(Pd/C-mediated)coupling-iodocyclization-coupling strategy in discovery of novel PDE4 inhibitors: A new synthesis of pyrazolopyrimidines

Article abstract of DOI:10.1039/c2md00273f

Pyranones fused with a pyrazolopyrimidine moiety were prepared via regioselective construction of pyranone ring using (Pd/C-mediated)coupling- iodocyclization followed by Sonogashira/Heck/Suzuki reactions. The pyrazolopyrimidine based reactant required was obtained via a new H ₃PO₃ mediated condensation reaction. This strategy has led to the discovery of a novel and potentially safe PDE4 inhibitor.

Full text of DOI:10.1039/c2md00273f

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CONCISE ARTICLE
(Pd/C-mediated)coupling–iodocyclization–coupling strategy in discovery of
novel PDE4 inhibitors: a new synthesis of pyrazolopyrimidines†
P. Mahesh Kumar,ab K. Siva Kumar,a Chandana L. T. Meda,c G. Rajeshwar Reddy,a Pradeep K. Mohakhud,a
K. Mukkanti,b G. Rama Krishna,d C. Malla Reddy,d D. Rambabu,c K. Shiva Kumar,c K. Krishna Priya,c
e
Keerthana Sarma Chennubhotla, Rakesh Kumar Banote, Pushkar Kulkarni, Kishore V. L. Parsa
e
e
c
*
c
*
and Manojit Pal
Received 26th October 2011, Accepted 1st April 2012
DOI: 10.1039/c2md00273f
Pyranones fused with a pyrazolopyrimidine moiety were prepared via regioselective construction of
pyranone ring using (Pd/C-mediated)coupling–iodocyclization followed by Sonogashira/Heck/Suzuki
reactions. The pyrazolopyrimidine based reactant required was obtained via a new H3PO3 mediated
condensation reaction. This strategy has led to the discovery of a novel and potentially safe PDE4
inhibitor.
Phosphodiesterase type 4 (PDE4), exists in four different iso-
forms (PDE4A, B, C and D), is one of eleven isozymes, and is
specific for the hydrolysis of cAMP to AMP.1–3 In inflammatory
and immune cells, the inhibition of cellular responses, including
the production and/or release of proinflammatory mediators,
cytokines, and active oxygen species, is associated with elevated
levels of cAMP. Thus, inhibition of PDE4 was expected to be
beneficial for the treatment of inflammatory and immunological
diseases including asthma and chronic obstructive pulmonary
disease (COPD).1,3–5 However, many PDE4 inhibitors have been
discontinued from clinical trials due to a narrow window
between efficacy and the undesired side effects such as nausea
and emesis.1,2,4–7 More recently, cardiovascular effects of PDE4
inhibitors have been reported.8 While roflumilast (Daxasꢀ,
Nycomed) was launched in Europe recently, the search for a new
chemical class for the evaluation of selective PDE4 inhibitory
properties with improved therapeutic indexes is desirable.
The PDE4B mRNA has been reported to be up-regulated due
to the exposure of monocytes to bacterial endotoxin LPS.9
Further, circulating leukocytes from PDE4B but not PDE4A
or PDE4D knockout mice displayed ꢀ90% reduction in LPS
induced TNF-a production indicating the key role of the PDE4B
subtype in promoting inflammation.10 Thus, targeting PDE4B
may be a more balanced approach to treat the inflammatory
component of COPD without causing or minimizing the emetic
side effects.1 Notably, design of PDE4D allosteric modulators
Fig. 1 Design of a novel chemical class D from A and B.
aCustom Pharmaceutical Services, Dr Reddy’s Laboratories Limited,
Bollaram Road Miyapur, Hyderabad 500 049, India
bChemistry Division, Institute of Science and Technology, Jawaharlal
Nehru Technological University, Hyderabad 500089, Andhra Pradesh,
India
cInstitute of Life Sciences, University of Hyderabad Campus, Gachibowli,
Hyderabad 500 046, Andhra Pradesh, India. E-mail: KishoreP@
ilsresearch.org; manojitpal@rediffmail.com
dDepartment of Chemical Sciences, Indian Institute of Science and
Research, Kolkata, West Bengal, 741252, India
eZephase Therapeutics Pvt. Ltd (An incubated company at the Institute of
Life Sciences), University of Hyderabad Campus, Gachibowli, Hyderabad
500 046, Andhra Pradesh, India
† Electronic supplementary information (ESI) available: Experimental
procedures, spectral data and (1H and 13C NMR, MS and HRMS)
spectra for all new compounds. CCDC 832934 and 832935. For ESI
and crystallographic data in CIF or other format see DOI:
10.1039/c2md00273f
Scheme 1 Preparation of the other starting compound 2.
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Table 2 Pd/C-mediated synthesis of 2-alynyl pyrazolo[1,5-a]pyrimidines (5)a
Scheme 2 Preparation of the starting pyrazole derivative 1.
Bromopyrazolo- Alkyne
(4; R3 ¼)
Time Product Yieldb
Entry pyrimidine (3)
(h)
(5)
(%)
1
2
3a
3a
4a; n-Hexyl
4b; n-Pentyl
15
15
5a
5b
70
65
4c;
4d;
4e;
3
4
5
3a
3a
3a
12
18
12
5c
5d
5e
65
68
60
Scheme 3 Synthesis of novel pyranopyrazolopyrimidine derivatives (7).
for enhancing cognition with improved safety has been reported
recently.11
Ibudilast A (Fig. 1), approved in Japan for the treatment of
asthma and stroke,12 is a pyrazole based PDE4 inhibitor.
Recently, we have reported 7-(hetero)aryl substituted pyr-
azolopyrimidine B (Fig. 1) derived from A as a potential scaffold
for the development of novel PDE4 inhibitors.13a In continuation
of this effort we became interested in design, synthesis and
evaluation of novel compounds represented by D (Fig. 1) for
their PDE4 inhibitory properties.13 The fused heterocyclic
structure D was arrived at via C by (i) connecting the two side
chains of A in an intramolecular fashion followed by introducing
an oxygen and a double bond in the newly formed six-membered
ring and (ii) introducing some of the structural features of B.
Since the new scaffold D possesses four potential positions
including the aryl group to introduce diversity a library genera-
tion based on D was therefore undertaken. Herein, we report
a (Pd/C-mediated)coupling–iodocyclization–coupling strategy
6
3a
4f;
10
5f
70
7
8
9
10
3a
3b
3b
3c
4g; –CH2CH2OH 10
4a; n-Hexyl
4e
4a
5g
5h
5i
67
70
65
60
15
12
15
5j
a
All the reactions were carried out by using 3 (1.0 mmol), terminal
alkyne (1.5 mmol), 1 : 4 : 2 ratio of Pd/C–PPh3–CuI and Et3N (3 equiv)
in DMF at 120 ꢁC. b Isolated yield.
a compound library based on D leading to the identification of
a novel inhibitor of PDE4B.
The synthesis of target compounds (7) is shown in Scheme 1.
The key compound (1) required for our synthesis was prepared
from ethyl 3-amino-1H-pyrazole-4-carboxylate via N-acylation
followed by bromination and subsequent deacylation
and
a new synthesis of pyrazolopyrimidine to generate
Table 1 Preparation of 2-bromopyrazolo[1,5-a]pyrimidines (3)a
Entry
Ketone (2) R1, R2
Time (h)
Product (3)
Yieldb (%)
1
2
3
H, H (2a)
CH3, H (2b)
H, CO2Et (2c)
4.0
4.0
5.0
3a
3b
3c
75
74
70
a
All the reactions were carried out by using compounds 1 (1.0 mmol), 2
(1.0 mmol) and H3PO3 (1.0 mmol) in ethanol at refluxing temperature.
Isolated yield.
b
Fig. 2 X-ray crystal structure of 5d (ORTEP diagram). Thermal ellip-
soids are drawn at 50% probability level.
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Table 3 (Contd.)
Table 3 Synthesis of 10H-pyrano[40,30:3,4]pyrazolo [1,5-a]pyrimidin-10-
one derivatives (6) via iodocyclization of alkynes 5a
2-Alkynyl
pyrazolo[1,5-a]-
Entry pyrimidine (5)
Yieldb
(%)
Product (6)
8
85
2-Alkynyl
pyrazolo[1,5-a]-
Entry pyrimidine (5)
Yieldb
(%)
a
Product (6)
All the reactions were carried out by using 5 (1.0 mmol), iodine (2.0
b
mmol) in DCM at 25 ꢁC for 2 h. Isolated yield.
1
2
92
91
(Scheme 2).14a Other reactants 2 were prepared from appropriate
aryl ketones following a known procedure (Scheme 3).14b 2-
Bromopyrazolo[1,5-a]pyrimidines14c (3) prepared via a new
H3PO3-mediated14d condensation reaction of 1 and 2 (Table 1)
were reacted with a variety of terminal alkynes (4) in the presence
of Pd/C under Sonogashira conditions (Table 2).15 A variety of 2-
alkynyl pyrazolopyrimidines (5) were prepared and the molec-
ular structure of a representative compound 5d was determined
unambiguously by X-ray crystallographic analysis (Fig. 2).17a
Intramolecular regioselective iodocyclization15,16 of alkyne 5
yielded corresponding 7-iodo derivatives (6, Table 3). The
molecular structure of 6e (6; R1, R2 ¼ H, R3 ¼ 1-cyclohexenyl)
was determined unambiguously by X-ray crystallographic anal-
ysis (Fig. 3).17b
3
4
5
92
90
93
The iodo derivatives (6) were used for further structural
elaboration under Sonogashira or Suzuki or Heck reaction
conditions to give 7-alkynyl, aryl and alkenyl derivatives (7)
(Table 4).
Most of the compounds (7) synthesized were evaluated for
their PDE4 inhibitory properties in vitro. The report11 that
PDE4D inhibition by allosteric inhibitors (maximum inhibition,
Imax 80–90%) did not cause emetic side effects has raised
a possibility that PDE4B inhibitors with partial but not complete
inhibition of PDE4D (Imax of ꢀ60 to 80%) could be developed to
treat COPD and asthma without causing emetic side effects.
Thus our goal was to identify inhibitors possessing such
a balanced selectivity towards PDE4B. Initially, PDE4B inhibi-
tory potential was assessed by using PDE4B enzyme isolated
6
88
7
90
Fig. 3 X-ray crystal structure of 6e (ORTEP diagram). Thermal ellip-
soids are drawn at 50% probability level.
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Table 4 Synthesis of 7-substituted 10H-pyrano[40,30:3,4] pyrazolo[1,5-a]
pyrimidin-10-ones (7) from compounds 6
Compound Time Yielda
Entry Product (7)
6
(h)
(%)
1
6a
3.0
93
7a
7b
7c
Fig. 4 Inhibition of PDE4B by compounds 7 (enzyme assay).
2
3
6c
6a
3.0
2.0
92
91
from Sf9 cells.18a Among all the compounds tested 7c, 7d, 7g, 7h
and 7i showed significant inhibition of PDE4B (i.e. 85, 93, 66, 74
and 74% respectively) at 30 mM (Fig. 4) whereas other
compounds e.g. 7e and 7f were inactive. The compounds 7a and
7b showed ꢀ50% inhibition of PDE4B at 30 mM. These obser-
vations suggested that the presence of an aryl group at C-3 of the
pyranone ring was important and a –CONH2 group attached to
this aryl ring (e.g. 7c and 7d) played a key role in PDE4B inhi-
bition. Notably, both aryl and alkyl groups were tolerated at C-2
of the pyranone ring (e.g. 7c vs. 7d). Compound 7d was then
tested in the cell based cAMP reporter assay in HEK 293 cells
and its TNF-a inhibitory activity19 was measured in lipopoly-
saccharide (LPS) stimulated RAW 264.7 cells.18b Rolipram,
a well known inhibitor of PDE4 was used as a reference
compound (Table 5). Based on the data generated (Table 5 and
Fig. 5–7, see ESI† also) compound 7d was identified as a prom-
ising inhibitor of PDE4.
4
5
6c
6a
2.0
2.0
91
91
7d
7e
Preliminary safety evaluation of compound 7d was carried out
in a zebrafish embryo model. Zebrafish embryos were examined
for mortality, scored for phenotypic effects and locomotor
activity. All embryos in the control group survived the entire
duration of the study and did not show any apparent effects on
phenotype or locomotor activity. One embryo was found dead in
both rolipram groups (10 and 20 mM) at 1 and 24 h time points,
respectively. All the other embryos in both the rolipram exposed
groups exhibited lordosis (inward curvature of the vertebrae),
and mild to moderate effects on locomotor activity during the
course of treatment. No mortality was observed at any time point
in the embryos exposed to compound 7d; though mild effects on
locomotor activity were noticed in 7d groups (10 and 20 mM) at 6
and 24 h, respectively. All the embryos were found dead in the
100 mM Terfenadine (positive control) showing severe defects in
6
6a
2.0
92
7f
7
8
6c
6c
2.0
2.0
86
82
7g
7h
Table 5 In vitro data of compound 7d
Compounds
Assay
PDE4 subtype Rolipram
7d
9
6c
2.0
80
Enzymatic assay (IC50, mM) PDE4B1
PDE4D2
Reporter assay (EC50, mM) PDE4A4
PDE4B1
PDE4D3
0.94 ꢂ 0.24 1.33 ꢂ 0.64
0.88 ꢂ 0.28 2.84 ꢂ 0.64
2.62 ꢂ 1.29 11.66 ꢂ 1.10
0.16 ꢂ 0.11 6.32 ꢂ 0.40
0.67 ꢂ 0.42 9.62 ꢂ 0.75
0.19 ꢂ 0.09 0.45 ꢂ 0.29
7i
a
Isolated yield.
TNF-a assay (IC50, mM)
670 | Med. Chem. Commun., 2012, 3, 667–672
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Fig. 5 PDE4B inhibition by 7d: (A) enzyme assay and (B) cell based
reporter assay.
Fig. 8 Evaluation of 7d and rolipram in zebrafish embryo toxicity
model. See ESI† for scoring guidelines. Kruskal–Wallis, Dunn’s multiple
comparison post hoc test. *p < 0.05, **p < 0.002, ***p < 0.0001.
In summary, a new chemistry strategy i.e. (Pd/C-mediated)
coupling–iodocyclization–coupling has been explored to
discover PDE4 inhibitors. This effort resulted in a new H3PO3
mediated facile synthesis of pyrazolopyrimidine under mild and
microwave free conditions. A pyranone ring fused with a pyr-
azolopyrimidine moiety has been investigated and exemplified as
a potential template for PDE4 inhibitors. The lead compound
was found to be less toxic and was better tolerated than rolipram
in zebrafish embryos. Overall, a novel and seemingly safer
PDE4B inhibitor has been discovered for the potential treatment
of COPD and asthma.
Fig. 6 PDE4D inhibition by 7d: (A) enzyme assay and (B) cell based
reporter assay.
locomotor activity at initial time points validating the test
procedure. Embryos exposed to either rolipram or compound 7d
did not display any cardiac arrhythmias under basal conditions
(data not shown).
PMK thanks Dr V. Dahanukar and DRL analytical group.
PK, KP and MP thank Prof. J. Iqbal for support and Dr Kir-
anam Chatti for his help in conducting study in zebrafish embryo
model.
Each embryo was assigned a lesion score (5-0) based on the
morphological score assessment guideline suggested by Panzica-
Kelly et al.20 and the results are shown in Fig. 8. Statistical
analysis of 24 h time point data was performed by the Kruskal–
Wallis one-way analysis of variance by ranks followed by Dunn’s
post hoc test. Incubation of embryos with rolipram (at 10 or 20
mM) but not with compound 7d for 24 h resulted in statistically
significant increase in the abnormal lesions of embryos (p <
0.002). Collectively, the data obtained demonstrate that
compound 7d was less toxic and was better tolerated than roli-
pram in zebrafish embryos under the experimental conditions
tested.
Notes and references
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K. Mukkanti, R. Kapavarapu, C. L. T. Meda, K. V. L. Parsa and
Fig. 7 TNF-a inhibition by (A) compound 7d and (B) rolipram.
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Crystallographic Data Centre as supplementary publication
numbers CCDC 832934; (b) Crystal data of 6e: molecular formula
¼
ꢀ
C21H16IN3O2, formula weight
monoclinic, space group ¼ P21/C, a ¼ 7.3912 (4) A, b ¼12.3586(6)
¼
469.27, crystal system
ꢀ
¼
14 (a) P. Toto, J. Chenault, A. E. Hakmaoui, M. Akssira and
G. Guillaumet, Synth. Commun., 2008, 38, 674; (b) R. SanMartin,
E. M. de Marigorta and E. A. Dominguez, Tetrahedron, 1994, 50,
2255; (c) L. Ming, W. Shuwen, W. Lirong, Y. Huazheng and
Z. Xiuli, J. Heterocycl. Chem., 2005, 42, 925; (d) To the best of our
knowledge this is the first example of H3PO3-mediated synthesis of
pyrazolo[1,5-a]pyrimidines.
15 (a) D. K. Barange, V. R. Batchu, D. Gorja, V. R. Pattabiraman,
L. K. Tatini, J. M. Babu and M. Pal, Tetrahedron, 2007, 63, 1775;
(b) M. Pal, Synlett, 2009, 2896.
3
ꢀ
ꢀ
A, c ¼ 20.4375 (10) A, V ¼ 1861.53 (9) A , T ¼ 296 K, Z ¼ 4, Dc ¼
1.264 Mg mꢃ3
measured, 4043 independent reflections, 3497 observed reflections [I
2.0s(I)], R1_obs 0.024, goodness of fit 0.834.
,
m(Mo-Ka)
¼
1.74 mmꢃ1
,
15 729 reflections
>
¼
¼
Crystallographic data (excluding structure factors) for 6e have been
deposited with the Cambridge Crystallographic Data Centre as
supplementary publication numbers CCDC 832935.
18 (a) P. Wang, J. G. Myers, P. Wu, B. Cheewatrakoolpong, R. W. Egan
and M. M. Billah, Biochem. Biophys. Res. Commun., 1997, 19, 320; (b)
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A. Balagopal, N. P. Mohapatra, M. D. Wewers, L. S. Schlesinger,
J. S. Gunn and S. Tridandapani, PLoS Pathog., 2006, 2, 681.
19 For our effort on identification of TNF-a inhibitors, see: (a)
K. S. Kumar, P. M. Kumar, K. A. Kumar, M. Sreenivasulu,
A. A. Jafar, D. Rambabu, G. R. Krishna, C. M. Reddy,
R. Kapavarapu, K. Shivakumar, K. K. Priya, K. V. L. Parsa and
M. Pal, Chem. Commun., 2011, 47, 5010; (b) K. S. Kumar,
P. M. Kumar, M. A. Reddy, Md. Ferozuddin, M. Sreenivasulu,
A. A. Jafar, G. R. Krishna, C. M. Reddy, D. Rambabu,
K. S. Kumar, S. Pal and M. Pal, Chem. Commun., 2011, 47, 10263.
20 J. M. Panzica-Kelly, C. X. Zhang, T. L. Danberry, A. Flood,
J. W. DeLan, K. C. Brannen and K. A. Augustine-Rauch, Birth
Defects Res., Part B, 2010, 89, 382.
16 For recent and leading examples, see: (a) R. Mancuso, S. Mehta,
B. Gabriele, G. Salerno, W. S. Jenks and R. C. Larock, J. Org.
Chem., 2010, 75, 897; (b) A. K. Verma, T. Aggarwal, V. Rustagi
and R. C. Larock, Chem. Commun., 2010, 46, 4064; (c) S. Mehta
and R. C. Larock, J. Org. Chem., 2010, 75, 1652; (d) C.-H. Cho,
B. Neuenswander and R. C. Larock, J. Comb. Chem., 2010, 12, 278.
17 (a) Crystal data of 5d: molecular formula ¼ C28H27N3O2, formula
weight ¼ 437.53, crystal system ¼ monoclinic, space group ¼ P21/
ꢀ
ꢀ
ꢀ
C, a ¼ 15.2480 (13) A, b ¼ 12.5719(10) A, c ¼ 14.0243 (12) A, V ¼
2423.6 (4) A , T ¼ 296 K, Z ¼ 2, Dc ¼ 0.10 Mg mꢃ3, m(Mo-Ka) ¼
3
ꢀ
0.08 mmꢃ1
,
14 662 reflections measured, 3278 independent
reflections, 2433 observed reflections [I > 2.0s(I)], R1_obs ¼ 0.042,
goodness of fit ¼ 2.591. Crystallographic data (excluding structure
factors) for 5d have been deposited with the Cambridge
672 | Med. Chem. Commun., 2012, 3, 667–672
This journal is ª The Royal Society of Chemistry 2012