 (hBChE) = 0.023 0.003 μM; IC50 (hBChE) = 0.131
b
0.003 μM)
Scheme 4 Selective Cbz or Boc
deprotection
diastereomers in 69–84% yields, with the cis-(S)-isomers
19 being the major diastereomer in approximately 62–87%
purity. The formation of the major cis-(S)-isomers 19 is
in accordance with the approach of the hydrogen from the
sterically less hindered Si-face of the double bond. In the
respective 13C-NMR spectra of the hydrogenated products
19, in all cases, more than two sets of the expected sig-
nals are observed, i.e. of the major cis-(S)-isomers 19 and
minor trans-(S)-isomers 19, even for the glycine deriva-
tives 19a and 19d. This could be due to the presence of
rotamers and/or because of the partial epimerization of the
amino acid part of the molecule, which would generate two
new minor diastereomers, i.e. the trans-(R)- and the cis-
(R)-isomers 19 (Scheme 4, Table 2). Compounds 19 have
therefore been tested for their inhibitory activities only as
mixtures of diastereomers. On the other hand, acid medi-
ated Boc deprotection of 16 and 17 using trifluoroacetic acid
(TFA) in CH2Cl2 furnished compounds 20a–f in 51–88%
yields, respectively. The formation of products of type 20 can
easily be explained by the initial protonation of the exocyclic
C=C bond, followed by the 1,2-methyl group migration and
the final deprotonation [23], which resulted in the formation
of thermodynamically most stable tetra-substituted alkenes
20a–f accompanied with the loss of one of the two stere-
ogenic centres (Scheme 4, Table 2).
The structures of novel compounds 4, 5, 9–11, 13, and 16–
20 were determined by spectroscopic methods (1H-NMR,
13C-NMR, 2D-NMR, IR, and HRMS) and by elemental
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Table 2 Yields and inhibitory
activities against hBChE and
mAChE of deprotected amides
19 and 20
Entry
1
Compound
Yield (%)
84
hBChE
IC50 SEM (μM) or
%RA at 10 μM
mAChE
RA at 10 μM SEM
SEM
n.a.a
n.a.a
19a
19b
2
3
79
74
n.a.a
n.a.a
n.a.a
n.a.a
19c
4
5
6
69
81
76
n.a.a
n.a.a
n.a.a
n.a.a
n.a.a
n.a.a
19d
19e
19f
20a
20b
7
8
51
84
3.32 0.28
1.09 0.04
n.a.a
n.a.a
9
81
78
0.80 0.05
12.03 1.35
n.a.a
n.a.a
20c
20d
10
11
63
88
0.248 0.02
n.a.a
n.a.a
20e
20f
12
1.71 0.14
a
n.a. not active (RA at 10 μM > 70 %)
analyses for C, H, and N. Compounds 8, 13c, f, j, 16g, and
17c, g, n were obtained in analytically pure form. The iden-
tities of compounds, not obtained in analytically pure form,
were confirmed by 13C-NMR and HRMS. The identities of
compounds13f, 13g, and17nwereadditionallyconfirmedby
single-crystal X-ray analyses (Figs. 2, 3, 4). For compounds
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Fig. 2 ORTEP representation
of single-crystal X-ray structure
of compound 13f
Fig. 3 ORTEP representation
of single-crystal X-ray structure
of compound 13g
Fig. 4 ORTEP representation
of single-crystal X-ray structure
of compound 17n
13d, 13g, 16g, and 17k, rotamers were observed. Com-
pounds 19a–f were isolated and characterized as mixtures
of stereoisomers; the major isomer being cis-(S)-isomers
19a–f.
Camphor-derived compounds have previously been re-
ported for their antimicrobial activity [1,2]; however, their
precise targets have not been established. Prepared amides
13, 16, 17, and their deprotected counterparts 19 and 20,
have therefore been assayed for their inhibitory potential
on several bacterial peptidoglycan biosynthesis enzymes
(d-Ala-d-Ala ligase B (DdlB), MurA, MurC, and MurD).
None of the analogues displayed notable inhibitory poten-
tial against these enzymes (residual activities at 100 μM >
50 %). These amides were also screened for hBChE inhibi-
tion (Tables 1 and 2), where several compounds displayed
IC50 values lower than 10 μM, with compounds 20c and 20e
being the most potent sub-micromolar inhibitors of hBChE
(IC50 values 0.80 0.05 μM and 0.25 0.02 μM, respec-
tively). These compounds are selective inhibitors of hBChE
with selectivity index (SI) >10 (compared to murine acetyl-
cholinesterase (mAChE), SI defined as ratio between IC50
against mAChE and hBChE). They represent a solid starting
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point for development of new selective BChE inhibitors that
could further be used as molecular probes in the investigation
of BChE role in Alzheimer’s disease.
DdlB: 50 mM Hepes, pH 8.0, 5 mM MgCl2, 6.5 mM(NH4)2
SO4, 10 mM KCl, 0.005% Triton X-114, 700 μM d-Ala,
500 μM ATP, purified DdlB, and 100 μM of each tested
compound dissolved in DMSO.
All compounds were soluble in the assay mixtures con-
taining 5% DMSO (v/v). After incubation for 15 min
at 37 ◦C, the enzyme reaction was terminated by adding
Conclusions
ꢀ
Biomol reagent (100 μL) and the absorbance was mea-
The Grob fragmentation of 10-iodocamphor (7) furnished the
keyintermediate(+)-isocampholenicacid(1)whichwasused
for the preparation of two novel chiral non-racemic amines 4
and 5. Amide derivatives 13, 16, and 17 were easily prepared
from camphor-derived building blocks 1, 4, and 5, respec-
tively, using robust activating agents like BPC and CDI. In
addition, amides 16 and 17 were selectively N-Boc, O-Boc,
or N-Cbz deprotected. Libraries of amides 13, 16, 17, 19, and
20 were assayed for their inhibitory potential against several
bacterial peptidoglycan biosynthesis enzymes (d-Ala-d-Ala
ligase B (DdlB), MurA, MurC, and MurD) that hold potential
for the development of new drugs to battle bacterial infec-
tions and Alzheimer’s disease. Two of them (20c and 20e)
showed encouraging selective sub-micromolar inhibition of
hBChE (IC50 values 0.80 0.05 μM and 0.25 0.02 μM,
respectively).
sured at 650 nm after 5 min. All of the experiments were run
in duplicate. Residual activities (RAs) were calculated with
respect to similar assays without the tested compounds and
with 5% DMSO.
Inhibition of hBChE and mAChE
The inhibitory activities were determined using the method
of Ellman [25]. Reagents (5,5ꢀ-Dithiobis (2-nitrobenzoic
acid), DTNB, butyrylthiocholine iodide, and acetylthio-
choline iodide) were purchased from Sigma-Aldrich. Stock
solutions of mAChE and recombinant hBChE at the con-
centrations of 4.6 mg/mL in 10 mM MES buffer, pH 6.5,
were kindly provided by Florian Nachon (IBS, Grenoble,
France). The reactions were carried out in a final vol-
ume of 300 μL of 0.1 M phosphate-buffered solution, pH
8.0, containing 370 μM DTNB, 500 μM butyrylthiocholine/
acetylthiocholine and 1 nM or 50 pM huBChE or mAChE,
respectively. The reactions were carried out at room temper-
ature. The formation of the yellow 5-thio-2-nitrobenzoate
anion was monitored for 1 min as the change in absorbance
at 412 nm, using a 96-well microplate reader (Synergytm
H4, BioTek Instruments, Inc., USA). The initial velocity (v0)
was calculated from the slope of the linear trend obtained,
with each measurement carried out in triplicate. To determine
the blank value (b), phosphate-buffered solution replaced
the enzyme solution. For the inhibitory screening, the com-
pounds were first diluted in DMSO at 1mM concentration
and added to each well at a final concentration of 10 μM. The
final content of the organic solvent (DMSO) was always 1%.
The enzyme and inhibitor have been preincubated for 300
s, to allow complete equilibration of the enzyme–inhibitor
complexes. The reactions were then started by addition of
the substrate. The initial velocities in the presence of the test
compounds(vi)werecalculated. Theinhibitorypotenciesare
expressed as the residual activities (RA = (vi −b)/(vo −b)).
For the IC50 determination, seven different concentrations
of each compound were used. The residual enzyme activi-
ties were plotted against the applied inhibitor concentrations,
and IC50 values determined by fitting the experimental data
to Equation:
Experimental
Biological evaluation
The inhibitory potency of these compounds against pure
enzymes was determined as described below. Suitable pos-
itive controls were used with inhibitory potencies in accor-
dance with reported values.
Inhibition of Mur enzymes and DdlB
The inhibition of Mur enzymes was monitored with the col-
orimetric malachite green method in which orthophosphate
generated during reaction is measured [24]. The mixtures
with final volume of 50 μL contained the following:
MurA: 50 mM Hepes, pH 7.8, 0.005% Triton X-114, 200 μM
UNAG, 100 μM PEP, purified MurA and 100 μM of each
tested compound dissolved in DMSO.
MurC: 50 mM Hepes, pH 8.0, 5 mM MgCl2, 0.005% Triton
X-114, 120 μM L-Ala, 120 μM UM, 450 μM ATP, purified
MurD, and 100 μM of each tested compound dissolved in
DMSO.
MurD: 50 mM Hepes, pH 8.0, 5 mM MgCl2, 0.005% Triton
X-114, 100 μM D-Glu, 80 μM UMA, 400 μM ATP, purified
MurD, and 100 μM of each tested compound dissolved in
DMSO.
Y = Bottom + (Top − Bottom)/(1 + 10ˆ((LogIC50 − X)
× HillSlope)),
123

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where X is the logarithm of the inhibitor concentration and
Y is the residual activity. For the fitting procedure, Gnuplot
software and an in-house python script were used.
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α
Cu K radiation (1.54184 Å) for 17n, respectively. The
diffraction data were processed using CrysAlis PRO soft-
ware [26]. All structures were solved by direct methods,
using SIR97 [27]. A full-matrix least-squares refinement on
F2 was employed with anisotropic displacement parameters
for all non-hydrogen atoms. H atoms were placed at calcu-
lated positions and treated as riding. SHELXL97 software
[28] was used for structure refinement and interpretation.
Drawings of the structures were produced using ORTEP-3
[29]. Structural and other crystallographic data have been
deposited with the Cambridge Crystallographic Data Cen-
tre as supplementary publication numbers CCDC 1437236 –
CCDC 1437238, for 13f, 13g, and 17n, respectively. These
data can be obtained free of charge via www.ccdc.cam.ac.uk/
conts/retrieving.html (or from the CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk).
Acknowledgments ThefinancialsupportfromBoehringer-Ingelheim
Pharma (Biberach, Germany) and from the Slovenian Research Agency
through grants P1-0179 and L1-6745 is gratefully acknowledged. We
thank Florian Nachon (IBS, Grenoble, France) for providing mAChE
and recombinant hBChE, Ian Chopra (University of Leeds, Leeds, UK)
for providing DdlB and Delphine Patine and Hélène Barreteau (I2BC,
Université Paris-Sud, Orsay, France) for providing MurA, MurC, and
MurD. We also thank to EN-FIST Centre of Excellence (Ljubljana,
Slovenia) for using the SuperNova diffractometer.
18. Lombar K, Grošelj U, Dahmann G, Stanovnik B, Svete J (2015)
Synthesis of 6-Alkyl-7-oxo-4,5,6,7-tetrahydropyrazolo[1,5-
c]pyrimidine-3-carboxamides. Synthesis 47:497–506. doi:10.
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19. Grošelj U, Pušavec E, Golobicˇ A, Dahmann G, Stanovnik B, Svete
J (2015) Synthesis of 1,5-disubstituted-4-oxo-4,5-dihydro-1H-
pyrazolo[4,3-c]pyridine-7-carboxamides. Tetrahedron 71:109–
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