Novel N-indolylmethyl substituted olanzapine derivatives: Their design, synthesis and evaluation as PDE4B inhibitors

Article abstract of DOI:10.1039/c3ob27424a

A new strategy for converting antipsychotic drug olanzapine into PDE4 inhibitors is described via the design and Pd/C mediated synthesis of novel N-indolylmethyl olanzapine derivatives. One compound showed good inhibition (IC₅₀ 1.1 μM) and >10 fold selectivity towards PDE4B over D that was supported by docking studies. This compound also showed significant inhibition of TNF-α and no major toxicities in cell lines and a zebrafish embryo model except the teratogenic effects to be re-assessed in rodents. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob27424a

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Novel N-indolylmethyl substituted olanzapine
derivatives: their design, synthesis and evaluation as
Cite this: Org. Biomol. Chem., 2013, 11,
2075
PDE4B inhibitors†
Dhilli Rao Gorja,a,b Soumita Mukherjee,a Chandana Lakshmi T. Meda,a
Girdhar Singh Deora,a K. Lalith Kumar,a Ankit Jain,a,c Girish H. Chaudhari,a,c
Keerthana S. Chennubhotla,a,c Rakesh K. Banote,a,c Pushkar Kulkarni,a,c
Kishore V. L. Parsa,*a K. Mukkantib and Manojit Pal*a
Received 31st October 2012,
Accepted 11th February 2013
DOI: 10.1039/c3ob27424a
www.rsc.org/obc
A new strategy for converting antipsychotic drug olanzapine into that hydrolyze cyclic nucleotides and thus play a key role in
PDE4 inhibitors is described via the design and Pd/C mediated regulating intracellular levels of the second messengers cAMP
synthesis of novel N-indolylmethyl olanzapine derivatives. One and cGMP, and hence cell function.1 PDE4 is a cAMP-specific
compound showed good inhibition (IC50 1.1 μM) and >10 fold PDE and predominant isoenzyme in the majority of inflamma-
selectivity towards PDE4B over D that was supported by docking tory cells, with the exception of platelets, implicated in inflam-
studies. This compound also showed significant inhibition of matory airways disease. Since elevated levels of cAMP play a
TNF-α and no major toxicities in cell lines and a zebrafish embryo major role in relaxation of vascular smooth muscle, inhibition
model except the teratogenic effects to be re-assessed in rodents.
of PDE4 is beneficial for the treatment of respiratory diseases
including asthma and COPD.1,2 Among the four isoforms e.g.
PDE4A, B, C, and D, the PDE4B plays a key role in inflamma-
tory cell regulation and its inhibition suppresses TNF-α pro-
duction via elevation of cAMP levels.6 The inhibition of PDE4D
on the other hand triggers the emetic response.7 Thus, selec-
tive inhibition of PDE4A and/or PDE4B was thought to provide
a means to achieve efficacy while mitigating the adverse effects
of the current PDE4 inhibitors. However, in spite of innumer-
able efforts, only a few PDE4B selective inhibitors have been
reported till date.8,9
Olanzapine (Zyprexa®, Eli Lilly) (1, Fig. 1) that belongs to
the thienobenzodiazepine class is a dopamine receptor anta-
gonist and has been used as a second generation atypical anti-
psychotic drug. It has proven efficacy against schizophrenia10
and has shown anti-emetic effects in the treatment of refrac-
tory nausea and vomiting in advanced cancer.11 The indole
derivatives 2 (Fig. 1), on the other hand, have been reported as
Chronic obstructive pulmonary disease (COPD), a major
health problem, is projected to be the 3rd leading cause of
death globally by 2020 according to the WHO.1 Asthma, on the
other hand, affects ∼300 million people worldwide at present.1
The search for improved therapies therefore is necessary.
Among the emerging agents to treat COPD or asthma, phos-
phodiesterase 4 (PDE4) inhibitors have attracted particular
attention.2 Additionally, evaluation of PDE4 inhibition for the
potential treatment of a wider range of CNS related diseases
including Parkinson’s disease,3 schizophrenia,4 and Alzhei-
mer’s disease5 has underlined the importance of development
of PDE4 inhibitors. The first-generation PDE4 inhibitor roli-
pram suffered from side effects including nausea and
emesis.1,2 Similar side effects delayed the market launch of the
second generation inhibitors cilomilast and roflumilast.
Encouragingly, roflumilast (Daxas®, Nycomed) was launched
in Europe for the treatment of chronic bronchitis in 2009 and
in US (Daliresp, Forest Lab) for exacerbations during COPD in
2012. Nevertheless, it is desirable to identify PDE4 inhibitors
having fewer side effects. PDEs are a diverse family of enzymes
aInstitute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad
500046, India. E-mail: manojitpal@rediffmail.com
bChemistry Division, Institute of Science and Technology, JNT University, Kukatpally,
Hyderabad 500072, India
cZephase Therapeutics Pvt. Ltd (An incubated company at the Institute of Life
Sciences), University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
†Electronic supplementary information (ESI) available: 1H and 13C NMR, MS
and HRMS data of all new compounds. See DOI: 10.1039/c3ob27424a
Fig. 1 Design of an olanzapine based novel PDE4 inhibitor.
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promising inhibitors of PDE4.12 We anticipated that a combi-
nation of the structural features of 1 and 2 in a single molecu-
lar species 3 (Fig. 1) could be helpful for the selective
inhibition of PDE4B with reduced emetic side effects. Our
design was primarily based on the fact that (i) the catalytic
pocket of PDE4B is more hydrophobic9a in nature than PDE4D
and (ii) the buried pocket volume available for an inhibitor to
bind within the PDE4B binding site is greater than that of
PDE4D.9b Thus the hydrophobic and bulky nature of 3 was
expected to be favorable for PDE4B selectivity. Furthermore,
docking studies performed using a series of molecules based
on 3 indicated a favorable effect of an N-sulfonyl moiety (Z =
SO2R), the oxygen of which may have H-bonding interaction
with the surrounding amino acids in the PDE4 binding pocket
thereby enhancing the binding affinity of the resulting mole-
cule towards PDE4. Herein, we report the design, synthesis
and pharmacological evaluation of a novel series of potential
PDE4B inhibitors13 based on the scaffold 3 derived from olan-
zapine. Notably, no PDE4 inhibitory properties have been
reported for olanzapine or its analogues. Moreover, since a
number of potent but non-selective inhibitors have been
reported earlier1,2 thus achieving PDE4B selectivity rather than
potency was the major goal of this research.
Table 1 Effect of reaction conditions on coupling of terminal alkyne 4 with 5aa
Entry
Pd-catalysts
Base
Time (h)
Yieldb (%)
1.
2.
3.
4.
5.
6.
10% Pd/C–PPh3
10% Pd/C–PPh3
10% Pd/C–PPh3
Et3N
Et3N
K2CO3
Et3N
Et3N
Et3N
4
6
10
12
8
81
89
15
c
PPh3
No product
65
14
Pd(PPh3)2Cl2 d
Pd(PPh3)2Cl2
12
a All reactions were carried out using 5a (1 equiv.), alkyne 4 (1 equiv.),
a Pd-catalyst (0.016 equiv.), PPh3 (0.125 equiv.), CuI (0.02 equiv.), and
a base (2 equiv.) in EtOH (5.0 mL), at 70 °C. b Isolated yield. c The
reaction was carried out without Pd/C. d The reaction was carried out
without CuI.
Table 2 Preparationa of compound 3 (Scheme 1) and its in vitro evaluation
against PDE4B and PDE4D
The designed compounds were conveniently prepared fol-
lowing a Pd/C-mediated coupling–cyclization strategy.14 The
key starting material i.e. the terminal alkyne 4 required for
accessing our target compound 3 (mainly 3a–j) was syn-
thesized by treating olanzapine (1) with propargyl bromide in
the presence of NaH in THF (Scheme 1).15 Initially, the alkyne
4 was reacted with N-(4-bromo-2-iodophenyl)-methanesulfona-
mide 5a in the presence of 10%Pd/C–PPh3–CuI and Et3N in
EtOH at 70 °C for 4 h (Table 1). While the expected product 3a
was isolated in 81% yield (Table 1, entry 1) the increase in reac-
tion time however improved the product yield (Table 1, entry
2). The use of K2CO3 in place of Et3N (Table 1, entry 3) or omis-
sion of Pd/C (Table 1, entry 4) was found to be ineffective. The
use of Pd(PPh3)2Cl2 (Table 1, entry 5) afforded satisfactory
yield (65%) whereas omission of CuI (Table 1, entry 6) reduced
% inhibition @ 30 μM
Entry
R1 =; Z = (5)
3b
Yieldc
PDE4B
PDE4D
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Br; Ms (5a)
H; Ms (5b)
NO2; Ms (5c)
CN; Ms (5d)
Me; Ms (5e)
COMe; Ms (5f)
H; Ts (5g)
Cl; Ts (5h)
Br; Ts (5i)
COMe; Ts (5j)
3a
3b
3c
3d
3e
3f
3g
3h
3i
89
90
87
85
81
82
72
85
76
78
68.9 1.7
87.1 0.6
72.6 1.1
86.7 2.0
61.9 1.5
69.5 0.8
37.0 6.9
13.5 1.9
30.0 1.5
22.4 0.8
35.3 6.2
70.0 1.2
47.8 2.0
64.1 1.7
41.2 0.8
ND
23.1 0.9
ND
ND
3j
ND
a All the reactions were carried out using 5 (1 equiv.), alkyne 4 (1
equiv.), 10% Pd/C (0.016 equiv.), PPh3 (0.125 equiv.), CuI (0.02 equiv.),
and Et3N (2 equiv.) in EtOH at 70 °C for 6 h. b Identified by 1H and 13
C
the yield to 14%. Thus, 10% Pd/C–PPh3–CuI and Et3N in EtOH NMR, IR, and MS. c Isolated yields. Ms = mesyl, Ts = p-tosyl, ND = not
determined.
was used to prepare other analogues of 3 (Scheme 1, Table 2).
A number of o-iodosulphanilides (5a–j) were reacted with
alkyne 4 under the optimized conditions. The reaction pro-
[e.g. CN (5d), NO2 (5c) and COCH3 (5f, 5j)] affording the
desired products in good yields.
ceeded well irrespective of the presence of an electron releas-
ing [e.g. Cl (5h), Br (5a, 5i) and Me (5e)] or withdrawing group
Most of the compounds synthesized (3a–j) were evaluated
for their PDE4B and PDE4D inhibitory properties in vitro using
enzyme based assays (Table 2). The PDE4B isolated from Sf9
cells and commercially available PDE4D were used for this
purpose. Rolipram, a well known inhibitor of PDE4, was used
as a reference compound. In general, compounds containing
an N-mesyl moiety showed significant inhibition (>60%) of
PDE4B than those possessing an N-tosyl group e.g. 3g–j when
tested at 30 μM. Compounds 3a–e showed superior inhibition
of PDE4B compared to PDE4D. Based on these initial data, a
dose–response study was performed with three representative
compounds e.g. 3a–c (and their precursor olanzapine). The
Scheme 1 Synthesis of N-indolylmethyl substituted olanzapine derivatives 3.
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Table 3 In vitro data of compounds 3a–c and their glide scores after docking
into the PDE4B and D
IC50 a (μM)
Glide score
PDE4B
Entry
Compound
PDE4B
PDE4D
PDE4D
1.
2.
3.
4.
3a
3b
3c
1.1 0.1
2.3 0.4
7.9 2.2
0.94 0.2
>10
−5.11
−5.47
−5.10
−6.66
−2.98
−2.90
−2.52
−5.0
8.2 0.9
51.7 11.6
0.88 0.3
Rolipram
a Data are represented as mean
experiments.
SD of at least 3 independent
IC50 values are presented in Table 3. While all these com-
pounds showed some selectivity towards PDE4B the com-
pound 3a was identified as most potent (IC50 ∼ 1.1 μM) and
selective (>10 fold towards PDE4B) among them (Fig. 2).
Notably, olanzapine did not inhibit PDE4B across the doses
tested. With respect to the initial in vitro data the compound
3a seemed to be comparable with phase 2 clinical candidate16a
CC-1088 (PDE4 IC50 = 1.1 μM) and possesses better PDE4B
selectivity than the marketed drug roflumilast [IC50 = 1.3 nM
(PDE4B) and 0.2 nM (PDE4D)]16b and other advanced com-
pounds17 e.g. cilomilast [IC50 = 86 nM (PDE4B) and 12 nM
(PDE4D)], oglemilast [IC50 = 2.5 nM (PDE4B) and 1.7 nM
(PDE4D)], etc. To understand the nature of interactions of 3a–c
with PDE4B and D, docking studies were performed (Table 3,
see also ESI†). All these compounds (i.e. 3a, 3b and 3c) showed
better interactions with PDE4B compared to D in silico.
Fig. 3 Cytotoxicity of 3a–c in RAW 264.7 cells.
The compound 3a was further evaluated for its anti-inflam-
matory effects. Consequently, pre-incubation of RAW 264.7
cells with 10 μM of compound 3a before stimulation with bac-
terial endotoxin caused ∼50% inhibition of TNF-α synthesis.
The toxicity of the 3a–c was also evaluated in cell lines and a
zebrafish embryo model. The compounds 3a and 3c did not
cause major toxicity in RAW 264.7 cells (Fig. 3) and HEK 293T
cells (Fig. S2 in ESI†). Further, compounds 3a and 3c were tol-
erated up to 10 and 50 μM, respectively, without any apparent
hepatotoxicity in 4 day old zebrafish embryos (Fig. 4). More-
over, 3a did not induce any changes in the heart rate.
However, 3a–c along with rolipram showed teratogenic effects
at 50 μM (lower concentrations for 3a) in zebrafish embryos
(Fig. S15A and S15B in ESI†). While its precursor i.e. olanzapine
is not an animal teratogen in two species (rats and rabbits) its
dose related embryo and fetal toxicity (decreased fetal weight
and related skeletal ossification) is known in the literature.18
Fig. 2 Dose dependent inhibition of PDE4B and PDE4D by 3a.
Fig. 4 Hepatotoxicity of 3a–c in zebrafish embryos.
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Thus observed teratogenic effects of 3a in zebrafish embryos
need to be assessed in appropriate animal models.
6 S.-L. C. Jin, L. Lan, M. Zoudilova and M. Conti, J. Immunol.,
2005, 175, 1523.
To assess the in silico binding mode of compounds 3a–c in
human D2 dopamine19 receptor docking studies were per-
formed using a homology model of the catalytic site of a
human D2 dopamine receptor. While these compounds
showed differences in the binding mode with that of olanza-
pine their docking scores (though lower than that of olanza-
pine) indicated significant interactions with the receptor (see
ESI†). While the larger volume of these molecules compared to
olanzapine was responsible for their lower docking scores
overall, these molecules appeared to have potential to bind
with a dopamine receptor. Further in vitro studies20 are in pro-
gress towards this direction.
In conclusion, a new strategy is presented for converting
the antipsychotic drug (and a non-PDE4 inhibitor) olanzapine
into PDE4 inhibitors via the design and synthesis of novel
N-indolylmethyl olanzapine derivatives. These compounds
were elegantly prepared by using a Pd/C mediated coupling–
cyclization strategy as a key step. Some of the synthesized com-
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The authors thank Prof. J. Iqbal and DBT (Grant BT/01/
COE/07/02) for support. DRG thanks DST, India for a Research
S. C. Joseph, K. S. Reddy, L. S. Reddy, P. M. Kumar,
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Associateship.
a
Research
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This journal is © The Royal Society of Chemistry 2013
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