Synthesis and SAR study of diphenylbutylpiperidines as cell autophagy inducers

Article abstract of DOI:10.1016/j.bmcl.2010.11.029

A novel series of diphenylbutylpiperidines as autophagy inducers was described and extensive SAR studies resulted in derivatives (15d-e, 15i-j) with 10-fold greater activity than the lead compounds 1 and 2. Meanwhile, a new synthetic route to diphenylbutyl bromide (6) from bromobenzene and γ-butyrolactone was also reported here.

Full text of DOI:10.1016/j.bmcl.2010.11.029

Bioorganic & Medicinal Chemistry Letters 21 (2011) 234–239
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR study of diphenylbutylpiperidines as cell autophagy inducers
Gang Chen a, Hongguang Xia a, Yu Cai a, Dawei Ma a, , Junying Yuan a,b, , Chengye Yuan a,
⇑
⇑
⇑
a State Key Laboratory of Bio-organic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
b Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2010
Revised 10 October 2010
Accepted 4 November 2010
Available online 11 November 2010
A novel series of diphenylbutylpiperidines as autophagy inducers was described and extensive SAR stud-
ies resulted in derivatives (15d–e, 15i–j) with 10-fold greater activity than the lead compounds 1 and 2.
Meanwhile, a new synthetic route to diphenylbutyl bromide (6) from bromobenzene and c-butyrolac-
tone was also reported here.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Autophagy
Diphenylbutylpiperidines
Synthesis
SAR study
Inducers
Autophagy, a cellular pathway involved in protein and organelle
turnover, has been proved to play an important role in human
physiology and several diseases such as cancer, cardiomyopathy
and neurodegenerative disorders (Alzheimer’s, Parkinson’s and
Huntington’s diseases, amyotrophic lateral sclerosis and prion
diseases).1
Due to its importance, the regulation of autophagy under star-
vation has been extensively studied. For example, as the target of
rapamycin in mammalian cells, mTOR kinase mediates a major
inhibitory signal that shuts off autophagy under nutrient-rich con-
ditions. Therefore, inhibition of mTOR can lead to the activation of
autophagy in response to starvation. Moreover, autophagy plays an
important role in cells under normal nutritional conditions by
mediating protein turnovers and loss of autophagy function in
the nervous systems.2 However, the regulation of autophagy under
normal nutritional conditions still remains unknown in large and
has been a great challenge to scientists ever since.
In our ongoing projects, we are interested in the design and syn-
thesis of biologically active small molecules that can induce
autophagy. In our initial studies, a high-throughput image-based
screen was carried out and seven FDA-approved drugs were found
to induce autophagy without causing cell death.3 It is interesting
that five of these compounds have been known to inhibit the intra-
cellular Ca2+ for decades. It should be noted that previous studies
have shown the possible involvement of intracellular Ca2+ in regu-
lating autophagy. For example, L-type Ca2+ channel antagonists,
the K+ATP channel opener minoxidil, and the Gi signaling activator
clonidine could induce autophagy in mTOR-independent manner.4
Recently, we also confirmed the activity of fluspirilene in inhibiting
Ca2+ flux and discovered that calpains play an important role in
controlling the levels of autophagy in normal living cells by regu-
lating the levels of ATG5.5
It is worthy to point out that three of these identified autophagy
inducers (Fig. 1), fluspirilene, Pimozide, and Trifluoperazine, are all
derivatives of diphenylbutylpiperidines (DPBPs). This class of com-
pounds, which were originally developed as antagonists of the D2
receptor, are now used clinically to treat various forms of psycho-
sis.6 Furthermore, recent evidence suggests that DPBPs are also po-
tent antagonists of calcium channels.7 In addition, Penfluridol, one
of the common DPBPs, which is not present in the library screened,
was discovered as a good autophagy inducer (EC50 = 3.2
well.
lM) as
Encouraged by these results, we began to switch our attention
onto the structural modification of diphenylbutylpiperidines.
Herein, we would like to report our recent SAR study of a new fam-
ily of DPBPs as cell autophagy inducers.
All of the DPBPs analogs described in this Letter were prepared
by nucleophilic substitution of diphenylbutyl halide with piperi-
dines according to literature.8 As a key intermediate of DPBPs,
diphenylbutyl bromide was synthesized via various routes.8,9
However, among these methods, relatively expensive as well as
the limited variety starting material forced us to find a more effec-
tive access. As shown in Scheme 1, treatment of Grignard reagents
of bromobenzene derivatives with
c-butyrolactone gave dihydr-
oxyl compound 3, which was then converted to dehydrated com-
pound 4 in acidic conditions. Compound 6 was synthesized from
4 through bromination followed by hydrogenation. In this way,
⇑
Corresponding authors. Fax: +86 21 5492 5379.
E-mail address: yuancy@mail.sioc.ac.cn (C. Yuan).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.029

G. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 234–239
235
F
F
N
N
N
NH
N
O
N
H
O
F
F
1
Fluspirilene
Pimozide
μΜ
EC50 = 2.4
F
N
N
N
CF3
Cl
OH
N
S
CF3
F
2
μΜ
EC50 = 3.2
Trifluoperazine
Penfluridol
Figure 1. DPBPs autophagy regulators.
R
X
R
X
a
b
a
O
MgBr
OH
HO
O
MgBr
O
OH
R
7
For X see Table 3
3
R=H,4-F,Cl,CF3,CH3,
OCH3, OCF3,3-F,CH3,CF3
X
X
d
b
c
R
R
c
d
Br
Br
9
8
OH
Br
R
R
4
R1
R2
5
X
N
R
R
e
14g-i, 15k-m
N
R1
R2
Scheme 2. Reagents and conditions: (a) EtOH, reflux; (b) MgBr2, Et2O, reflux; (c)
Pd–C, H2, EtOH, rt; (d) piperidines, Na2CO3, Kl, CH3CN, reflux.
Br
R
R
6
14a-f, 15a-j
compounds unique compared to others, our efforts were then ex-
erted on the synthesis of its derivatives. As shown in Scheme 3
and 4, Grignard reaction of bromobenzene derivatives with N-Boc
piperidone gave corresponding compound 10. However, removal
of Boc group of 10 with trifluoroacetic acid only led to the dehy-
drated products. After several trials, piperidine 11 could be synthe-
sized from 10 in the mixture of 3 M aqueous HCl/and ethyl acetate
at room temperature. Subsequent protection of hydroxyl group of
resulting molecules 17a–f, j by acetyl group gave compounds
18a–g10 and substitution with acetamide group by Ritter reaction
provided corresponding compounds 19a–g.8
Scheme 1. Reagents and conditions: (a) THF, reflux; (b) EtOH, cond HCl, reflux; (c)
CBr4, Ph3P, CH2Cl2, 0 °C to rt; (d) Pd–C, H2, EtOH, rt; (e) piperidines, Na2CO3, Kl,
CH3CN, reflux.
various structures of DPBPs could be obtained by simple
transformations.
Unlike the synthetic strategy above, DPBPs compounds with tri-
cyclic system were prepared as outlined in Scheme 2. Grignard reac-
tionofbromocyclopropanewithtricyclicketoneafforded compound
7, which then rearranged to compound 8 by magnesium bromide.
Subsequent hydrogenation of 8 gave the target molecule 9.
Considering that there is a tertiary hydroxyl group in the 4-po-
sition of piperidine ring, which makes the structure of this type of
With these compounds in hand, we began to estimate them as
follows: H4-LC3 cells were cultured in the presence of indicated
compounds for 4 h, fixed with 4% paraformaldehyde (Sigma) and
stained with 3 lg/ml DAPI (Sigma). Images data were collected

236
G. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 234–239
Table 1
Boc
N
EC50 induction for left-hand side modification
MgBr
R
Boc
N
a
b
OH
OH
R
N
Cl
CF3
N
R
N
O
R
NH
10
O
12a-k
R = H, 4-F, Cl, Br, CH3, CF3,
3-F, CH3, CF3, 4-Cl-3-CF3
13a-f
R
Compound/EC50
M)
Compound/EC50
(lM)
(
l
H
N
R'
F
N
c
OH
OH
R
12a (1)/2.4
13a (2)/3.2
R
R'
11
17a-j
F
Scheme 3. Reagents and conditions: (a) THF, reflux; (b) EtOAc, 3 M HCl rt; (c)
diphenylbutyl bromide, Na2CO3, KI, CH3CN, reflux.
12b/14.8
12c/24.9
13b/3.2
CN
F
N
OH
N
—
F
R
F
F
17a-f, j
R = H, 4-F, Cl, Br, CH3, CF3, 4-Cl-3-CF3
12d/6.2
13c/4.3
b
a
F
F
O
F
O
N
N
O
O
NH
12e/>50
13d/>50
F
R
F
R
18a-g
19a-g
F
Scheme 4. Reagents and conditions: (a) acetyl chloride, CHCl3, 0 °C to rt; (b) concd
H2SO4, CH3CN, rt.
O
with an ArrayScan HCS 4.0 Reader with a 20Â objective (Cellomics)
for DAPI-labeled nuclei and GFP-tagged intracellular proteins. The
Spot Detector BioApplication was used to acquire and analyze the
images after optimization. Images of 1000 cells for each compound
treatment were analyzed to obtain the average cell number per
field, fluorescence spot number, area, and intensity per cell. The
EC50 was analyzed using GraphPad Prism 4. DMSO and rapamycin
were used as negative or positive control, respectively. The per-
centages of changes of LC3-GFP were calculated by dividing with
that of DMSO-treated samples. Each treatment was done in tripli-
cate to obtain the mean SD. The images were also analyzed by
using a conventional fluorescence microscope for visual inspection.
The experiments were repeated three times with consistent
results.
12f/>50
12g/>50
12h/>50
À
À
À
F
HO
F
F
As shown in Table 1, the impact of the modification of the left-
hand sides on the cell autophagy inducing activity is well studied.

G. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 234–239
237
Table 3
Table 1 (continued)
EC50 induction for different tricyclic moieties
R
Compound/EC50
M)
Compound/EC50
(lM)
(
l
Cl
OH
CF3
N
12i/>50
À
R
N
N
NH
R
F
F
O
14g-i
15k-m
R
Compound/EC50
(lM)
Compound/EC50 (lM)
12j/4.7
13e/2.7
14g/7.9
15k/2.6
F
F
12k/2.1
13f/4.5
14h/>50
14i/5.1
15l/14.4
15m/2.6
S
F
Firstly, changing the linker of the diphenyl ring and alkyl chain
(12b,11 12c, and 12d12) leads to the decrease of bioactivity, indicat-
ing that appropriate space is needed. Interestingly, compound 13b
(CN vs H) showed the same strength of bioactivity as compound
13a. Secondly, compounds 12e13 and 13d were inactive, which
can be rationalized by the fact that the amide linkage could be eas-
ily hydrolyzed in vivo. Thirdly, compounds 12f–i14 with one phenyl
ring removed showed no activity, indicating the importance of the
diphenyl part. Lastly, we also changed the lengths of the spacer
linkers. Compounds 12j15 and 13e, with one more methylene
group compared with compounds 12a and 13a, demonstrated
twice weaker bioactivity of 12j but a little more activity of 13e.
In contrast, one less methylene group gave no change of activity
(12k),16 while compound 12f showed 1.5-fold decreased activity.
These results indicated that there may be some tolerance when it
comes to the space length (2–4 carbon chain).17
In addition, the impact of substituents on the diphenyl ring was
examined as a part of the SAR studies. As shown in Table 2, com-
pounds 14a and 15a with no substituent on the diphenyl ring
Table 2
EC50 induction for diphenyl ring substitution
R
R
N
N
N
OH
NH
O
Cl
R
R
15a-j
CF3
14a-f
R
Compound/EC50
(l
M)
Compound/EC50 (lM)
H
4-F
4-Cl
4-CF3
4-CH3
4-OCH3
4-OCF3
3-F
14a/34.6
14b (1)/2.4
14c/4.2
14d/3.7
14e/3.1
14f/4.6
—
—
—
—
15a/5.1
15b (2)/3.2
15c/2.4
15d/0.28
15e/0.29
15f/1.2
15g/0.97
15h/2.2
15i/0.65
15j/0.29
3-CF3
3-CH3

238
G. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 234–239
Table 4
EC50 induction for right-hand side modification
F
R
16a-j
F
Compound
R
EC50
>50
(lM)
16a
N
N
OH
16b
>50
N
N
N
N
O
16c
16d
16e
16f
>50
>50
>50
>50
Cl
N
N
N
CH3
CH3
CH3
16g
16h
6.4
N
N
OH
O
CH3
CH3
4.4
O
O
N
N
16i
16j
2.2
NH
10.5
CN
demonstrated the lowest activity in this series. On the other hand,
compounds 14c–f with para-substituted diphenyl ring still showed
no remarkable improvement in activity compared with compound
14b. By contrast, compounds 15c–j with various para or meta-sub-
stituents exhibited promising results. Among them, compound 15d
(R = 4-CF3), 15e (R = 4-CH3), and 15j (R = 3-CH3) were ten times
more potent when compared with compound 15b, and compound
15i (R = 3-CF3) showed fivefold improved bioactivity. On the
whole, the electronic effect of substituents on the diphenyl has
no significant effect on the bioactivity (15d vs 15e, 15i vs 15j).
These results suggest that appropriate steric hindrance on the di-
phenyl ring will exert a positive effect on the bioactivity.
As showed in Table 3, incorporation of the two phenyl rings in
tricyclic moieties which brought more rigidity, afforded different
results. Compounds 14g and 14i showed lower activity compared
with compound 14b. However, for compounds 15k and 15m, the
bioactivity enhanced a little bit. Probably due to the poor solubility
when a sulfur atom was introduced, compound 14h lost bioactivity
completely while compound 15l showed less activity.
After the modification of the left-hand sides of DPBPs, we decided
to examine the effects of the piperidine ring on the bioactivity. As
shown in Table 4, compounds 16a–c with a piperazine ring as well
ascompound16dwitha morpholineringlackedactivitycompletely.
Compound 16e18 with an unsaturated piperidine and 16f18 with no
hydroxyl group also showed no inducing activity. As previously
reported in the literature,8 changing of hydroxyl group, either pro-
tected by acetyl group (16h) or substituted with acetamide group
(16i), showed good results. However, compounds 16j19 with cya-
no-group instead of the hydroxyl group showed tiny autophagy
inducing activity. These results above indicated that the six-mem-
bered ring in the right side need appropriate conformation and the
hydroxyl group or related groups may interact with the target pro-
tein through hydrogen bonding.
Encouraged by the results of Table 4, we changed substituents on
the piperidine phenyl ring and groups at R1 to examine their effects
on the bioactivity. As shown in Table 5, when R1 = OH, compounds
17b, 17e–f with para-substituted on the phenyl ring showed better
results than compounds 17g–i with meta-substituents, while

G. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 234–239
239
Table 5
EC50 induction for variations at R1 and R2
F
N
R2
R1
17a-j, 18a-g, 19a-g
F
R2
R1
O
O
OH
N
H
CH3
O
CH3
Compound/EC50
(lM)
Compound/EC50
(
lM)
Compound/EC50 (lM)
H
4-F
4-Cl
4-Br
4-CF3
4-CH3
3-F3
3-CF3
3-CH3
4-Cl-3-CF3
17a/12.0
17b/6.1
17c/7.7
17d/2.2
17e/4.2
17f/6.4
17g/10.1
17h/6.7
17i/24.6
17j (2)/3.2
18a/>50
18b/5.4
18c/5.0
18d/2.6
18e/4.5
18f/4.4
—
19a/6.2
19b/3.0
19c/3.0
19d/2.5
19e/3.5
19f/2.2
—
—
—
—
—
18g/6.3
19g/4.0
2. (a) Lum, J. J.; DeBerardinis, R. J.; Thompson, C. B. Nat. Rev. Mol. Cell. Biol. 2005, 6,
439; (b) Hara, T.; Nakamura, K.; Matsui, M.; Yamamoto, A.; Nakahara, Y.;
Suzuki-Migishima, R.; Yokoyama, M.; Mishima, K.; Saito, I.; Okano, H.;
Mizushima, N. Nature 2006, 441, 885.
3. Zhang, L.; Yu, J.; Pan, H.; Hu, P.; Hao, Y.; Cai, W.; Zhu, H.; Yu, A.; Xie, X.; Ma, D.;
Yuan, J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 19023.
4. Williams, A.; Sarkar, S.; Cuddon, P.; Ttofi, E. K.; Saiki, S.; Siddiqi, F. H.; Jahreiss,
L.; Fleming, A.; Pask, D.; Goldsmith, P.; O’Kane, C. J.; Floto, R. A.; Rubinsztein, D.
C. Nat. Chem. Biol. 2008, 4, 295.
5. Xia, H.; Zhang, L.; Chen, G.; Zhang, T.; Liu, J.; Jin, M.; Ma, X.; Ma, D.; Yuan, J.
Autophagy 2010, 6, 1.
6. Seeman, P.; Lee, T.; Chau-Wong, M.; Wong, K. Nature 1976, 261, 717.
7. (a) Gould, R. J.; Murphy, K. M. M.; Reynolds, I. J.; Snyder, S. H. Proc. Natl. Acad.
Sci. U.S.A. 1983, 80, 5122; (b) Grantham, C. J.; Main, M. J.; Cannell, M. B. Br. J.
Pharmacol. 1994, 111, 483; (c) Sah, D. W. Y.; Bean, B. P. Mol. Pharmacol. 1994, 45,
84; (d) Enyeart, J. J.; Biagi, B. A.; Day, R. N.; Sheu, S. S.; Maurer, R. A. J. Biol. Chem.
1990, 265, 16373; (e) Galizzi, J. P.; Fosset, M.; Romey, G.; Laudron, P.;
Lazdunski, M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 7513; (f) King, V. F.; Garcia, M.
L.; Shevell, J. L.; Slaughter, R. S.; Kaczorowski, G. J. J. Biol. Chem. 1989, 264, 5633.
8. Hulshof, J. W.; Vischer, H. F.; Verheij, H. F.; Fratantoni, M. H. P.; Smit, M. J.; de
Esch, I. J. P.; Leurs, R. Bioorg. Med. Chem. 2006, 14, 7213.
9. (a) Sindelar, K.; Rajsner, M.; Cervena, I.; Valenta, V.; Jilek, J. O.; Kakac, B.;
Holubek, J.; Svatek, E.; Miksik, F.; Protiva, M. Collect. Czech. Chem. Commun.
1973, 38, 3879; (b) Botteghi, C.; Marchetti, M.; Paganelli, S.; Persi-Paoli, F.
Tetrahedron 2001, 57, 1631; (c) Hulshof, J. W.; Casarosa, P.; Menge, M. P. B.;
Kuusisto, L. M.; van der Goot, H.; Smit, M. J.; de Esch, I. J. P.; Leurs, R. J. Med.
Chem. 2005, 48, 6461.
compound 17a with no substituent on the phenyl ring showed de-
creased bioactivity.
Since it has been found that the presence of a hydroxyl group at 4-
position of the piperidine ring may lead to potential metabolic tox-
icity,20 introduction of an ester group and amide group was then
achieved to eliminate the negative effect (Table 5). Unfortunately,
18a–f showed no improved activity when compared with 17a–f.
On the other hand encouraging results were obtained when an
amide group was introduced (19a–f). However, both compounds
18g and 19g displayed decreased inducing activity compared with
compound 17j, indicating appropriate space on the piperidine ring
is responsible to the activity of compound under investigation.
In summary, we have identified
a novel class of diph-
enylbutylpiperidines as effective autophagy inducers. An effective
synthetic route to diphenylbutyl bromide has also been developed.
Structural modifications of compound 1 and 2 bring in significant
increase of activity (compounds 15d, 15e, 15i, 15j). Besides this, ef-
fects of structural variations on either the left-hand or the right-
hand were studied as well. Moreover, substituents on the piperi-
dine phenyl ring of compound 2 were investigated in the same
way. Further SAR exploration to improve the overall biological
activity profiles and structural modifications of Pimozide will be
reported in due course.
10. Rajsner, M.; Dlabac, A.; Protira, M. Collect. Czech. Chem. Commun. 1981, 46,
1530.
11. For the synthesis of the left side of compound 12b, see Ref.: Kulp, S. S.; Fish, V.
B.; Easton, N. R. J. Med. Chem. 1963, 6, 516.
12. The structure of the left side of compound 12d is the same as compound 5
(R = 4-F).
Acknowledgments
13. The carboxylic acid group in the left of compound 12e can be prepared from
compound 4 (R = 4-F) by hydrogenation, Jones oxidation.
14. For the synthesis of these left sides of compounds 12f–i, see Ref.: Janssen, P. A.
J.; van der Westeringh, C.; Jageneau, A. H. M.; Demoen, P. J. A.; Hermans, B. K.
F.; van Daele, G. H. P.; Schellekens, K. H. L.; van der Eycken, C. A. M.;
Niemegeerx, C. J. E. J. Med. Chem. 1959, 1, 281.
This work was supported in part by grants from the National
Natural Science Foundation of China (No. 21072212 to C.Y.) and
the National Institute of Health of US (to J.Y.).
15. The left side of compound 12j can be synthesized from the same route as
Supplementary data
showed in Scheme 1 with the starting material d-valerolactone instead of
butyrolactone.
c-
16. Kashali, N.; Polat, M. F.; Altundas, R.; Kara, Y. Helv. Chim. Acta 2008, 91, 67.
17. In this Letter we mainly used the left side with three carbon chain due to its
easy preparation.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.11.029.
18. The right side of compound 16e can be prepared from compound 10 (R = CH3)
by removing the Boc group with trifluoroacetic acid, and hydrogenation of
compound 16e can give compound 16f.
References and notes
19. The right side of compound 16j is commercially available.
20. Castagnoli, N., Jr.; Rimoldi, J. M.; Bloomquist, J.; Castagnoli, K. P. Chem. Res.
Toxicol. 1997, 10, 924.
1. (a) Shintani, T.; Klionsky, D. J. Science 2004, 306, 990; (b) Mizushima, N.; Levine,
B.; Cuervo, A. M.; Klionsky, D. J. Nature 2008, 451, 1069.