Allobetulin derived seco-oleananedicarboxylates act as inhibitors of acetylcholinesterase

Article abstract of DOI:10.1016/j.bmcl.2015.04.086

Ring opening of allobetulone gave either seco-acid 8 or di-acid 4. These acids were converted into esters that were screened by Ellman's assay. A dibutenylester of low cytotoxicity (NIH 3T3 murine embryonic fibroblasts) was shown to be a good mixed-type inhibitor (K_i = 3.39, K_i′ = 2.26 μM) for acetylcholinesterase.

Full text of DOI:10.1016/j.bmcl.2015.04.086

Accepted Manuscript
Allobetulin derived seco-oleananedicarboxylates act as inhibitors of acetylcho-
linesterase
Lucie Heller, Stefan Schwarz, Anja Obernauer, René Csuk
PII:
S0960-894X(15)00418-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.04.086
BMCL 22669
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
26 February 2015
23 April 2015
24 April 2015
Please cite this article as: Heller, L., Schwarz, S., Obernauer, A., Csuk, R., Allobetulin derived seco-
oleananedicarboxylates act as inhibitors of acetylcholinesterase, Bioorganic & Medicinal Chemistry Letters (2015),
doi: http://dx.doi.org/10.1016/j.bmcl.2015.04.086
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Allobetulin derived seco-oleananedicarboxylates act as inhibitors of acetylcholinesterase.
Lucie Heller, Stefan Schwarz, Anja Obernauer and René Csuk*
Martin-Luther-Universität Halle-Wittenberg, Bereich Organische Chemie, Kurt-Mothes-Str.
2, D-06120 Halle (Saale), Germany; e-mail: rene.csuk@chemie.uni-halle.de
Abstract – Ring opening of allobetulone gave either seco-acid 8 or di-acid 4. These acids
were converted into esters that were screened by Ellman’s assay. A dibutenylester of low
cytotoxicity (NIH 3T3 murine embryonic fibroblasts) was shown to be a good mixed-type
inhibitor (Ki = 3.39, Ki’ = 2.26 µM) for acetylcholinesterase.
Worldwide approximately 35 million individuals suffer from some kind of dementia; 20
million of them are affected by Alzheimer’s disease (AD), and within the next 30 years this
number will double.1-4 AD is a form of pre-senile dementia mainly diagnosed in people whose
ages are over 65, but is not exclusively an aging-related disease.5,6 A significant number of all
AD cases is known as the early onset familial AD (fAD),5 and their symptoms appear in
young age. But the vast majority of AD cases, however, are classified as sporadic AD (sAD)
being triggered by genetic as well as non-genetic factors, and its prevalence increases with
age.7-9 Regardless of their age, all of these AD patients lose their short-term memory, there is
a progressive decline in attention, languages and spatial reasoning, and finally all of these
patients will withdraw from social life, and they will be no longer able to execute daily life
activities at all.1-5
The impairment on the cholinergic function seems of critical importance in AD (“cholinergic
hypothesis”),1 and the levels of the neurotransmitter acetylcholine (ACh) are decreased in AD
brains. In addition, during the development of AD an increased activity of
butyrylcholinesterase (BChE) was found in the hippocampus and the temporal cortex.1

Several cholinesterase inhibitors are commercially available to alleviate the symptoms of AD,
and drugs such as galantamine, tacrine, rivastigmine and donepezil and derivatives thereof are
successful in slowing down the process of cognitive impairment.1
Recently, several triterpenes came into the focus of scientific interest concerning the treatment
10-18
of AD related symptoms.
In search of novel inhibitors of AChE we used Ellman’s assay
to screen our library of triterpenoids, containing > 1400 derivatives of triterpenoic acids.
While many compounds showed low or no activity at all, several triterpenoids 10, 19 especially
20
derivatives derived from allobetulin (1, Scheme 1) gave some promising results; 1 can be
regarded as a “re-arranged” betulin derivative.21 The hits from this primary screening,
however, possessed only low solubility. Probably due to this low solubility usually observed
with derivatives of allobetulin, the number of biological investigations of allobetulin remained
small over the years.22-31 Hence, we decided to search for allobetulin derivatives of improved
solubility, low cytotoxicity and significant ability to inhibit AChE.
Scheme 1. Structure of betulin and synthesis of allobetulin (1): a) p-TSA, CHCl3, reflux 1 hour, according to ref. 32, 75 %.
Parent allobetulin (1, Scheme 1) can be obtained in 75 % isolated yield from betulin by its
reaction with p-TSA.32 From the reaction of 1 with acetic acid and nitric acid a mixture of
33
mononitro-2 and dinitro-3 was obtained (Scheme 2); this mixture was easily separated by
chromatography. Compound 2 was characterized in its 13C NMR spectrum by two signals at δ
= 123.0 and 176.3 ppm that were assigned to enolic carbons C-2 and C-3, respectively. In its
IR spectrum the signals at ν = 1513 and 1376 cm-1 were attributed to the NO2 valence
vibrations. In the dinitro compound 3, the carbonyl group at C-3 was detected at δ = 197.4
ppm, while the signal of the dinitro substituted carbon C-2 was found at δ = 119.4 ppm.

Scheme 2. Synthesis of nitro-compounds 2 and 3, seco-diacid 4 and esters 5-7: a) HOAc, HNO3, 25 °C, 1 h, 70 % (of 2) and 8 % (of 3); b)
MeOH, H2O2, K2CO3, 25 °C, 5 days, acidic workup, 78 %; c) K2CO3, DMF, R-X, 25 °C, 12 h.
Reaction of 2 with potassium carbonate and hydrogen peroxide in methanol for five days gave
33, 34
seco-diacid 4
in an isolated yield of 78 %. This compound is highly insoluble in most
organic solvents. Its esterification with alkyl halides in the presence of finely powdered
potassium carbonate yielded the esters 5-7 showing improved solubility.
For comparison, the mono-acid 8 and the mono esters 10-12 were prepared starting from 1.
34
Thus, Jones oxidation of 1 furnished allobetulone (9, Scheme 3)
whose treatment with
®
oxone and conc. sulfuric acid in ethanol furnished ring opened seco-ethyl ester 10. 35 The
formation of this seco compound can be explained by a Bayer-Villiger re-arrangement of 9
followed by a transesterification of the transient lactone and an elimination reaction of a
tertiary alcohol.

Scheme 3. Synthesis of compounds 8-12: a) CrO3, H2SO4, acetone, 81 %; b) oxone®, EtOH, H2SO4, 25 °C, 7 days, 65 %; c) MeOH, KOH,
25 °C, 7 d, 87 %; d) R-X, K2CO3, DMF, 25 °C, 12 h.
Reaction of this ester 10 with methanolic potassium hydroxide for one week gave acid 8,
whose esterification with propargyl bromide or 4-bromo-1-butene in DMF as described above
gave the esters 11 and 12, respectively.
Allobetulin, monoacid 8, diacid 4 and esters 5-7 and 10-12 were subjected to Ellman’s assay
36, 37
to determine their inhibitory activity for the enzymes AChE (from Electrophorus
electricus) and BChE (from equine serum). Pre-screening showed all of these compounds (as
well as of other monoalkyl- and dialkyl-esters that were easily accessed by esterification of 4
and 8, respectively) as weak inhibitors of AChE and BChE, hence we refrained from a more
extensive screening with these enzymes. The ethyl esters 5 and 10 were weak inhibitors of
AChE; the solubility of 12 in aqueous buffers was low. However, these experiments showed 8
as a moderate competitive inhibitor for AChE with a Ki = 31.78 4.28 µM (IC50 = 103.5
9.7 µM) , while the di-propargyl ester 6 was a mixed-type inhibitor for this enzyme with Ki =
10.45 3.71 µM (competitive part) and Ki’ = 4.63 0.63 µM (uncompetitive part) with IC50
= 6.3 0.2 µM. Better results were obtained for the mono-propargyl ester 11 (Ki = 3.33
1.15 mM, Ki’ = 2.48 0.06 µM; IC50 = 6.7 0.1 µM) and the di-butenyl ester 7 showing
inhibition constants in the single digit range of Ki = 3.39 1.13 µM and Ki’ = 2.26 0.36 µM
(IC50 = 8.7 0.2 µM). For comparison, for standard compound serine IC50 values of. 0.015
µM – 0.11 µM (for AChE) and 0.016 µM – 0.218 µM (for BChE) have been reported.38-40

A low cytotoxicity seems mandatory for compounds to treat AD. Thus, EC50 values were
31-44
measured using the photometric sulforhodamine B assay
fibroblasts (NIH 3T3).
employing murine embryonic
The results from the SRB assay gave an EC50 for 8 of > 30 µM but also the best inhibitors of
AChE (mono-propargyl 11 and di-butenyl 7) of rather low cytotoxicity (> 30 µM, cut-off of
the assay), while the di-propargyl ester 6 (EC50 = 7.0 2.5 µM) seems not suited due to high
cytotoxicity.
In conclusion, we report the synthesis of a small series of allobetulin derived seco
compounds. The results from Ellman’s-assays showed these compounds as good inhibitors of
acetylcholinesterase. Their low cytotoxicity qualifies some of these compounds (and
derivatives thereof) for further studies in the field of AD research inasmuch as the
development of AChE/BChE selective inhibitors might reduce udesired side effects as
associated with dual inhibitors and slow down AD progress especially at the beginning of the
disease. In addition, the administration of inhibitors with different AChE/BChE selectivity
might be useful as AD progresses.45
Acknowledgments
Many thanks are due to Dr. D. Ströhl for recording the NMR spectra, to Dr. R. Kluge for the
ESI-MS spectra, to A. Loesche for assistance with the assays and to J. Wiese for her help with
instrumental analysis. Support by the “Gründerwerkstatt – Biowissenschaften” is gratefully
acknowledged. The cell line (NiH 3T3) was kindly provided by Dr. T. Müller (Dept. of
Haematology / Oncology, Univ. Halle).
Supplementary data
Supporting information is available (experimental procedures and full analytical data of all
compounds). This material is available free of charge via the internet at
http://sciencedirect.com.
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Graphical Abstrac
Leave this area blank for abstract info.
Allobetulin derived seco-oleanane-
dicarboxylates act as inhibitors of
acetylcholinesterase
L. Heller, S. Schwarz, A. Obernauer and R. Csuk*