Chinese Chemical Letters 27 (2016) 1626–1629
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Design and synthesis of 5-cyclopropyl substituted cyclic acylguanidine
compounds as BACE1 inhibitors
Jia-Kuo Liu, Wei Gu, Xiao-Rui Cheng, Jun-Ping Cheng, Ai-Hua Nie *, Wen-Xia Zhou *
Beijing Institute of Pharmacology and Toxicology , Beijing, 100850, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 1 March 2016
Received in revised form 6 April 2016
Accepted 14 April 2016
Available online 11 May 2016
By taking compound 1 as a lead, a series of 5-cyclopropyl substituted cyclic acylguanidine compounds
were designed and synthesized as BACE1 inhibitors, compound 4d exhibited 84-fold improved
inhibition efficiency than lead compound 1. The diphenyl fragment at the P3 position and the
substituents at the second phenyl ring were essential for the compounds to achieve improved inhibition
efficiency. This SAR studies provides new insights into the design and synthesis of more promising
BACE1 inhibitors for the potential treatment of AD.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Keywords:
Alzheimer’s disease
BACE1 inhibitor
b-Secretase
Drug discovery
Cyclic acylguanidine
1. Introduction
Alzheimer’s disease (AD) is a chronic neurodegenerative
disorder. According to the amyloid cascade hypothesis [1],
abnormal accumulation of amyloid peptides (Ab) in the brain
resulting in neuronal toxicity is the main cause of AD [2]. Although
the amyloid cascade hypothesis remains polemic as it has not been
fully validated [3], it still represents a widely supported theory,
substantiated by genetic evidence from the various mutations of
amyloid precursor protein (APP) [4]. BACE1 (b-secretase) is a ratelimiting enzyme that hydrolyzes b-amyloid precursor protein (bAPP) to produce amyloid peptides (Ab) while most amino-acid
mutations close to the cleavage site of APP result in faster
proteolysis and increased rate of disease progression [5]. In
currently, BACE1 is considered as an attractive therapeutic target
for the treatment of AD [6]. To date, different structural classes
BACE1 inhibitors have been designed and developed [7], but it is
still a challenge to discover brain penetration BACE1 inhibitor with
low molecule weight, potent activity and high selectivity. A cyclic
acylguanidine compound, compound 1, which was discovered
each other independently by Schering-Plough [8], Wyeth [9] and
Pfizer [10], is a weak BACE1 inhibitor (IC50 = 7.1 mmol/L) [8] with
* Corresponding authors.
E-mail addresses: [email protected] (A.-H. Nie), [email protected]
(W.-X. Zhou).
brain penetration. This finding is a milestone to the development of
BACE1 inhibitor based on the amyloid cascade hypothesis. Many
excellent BACE1 inhibitors of this type that were effective in vivo
have been discovered, some of which have entered clinical trial
[11,12].
In our primary research, by taking compound 1 as a lead, we had
design and synthesized a series of new cyclic acylguanidine
compounds as BACE1 inhibitors and compound 2 exhibited submicromolar activity in vitro [13]. In this report, based on the SAR
studies revealed in our previous paper [13], we then designed
another type of compounds (3 and 4) (Fig. 1). The cyclopropyl
fragment was introduced to make the molecule more flexible and
lower the molecular weight. It is hoped that this could help the
molecule to adjust its conformation to better fill the BACE1 active
binding site to form the interactions essential for its biological
activity.
2. Experimental
The synthetic route of target compounds 3 was shown in
Scheme 1. To the mixture of cyclopropyl acetylene and 3bromophenol in THF was added TBAF/PdCl2(PPh3)2, the resulting
mixture was heated to 80 8C for 8 h to get acetylene intermediate 6
(yield 92%). Then 6 was dissolved in a co-solvent of acetone and
H2O to which MgSO4/Na2CO3/KMnO4 was added. After reacting at
r.t. for 2 h, dione intermediate 7 was obtained in a yield of 63%. A
mixture of 7, 1-methylguanidine hydrochloride and Na2CO3 in
http://dx.doi.org/10.1016/j.cclet.2016.05.002
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
[(Fig._1)TD$IG]
J.-K. Liu et al. / Chinese Chemical Letters 27 (2016) 1626–1629
1627
Fig. 1. Structures of lead and the designed compounds.
EtOH/H2O was refluxed for 2 h to give compound 8 (yield 42%). The
phenol group then reacted with ethyl methyl-carbamic chloride to
give one of the final product 3a (yield 21%). The synthetic route of
the other target compound 3b also started from cyclopropyl
acetylene (Scheme 1), which reacted with 1,3-dibromobenzene to
give intermediate 9 (yield 70%). One of the acetylene fragments of 9
was then oxidized into dione intermediate 10 by MgSO4/Na2CO3/
KMnO4 in acetone/H2O (yield 20%). Cyclization and rearrangement
of 10 with 1-methylguanidine hydrochloride at the presence of
Na2CO3 gives final product 3b (yield 24%).
[(Schem_1)TD$FIG]
The general synthetic route of compounds 4 (Scheme 2) also
started from cyclopropyl acetylene, which reacted with 1,3dibromobenzene at the presence of TBAF/PdCl2(PPh3)2 in THF to
get acetylene intermediate 11 (yield 72%). Oxidization of 11 with
MgSO4/Na2CO3/KMnO4 gives dione intermediate 12 (yield 65%).
K2CO3 and PdCl2(dppf) were added to a solution of 12 and 3hydroxyphenyl boronic acid in 1,4-dioxane at room temperature,
then H2O was added until a clear solution was made, the resulting
mixture was then heated to 90 8C for 12 h to afford 13 (yield 95%).
The dione fragment then cyclized with 1-methylguanidine
Scheme 1. Synthetic route of the designed compounds 3a,3b. Reagents and conditions: a) 3-bromophenol, TBAF, PdCl2(PPh3)2, reflux; b) KMnO4, MgSO4, Na2CO3; c) 1methylguanidine hydrochloride, Na2CO3, EtOH/H2O; d) DIPEA/THF, reflux; e) 1,3-dibromobenzene, TBAF, PdCl2(PPh3)2, reflux; f) KMnO4, MgSO4, Na2CO3; g) 1methylguanidine hydrochloride, Na2CO3, EtOH/H2O.
[(Schem_2)TD$FIG]
Scheme 2. Synthetic route of the designed compounds 4a–4l. Reagents and conditions: a) TBAF, PdCl2(PPh3)2, reflux; b) KMnO4, MgSO4, Na2CO3; c) 3-hydroxyphenyl boronic
acid, K2CO3 and PdCl2(dppf); d) 1-methylguanidine hydrochloride, Na2CO3, EtOH/H2O; e) acetone/K2CO3; f) 3-aminobenzeneboronic acid, K2CO3 and PdCl2(dppf); g) i:
1-methylguanidine hydrochloride, Na2CO3, EtOH/H2O, ii: 3-bromopropyne, acetone/K2CO3; h) i: 3-bromobenzeneboronic acid, ii: TBAF, PdCl2(PPh3)2, reflux; i)
1-methylguanidine hydrochloride, Na2CO3, EtOH/H2O; j) phenylboronic acid, K2CO3 and PdCl2(dppf); k) 1-methylguanidine hydrochloride, Na2CO3, EtOH/H2O.
J.-K. Liu et al. / Chinese Chemical Letters 27 (2016) 1626–1629
1628
hydrochloride in EtOH/H2O under reflux to give cyclic acylguanidine intermediate 14 (yield 62%). Then 14 was dissolved in acetone
to which K2CO3 and the corresponding carbamic chloride or 3bromopropyne was added. After reacting at r.t. for 36 h, the
mixture was filtered and the filtrate was directly separated on a
silica column to give final products 4a–4i (yield 19%–34%). Target
compound 4j was synthesized from 12. K2CO3/PdCl2(dppf)
catalyzed Suzuki-coupling of 12 with 3-aminobenzeneboronic
acid in 1,4-dioxane and H2O gives intermediate 17. Then 17 was
cyclized with 1-methylguanidine hydrochloride to get the
acylguanidine fragment. The amino group then reacted with 3bromopropyne in acetone at r.t. for 36 h to give target compound
4j. The bromobenzene group of 12 reacted with 3-bromobenzeneboronic acid to give the diphenyl fragment, the product was then
mixed with cyclopropyl acetylene and TBAF/PdCl2(PPh3)2 in THF.
After refluxing for 24 h, intermediate 15 was obtained in a yield of
50%. The dione group of 15 cyclized with 1-methylguanidine
hydrochloride to give target compound 4k (yield 10%). Suzuki
coupling of 12 and phenylboronic acid gives intermediate 16,
which reacted with 1-methylguanidine hydrochloride to give
target compound 4l.
TruPointTM Beta-Secretase Assay Kit is used for the BACE1
inhibition activity evaluation. A 15 mL of human recombinant
BACE1 solution (0.67 mU/mL in reaction buffer) was mixed with
15 mL reaction buffer on a 384-well optiplate. To this mixture was
added 2 mL of the tested compounds in DMSO (three concentrations for each compound 2.1 105, 2.1 106 and
2.1 107 mol/L). After a pre-incubation of 30 min at r.t., BACE1
substrate Eu-CEVNLDAEFK-Qsy 7 (400 nmol/L in reaction buffer)
15 mL was added. This incubation lasts 6 h at r.t., and then 10 mL
stop solution was added. The fluorescence of the product was
measured on an Envision Multilabel Plate Reader with an
excitation wavelength of 340 nm and an emission wavelength
of 615 nm. Blank control and negative control experiments
were also conducted for the calculation of inhibition rates.
The compounds that exhibited obviously improved inhibition
efficiency were further tested to determine their IC50 value (five
concentrations for each compound 2.1 105, 2.1 106,
2.1 107, 2.1 108 and 2.1 109 mol/L to make the IC50
accurate).
The structure of the new compounds was characterized by 1H
NMR and MS. Detailed experimental procedures, characterization
data and 1H NMR and MS spectra of target compounds are available
in supporting information.
3. Results and discussion
In the previous work [13], attaching the rivastigmine pharmacophore phenyl ester group to the lead compound 1 to interact
with the unoccupied S3 pocket, we have successfully discovered
compound 2 with much improved inhibition efficiency. Encouraged by this result, we then designed and synthesized compounds
3 and 4. The cyclopropyl was introduced to replace one phenyl ring
of the lead compound 1 to obtain compounds 3a and 3b. The
phenyl ester 3a and cyclopropyl acetylene 3b fragments were
applied to interact with the unoccupied S3 pocket or S3 subpocket. It is hoped that this additional interactions would improve
the inhibition efficiency and druggability. However, BACE1
inhibition tests showed that these compounds did not exhibit
much improved inhibition efficiency as expected (Table 1). As can
be seen, compound 3a showed decreased inhibitory activity than
lead compound 1, while compound 3b showed an IC50 of
0.807 mmol/L. It seems that when the cyclopropyl was introduced,
steric rigidified-G was preferred to give compounds with relatively
higher inhibition efficiency. This is in accordance with the SAR we
have previously concluded. Docking results of 3b with BACE1
active binding site (Fig. 2) revealed that only part of the terminal
cyclopropyl fragment was suited in the S3 sub-pocket to form
hydrophobic interactions. This is believed to be an important cause
to prevent the enzymatic inhibitory activity of 3b from further
improving.
Table 1
Inhibitory activity of compounds 3 and 4 against BACE1 at the concentration of 1 mmol/L.a,b
Structure
Compound
G or Y
3a
Inhibition rate (%)c
IC50 (mmol/L)
6.5
–
69.9
0.807
–6.9
–
8.9
–
[TD$INLE]
[TD$INLE]
3b
[TD$INLE]
4a
[TD$INLE]
[TD$INLE]
4b
[TD$INLE]
a
b
4c
[TD$INLE]
91
0.134
4d
4e
4f
4g
4h
4i
4j
–OCH2CH2F
–OC2H5
–O(CH2)2CH2Cl
–O(CH2)2CH3
–O(CH2)3CH3
–O(CH2)2CH2F
94.5
88.8
69.1
69.4
44.6
76.9
55.7
0.0847
0.303
0.603
0.541
1.44
0.331
1.15
4k
[TD$INLE]
90.8
0.145
4l
–H
63.9
0.685
[TD$INLE]
BACE1 inhibition rate of lead compound 1 against BACE1 is 12.0% and 65.8% at the concentration of 1 mmol/L and 10 mmol/L, respectively.
A BACE1 inhibitor VIa was chosen as the positive control, see reference [14].
[(Fig._2)TD$IG]
J.-K. Liu et al. / Chinese Chemical Letters 27 (2016) 1626–1629
1629
Fig. 2. Docking result of compound 3b (left) and 4k (right) with BACE1 active binding site (PDB: 4DJY).
To further improving the inhibitory activity, we then designed
and synthesized compounds 4. In compounds 4 the diphenyl
fragment was applied to form enhanced interactions as the second
phenyl ring has been proved to be a better substitute to bind the S3
pocket. The–Y fragment was attached at the meta-position of this
phenyl ring to interact with the S3 sub-pocket. Compounds 4a–k
were synthesized and tested their enzymatic inhibition efficiency
against BACE1 (Table 1). As can be seen, these compounds showed
generally much improved inhibitory activity than lead compound
1 except 4a and 4b, which means the phenyl ester group is not well
suited in the S3 sub-pocket as they are expected. While the
propyne (4c, 4j) or linear paraffin (4d–4i) or cyclopropyl acetylene
(4k) helps improving the bioactivity of the compounds. Linker
atom of -Y plays the role of a certain degree of influencing the
bioactivity, as 4j showed relatively higher IC50 value
(IC50 = 1.15 mmol/L) when the basic N atom was used. Linear
paraffin -Y is important for the compound to maintain its
bioactivity. Ethyl seems to be the most suitable (4e
IC50 = 0.303 mmol/L), when the chain gets longer (4g, 4h), the
bioactivity decreases sharply. Introduction of the F atom at the
terminal of ethyl of 4e gives the most potent compound 4d with an
IC50 of 0.0847 mmol/L. This is believed to be due to the polarization
effect induced by the strong electron-attracting ability of the F
atom. Compound 4i also showed higher inhibition efficiency than
the corresponding 4g. To better understand the binding mode of
this series of compounds with BACE1, compound 4k was docked
into BACE1 active binding site (Fig. 2) and this confirmed our
hypothesis that the second phenyl ring interact well with the S3
pocket while the linear cyclopropyl acetylene fragment nicely
fitted the S3 sub-pocket. This also explained why compound 4a
and 4b showed sharply reduced inhibitory activity. Efforts to
discovery compounds with further improved inhibition efficiency
are ongoing.
4. Conclusion
In conclusion, based on the work we previously reported, we
have design and synthesized a series of new compounds as BACE1
inhibitors. By introducing the cyclopropyl to lower the molecule
weight and make the molecule more flexible, compounds with
sub-micromolar activity were obtained, the most potent 4d
exhibited 84-fold improved inhibition efficiency than lead
compound 1. SAR studies revealed that it is important for the
molecule to maintain the rigid conformation at the P3 position
exemplified by the diphenyl fragment. The -Y fragment was
attached at the meta-position of the second phenyl ring and this
was important to guide this branch into the S3 sub-pocket to form
further enhanced interactions. Introduction of F atom to the ethyl
of 4e gives the most potent 4d, this illustrates that short polarizedY is essential for the molecule to maintain the bioactivity. This SAR
studies provide new insights into the design and synthesis of more
promising BACE1 inhibitors for the potential treatment of AD.
Acknowledgment
This work was supported by grants from the National Natural
Science Foundation of China (No. 81172924) and Beijing Municipal
Natural Science Foundation (No. 7112106).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2016.05.
002.
References
[1] J.A. Hardy, G.A. Higgins, Alzheimer’s disease: the amyloid cascade hypothesis,
Science 256 (1992) 184–185.
[2] E. Hubin, N.A.J. van Nuland, K. Broersen, et al., Transient dynamics of Ab contribute to toxicity in Alzheimer’s disease, Cell. Mol. Life Sci. 71 (2014) 3507–3521.
[3] E. Karran, M. Mercken, B. De Strooper, The amyloid cascade hypothesis for
Alzheimer’s disease: an appraisal for the development of therapeutics, Nat.
Rev. Drug Discov. 10 (2011) 698–712.
[4] T. Jonsson, J.K. Atwal, S. Steinberg, et al., A mutation in APP protects against
Alzheimer’s disease and age-related cognitive decline, Nature 488 (2012) 96–99.
[5] R. Vassar, D.M. Kovacs, R. Yan, et al., The beta-secretase enzyme BACE in health
and Alzheimer’s disease: regulation, cell biology, function, and therapeutic potential, J. Neurosci. 29 (2009) 12787–12794.
[6] G. Evin, C. Hince, BACE1 as a therapeutic target in Alzheimer’s disease: rationale
and current status, Drugs Aging 30 (2013) 755–764.
[7] A.K. Ghosh, H.L. Osswald, BACE1 (b-secretase) inhibitors for the treatment of
Alzheimer’s disease, Chem. Soc. Rev. 43 (2014) 6765–6813.
[8] Z. Zhu, Z. Sun, Y. Ye, et al., Discovery of cyclic acylguanidines as highly potent and
selective b-site amyloid cleaving enzyme (BACE) Inhibitors: Part I; inhibitor
design and validation, J. Med. Chem. 53 (2010) 951–965.
[9] M.S. Malamas, J. Erdei, I. Gunawan, et al., Design and synthesis of 5, 50 -disubstituted aminohydantoins as potent and selective human b-secretase (BACE1)
inhibitors, J. Med. Chem. 53 (2010) 1146–1158.
[10] M.S. Malamas, A. Robichaud, J. Erdei, et al., Design and synthesis of aminohydantoins as potent and selective human b-secretase (BACE1) inhibitors with
enhanced brain permeability, Bioorg. Med. Chem. Lett. 20 (2010) 6597–6605.
[11] D. Oehlrich, H. Prokopcova, H.J.M. Gijsen, The evolution of amidine-based brain
penetrant BACE1 inhibitors, Bioorg. Med. Chem. Lett. 24 (2014) 2033–2045.
[12] R. Vassar, BACE1 inhibitor drugs in clinical trials for Alzheimer’s disease, Alzheimers Res. Ther. 6 (2014) 89–93.
[13] J.K. Liu, W. Gu, X.R. Cheng, et al., Design and synthesis of cyclic acylguanidines as
BACE1 inhibitors, Chin. Chem. Lett. 26 (2015) 1327–1330.
[14] X.R. Cheng, Y. Zhou, W. Gu, et al., The selective BACE1 inhibitor VIa reduces
amyloid-b production in cell and mouse models of Alzheimer’s disease, J.
Alzheimers Dis. 37 (2013) 823–834.