Bioorganic & Medicinal Chemistry Letters 24 (2014) 1166–1171

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Synthesis of 2H-1,3-benzoxazin-4(3H)-one derivatives containingindole moiety: Their in vitro evaluation against PDE4B

0960-894X/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.12.117

⇑ Corresponding authors. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.E-mail addresses: vbrmandava@yahoo.com (M.V.B. Rao), manojitpal@rediffmail.

com (M. Pal).

Raja Mohan Rao a, Bethala Jawahar Luther b, Chekuri Sharmila Rani c, Namburi Suresh c,Ravikumar Kapavarapu d, Kishore V. L. Parsa a, Mandava V. Basaveswara Rao c,⇑, Manojit Pal a,⇑a Dr. Reddys Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, Indiab Department of Chemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh, Indiac Department of Chemistry, Krishna University, Krishna Dist., Andhra Pradesh, Indiad Doctoral Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 November 2013Revised 26 December 2013Accepted 28 December 2013Available online 7 January 2014

Keywords:2H-1,3-Benzoxazin-4(3H)-oneIndoleBenzofuranPd/CPDE4

A number of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole or benzofuran moieties weresynthesized by using Pd/C–Cu mediated coupling-cyclization strategy as a key step. The o-iodoanilidesor o-iodophenol were coupled with 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-oneusing 10%Pd/C–CuI–PPh3 as a catalyst system and Et3N as a base to give the target compounds. All thesynthesized compounds were tested for their PDE4B inhibitory potential in vitro using a cell based cAMPreporter assay. Some of them showed fold increase of the cAMP level when tested at 30 lM. A represen-tative compound showed encouraging PDE4B inhibitory properties that were supported by its dockingresults.

� 2014 Elsevier Ltd. All rights reserved.

While asthma affects �300 million people worldwide at pres-ent, chronic obstructive pulmonary disease (COPD) is projectedto be the 3rd leading cause of death globally by 2020.1 Inhibitorsof phosphodiesterase 4 (PDE4) are considered as beneficial to treatCOPD or asthma.1,2 However, the first-generation (e.g., rolipram) aswell as second generation PDE4 inhibitors (e.g., cilomilast and rof-lumilast) suffered from side effects like nausea and emesis.1,2

Encouragingly, roflumilast (Daxas�, Nycomed) has been launchedin Europe for the treatment of chronic bronchitis in 2009 and inUS (Daliresp, Forest Lab) for exacerbations during COPD in 2012.Nevertheless, it is desirable to devote continued efforts towardsidentification of newer class of PDE4 inhibitors having fewer sideeffects.

In the inflammatory cells cAMP (cyclic adenosine monophos-phate) plays the role of a negative regulator of the primary activat-ing pathways such as cytokine release by T-cells. Levels of cAMP onthe other hand are regulated by cAMP-specific phosphodiesterases(PDE) isozyme for example, PDE4 predominantly expressed ininflammatory and immune cells in addition to brain.1,2 PDEs thesuper family of enzymes (that hydrolyze the phosphodiester bondof cAMP and cGMP) can be subdivided into 11 different groups or

isozymes, for example, PDE1 to PDE11. Inhibition of the PDE4effectively elevates the intracellular cAMP levels, thereby activat-ing specific protein phosphorylation cascades that in turn inhibitsthe release of inflammatory mediators such as cytokines [tumornecrosis factor-a (TNF-a), interleukin-2 (IL-2), interleukin-12(IL-12), leukotriene B4 (LTB4), interferon-c (IFN-c)], as well as acti-vation of inflammatory cells.3 The cAMP specific PDE4 isozymes4

(which require a divalent metal ion, for example, Zn for catalysis)are encoded by four genes (A-D) that give rise to four isoforms,for example, PDE4A to PDE4D.5 Since knockout mice studies haverevealed that PDE4B ablation suppresses TNF-a production, henceinhibition of PDE4B has been proposed to be beneficial for thedevelopment of more effective anti-inflammatory drugs to combatwith COPD and asthma.

Due to their wide range of pharmacological properties 2-substi-tuted indoles have been explored as a number of potentialtherapeutic agents6 including PDE4 inhibitors.7 The 2H-benzo[e][1,3]oxazin-4(3H)-one nucleus on the other hand has been foundto be integral part of several bioactive agents.8,9 Thus, combinationof both in a single entity via linking them through an appropriatelinker should provide a new framework from which the moleculesderived might show interesting pharmacological activities.Prompted by this idea we designed the template C (when X = NRor O) from A and B. Initially, a number of virtual molecules derivedfrom C were tested in silico against several drug targets including

R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171 1167

PDE4B without performing their actual chemical synthesis.Accordingly, the compound D emerged as a virtual hit againstPDE4B in this study. Docking studies (Fig. 2, see also Fig. S-1 inESI) [performed using Chemical Computing Group’s MolecularOperating Environment (MOE) software 2008.10 Version, ‘DOCK’application module, see the ESI] indicated that compound D uti-lized the conserved active binding site with its benzooxazinonecarbonyl and N-methanesulfonyl oxygen participated in H-bond-ing interactions with the PDE4B protein. The carbonyl oxygenformed H-bond with the His234 of metal binding pocket and sul-fonyl oxygen formed H-bond with the Gln443 of Q pocket in theactive site. These interactions were similar to that of the knowninhibitor rolipram (see Fig. S-2 in ESI). Indeed, the dock score ofcompound D (�19.79 K cal/mol) was comparable with that of roli-pram (�24.62 K cal/mol). The compound D has also shown anarene-arene interaction with the Phe446 residue of PDE4B(Fig. 2, see also Fig. S-1 in ESI). Encouraged by these observationswe then decided to synthesize a library of small molecules basedon C and screen them against PDE4B in vitro. As part of our ongo-ing effort on the identification of novel PDE4 inhibitors10 we nowreport the results of our recent study. To the best of our knowl-edge the in vitro pharmacological properties of these 2H-1,3-benz-oxazin-4(3H)-one derivatives (C) containing indole or benzofuranmoieties have not been explored earlier.

Figure 2. Binding mode of D in t

XO N

O

ON

N

O

O

A

B

C

group

group

X = NR, O

Figure 1. Design of novel and potential bioa

Several elegant and attractive methods11–13 have been reportedfor the construction of indole ring including the transition metalmediated methods of which the palladium catalyzed reactionsgained particular attention. As a less expensive catalyst systemthe use of Pd/C–CuI–PPh3 has also gained considerable interestfor the efficient synthesis of various heterocyclic structures14

including indoles.15,16 Compared to other Pd catalysts Pd/C is sta-ble, easy to handle and separable from the product and is recycla-ble.14 The use of Pd/C catalyzed reaction therefore is advantageous.All these features prompted us to explore a Pd/C mediated con-struction of indole ring as a key step for the synthesis of our targetcompounds C and D (or 3) (Scheme 1).

The starting alkyne that is, 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-one (2) required for the synthesis ofour target compound 3 was prepared according to a reportedprocedure as shown in Scheme 2.8 Thus, the methyl salicylate 5obtained from 4 was converted to 2-hydroxy-N-(2-hydroxyethyl)benzamide (6) which was then treated with paraformaldehydeunder acidic conditions followed by the hydrolysis of the interme-diate formed to give 3-(2-hydroxyethyl)-2H-benzo[e][1,3]oxazin-4(3H)-one (7). The subsequent propargylation of 7 afforded thedesired terminal alkyne 2 that was used for the coupling-cyclization with a range of o-iodoanilides and o-iodophenol (1) inthe presence of 10% Pd/C, PPh3, CuI and Et3N in MeOH (Table 1).

he PDE4B (PDB code-1XMY).

NO N

O

O

SD

O2N

O O

ctive molecules C (and D) from A and B.

XO N

O

ORI

XH

R 10% Pd/C, PPh3CuI, Et3N,

MeOH,reflux4-6h

R = Me, Cl, H, CF3, I,COCH3, F, NO2

, CN.

O

N

OO

+

1 2 3

X = NSO2Me, O

Scheme 1. Pd/C-mediated synthesis of 3-[(indol-2-ylmethoxy)ethyl]-2H-1,3-benzoxazin-4(3H)-one derivatives (3).

OH

O

OH

OH

O

OMeOxalyl chlorideMeOH

80o C 170o C OH

O

NH

OH(CH2O)n, AcOHHCl, CHCl3

Na2CO3,CH3OH

O

N

OOH

O0 C -RT40%

O

N

OO

K2CO3Acetonert, 5-6 h

90% 70%

72%

H2NOH

4 5 6

7

Br

2

Scheme 2. Synthesis of 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-one (2).8

Table 1Synthesis of 3-[(indol-2-ylmethoxy)ethyl]-2H-1,3-benzoxazin-4(3H)-ones/3-[(benzofuran-2-ylmethoxy)ethyl]-2H-1,3-benzoxazin-4(3H)-one (3) (Scheme 1)a

Entry o-Iodoanilide/o-iodophenol (1) Time (h) Product (3) Yieldb (%)

1

NH

I

SO

O

1a4

NS

ON

O

O

O

O

3a

65

2

NH

I

SO

O

Cl

1b4.5

NS

ON

O

O

O

O

Cl

3b

62

3

NH

I

SO

O

1c4

NS

ON

O

O

O

O

3c

68

4

NH

I

SO

O

F3C

1d5

NS

ON

O

O

O

O

F3C

3d

64

1168 R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171

Table 1 (continued)

Entry o-Iodoanilide/o-iodophenol (1) Time (h) Product (3) Yieldb (%)

5

NH

I

SO

O

I

1e4

NS

ON

O

O

O

O

I

3e

60

6NH

I

SO

O

O

1f

4.5NS

ON

O

O

O

O

O

3f

64

7

NH

I

SO

O

F

1g 5.5

NS

ON

O

O

O

O

F

3g

69

8

NH

I

SO

O

O2N

1h6

NS

ON

O

O

O

O

O2N

3h

61

9

NH

I

SO

O

NC

1i6

NS

ON

O

O

O

O

NC

3i

70

10OH

I

1j 4

O

ON

O

O

3j

69c

a All the reactions were performed using o-iodoanilide (1, 2 mmol), alkyne (2, 2 mmol), 10% Pd/C (0.02 mmol), CuI (0.02 mmol), PPh3 (0.04 mmol) and Et3N (0.5 mL) inMeOH (5.0 mL) at refluxing temp under a nitrogen atmosphere.

b Isolated yield.c o-Iodophenol (1j, 2 mmol) was used in place of o-iodoanilide.

R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171 1169

The presence of various groups for example, Me, F, Cl, I, CF3, COMe,NO2 and CN (entries 1–9, Table 1) on the anilide ring were toler-ated as the reactions proceeded well in all these cases affordingthe desired indoles 3 in acceptable yields. The use of o-iodophenol(1j) in place of o-iodoanilide however afforded the correspondingbenzofuran derivative 3j (entry 10, Table 1) following thecoupling-cyclization pathway.

Mechanistically (Scheme 3), the one-pot synthesis of compound3 under the catalysis of Pd/C–CuI–PPh3 seemed to proceed via (i)the generation of actual catalytic species, (ii) alkynylation of iodo

compound 1 and (iii) intramolecular cyclization of the resultinginternal alkyne. Thus, in the initial step an active Pd(0) species isgenerated via a Pd leaching process14 into the solution [from theminor portion of the bound palladium (Pd/C)] followed by interac-tions with the phosphine ligands. The dissolved Pd(0)–PPh3 com-plex then produces the organo-Pd(II) species E-1 via oxidativeaddition with 1. The E-1 then undergoes trans organometallationwith copper acetylide generated in situ from CuI and terminal al-kyne 2 followed by reductive elimination of Pd(0) to afford theinternal alkyne E-2. The E-2 thus formed subsequently undergoes

Pd/C Pdleaching

Pd insolution

PPh3

Pd(0)-PPh3complex insolution

Cu

2

CuI/Et3N+ Pd(0)

Precipitation ofPd on C at the endof catalytic cycle

CuI, BH+

BH+

B

BH+

BH+I-

BH+I-

CuI, B

3

E-2

Generationof actualcatalyticspecies

The catalytic cycleIntramolecularcyclization

1

(B = Et3N)

E-1

B

I

X

R

BH+

Pd

X

R I

BH+

Pd

X

R

BH+X

R

X

R Cu

Scheme 3. Proposed mechanism for the Pd/C-Cu catalyzed one-pot synthesis of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole or benzofuran moieties (3).

[3h]µM

0.001 0.01 0.1 1 10 1000

2

4

6

8

10

Fold

incr

ease

ove

r con

trol

Figure 4. Dose-dependent fold increase of the cAMP level by compound 3h (PDE4BHEK 293 cell based reporter assay).

1170 R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171

Cu-mediated 5-exo-dig ring closure in an intramolecular fashion togive the product (3). Overall, the catalytic cycle seemed to work insolution rather than on the surface and at the end of the reactionPd was re-precipitated on the charcoal surface.

All the synthesized compounds were tested for their PDE4Binhibitory potential in vitro. In a cell based cAMP reporter assay17

fold increase of the cAMP level caused by the test compounds overforskolin control was determined. The known inhibitor rolipramwas used as a reference compound in this assay. Among the com-pounds tested indole derivatives 3b, 3d, 3g, 3i and the benzofuranderivative 3j showed fold increase when tested at 30 lM (Fig. 3).However, the compound 3h showed >5 fold increase over controlwhen tested at the same concentration which is comparable tothe rolipram’s >7 fold increase in the same assay. It is evident thatthe presence of an electron withdrawing group for example, Cl, CF3,F, NO2 or CN at the C-5 of the indole ring seemed to be favorable forthe activity and the NO2 group was found to be the most effectiveamong them. Notably, an acetyl group at the same position (e.g.,compound 3f) was found to be detrimental for activity perhapsdue to its bulkier size than other electron withdrawing groupstested. In contrast the benzofuran derivative showed significantfold increase in spite of not having any electron withdrawing groupat the C-5 position of the ring. Nevertheless, the compound 3h wasidentified as the best among compounds tested. In a dose responsestudy compound 3h showed dose-dependent fold increase of thecAMP level (Fig. 4) indicating its potential as an inhibitor. The ob-served PDE4B inhibitory properties of compound 3h (or compound

Figure 3. PDE4B HEK 293 cell based reporter screen of compound 3 (Y-axis: foldelevation of cAMP over forskolin control).

D, Fig. 1) was also supported by the docking studies as presented inFig. 2 that showed good binding of 3h with the PDE4B.

In conclusion, a number of 2H-1,3-benzoxazin-4(3H)-onederivatives containing indole or benzofuran moieties were initiallydesigned as a novel class of bioactive compounds. In silico studiesof these compounds against a number of drug target indicated thatthey could be explored as potential inhibitors of PDE4. Accordingly,compounds were synthesized by using Pd/C-Cu mediated coupling-cyclization strategy as a key step. The o-iodoanilides oro-iodophenol were coupled with 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-one using 10%Pd/C–CuI–PPh3 as acatalyst system and Et3N as a base to give the target compounds.All the synthesized compounds were tested for their PDE4B inhibi-tory potential in vitro using a cell based cAMP reporter assay. Someof them showed fold increase of the cAMP level when tested at30 lM. A representative compound 3h showed encouraging PDE4Binhibitory properties and was supported by its docking resultsobtained prior to its synthesis. Overall, the newly designed

R. M. Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1166–1171 1171

framework presented here seems to provide a useful basis for thedevelopment of novel future PDE4 inhibitors for the potentialtreatment of asthma and COPD.

Acknowledgments

Authors thank management of DRILS for encouragement andsupport.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.117.

References and notes

1. Kodimuthali, A.; Jabaris, S. L.; Pal, M. J. Med. Chem. 2008, 51, 5471.2. Houslay, M. D.; Schafer, P.; Zhang, K. Y. J. Drug Discov. Today 2005, 10, 1503.3. Jin, S.-L. C.; Lan, L.; Zoudilova, M.; Conti, M. J. Immun. 2005, 175, 1523.4. Houslay, M. D.; Sullivan, M.; Bolger, G. B. Adv. Pharmacol. 1998, 44, 225.5. Jeon, Y. H.; Heo, Y.-S.; Kim, C. M.; Hyun, Y.-L.; Lee, T. G.; Ro, S.; Cho, J. M. Cell

Mol. Life Sci. 2005, 62, 1198.6. Holenz, J.; Pauwels, P. J.; Díaz, J. L.; Mercè, R.; Codony, X.; Buschmann, H. Drug

Discov. Today 2006, 11, 283.7. Hulme, C.; Moriarty, K.; Miller, B.; Mathew, R.; Ramanjulu, M.; Cox, P.; Souness,

J.; Page, K. M.; Uhl, J.; Travis, J.; Huang, F.-C.; Labaudiniere, R.; Djuric, S. W.Bioorg. Med. Chem. Lett. 1998, 8, 1867.

8. Benedini, F.; Bertolini, G.; Cereda, R.; Dona, G.; Gromo, G.; Levi, S.; Mizrahi, J.;Sala, A. J. Med. Chem. 1995, 38, 130.

9. Madhavan, G. R.; Chakrabarti, R.; Reddy, K. A.; Rajesh, B. M.; Balraju, V.; Rao, P.B.; Rajagopalan, R.; Iqbal, J. Bioorg. Med. Chem. 2006, 14, 584.

10. For our earlier effort, see: (a) Kumar, K. S.; Kumar, P. M.; Kumar, K. A.;Sreenivasulu, M.; Jafar, A. A.; Rambabu, D.; Krishna, G. R.; Reddy, C. M.;Kapavarapu, R.; Kumar, K. S.; Priya, K. K.; Parsa, K. V. L.; Pal, M. Chem. Commun.2011, 5010; (b) Reddy, G. R.; Reddy, T. R.; Joseph, S. C.; Reddy, K. S.; Reddy, L. S.;Kumar, P. M.; Krishna, G. R.; Reddy, C. M.; Rambabu, D.; Kapavarapu, R.;Lakshmi, C.; Meda, T.; Priya, K. K.; Parsa, K. V. L.; Pal, M. Chem. Commun. 2011,7779; (c) Kumar, P. M.; Kumar, K. S.; Mohakhud, P. K.; Mukkanti, K.;Kapavarapu, R.; Parsa, K. V. L.; Pal, M. Chem. Commun. 2012, 431; (d) Sunke,R.; Adepu, R.; Kapavarapu, R.; Chintala, S.; Meda, C. L. T.; Parsa, K. V. L.; Pal, M.Chem. Commun. 2013, 3570; (e) Prasad, B.; Sreenivas, B. Y.; Krishna, G. R.;Kapavarapu, R.; Pal, M. Chem. Commun. 2013, 6716.

11. (a) Humphrey, G. R.; Kuethe, J. T. Chem. Rev. 2006, 106, 2875; (b) Cacchi, S.;Fabrizi, G. Chem. Rev. 2005, 105, 2873; (c) Gribble, G. W. in ComprehensiveHeterocyclic Chemistry II, ed. Katrizky, A. R.; Rees C. W.; Scriven, E. F. V.Pergamon, Oxford, 1996, vol. 2, p. 20.

12. Krüger, K.; Tillack, A.; Beller, M. Adv. Synth. Catal. 2008, 350, 2153.13. Ackermann, L. Synlett 2007, 507.14. Pal, M. Synlett 2009, 2896.15. (a) Pal, M.; Subramanian, V.; Batchu, V. R.; Dager, I. Synlett 2004, 1965; (b)

Layek, M.; Lakshmi, U.; Kalita, D.; Barange, D. K.; Islam, A.; Mukkanti, K.; Pal, M.Beilstein J. Org. Chem. 2009, 5, No. 46. http://dx.doi.org/10.3762/bjoc.5.46.

16. (a) Alinakhi Prasad, B.; Reddy, U.; Rao, R. M.; Sandra, S.; Kapavarapu, R.;Rambabu, D.; Krishna, G. R.; Reddy, C. M.; Ravada, K.; Misra, P.; Iqbal, J.; Pal, M.Med. Chem. Commun. 2011, 2, 1006; (b) Rao, R. M.; Reddy, U.; Alinakhi, C. H.;Mulakayala, N.; Alvala, M.; Arunasree, M. K.; Poondra, R. R.; Iqbal, J.; Pal, M. Org.Biomol. Chem. 2011, 9, 3808.

17. Wang, P.; Myers, J. G.; Wu, P.; Cheewatrakoolpong, B.; Egan, R. W.; Billah, M. M.Biochem. Biophys. Res. Commun. 1997, 234, 320.