Green, unexpected synthesis of bis-coumarin derivatives as potent anti-bacterial and anti-inflammatory agents

Accepted Manuscript Green, unexpected synthesis of bis-coumarin derivatives as potent anti-bacterial and anti-inflammatory agents Bahubali M. Chougala, S. Samundeeswari, Megharaja Holiyachi, Nirmala S. Naik, Lokesh A. Shastri, Suneel Dodamani, Sunil Jalalpure, Sheshagiri R. Dixit, Shrinivas D. Joshi, Vinay A. Sunagar PII:

S0223-5234(17)30874-7

DOI:

10.1016/j.ejmech.2017.10.072

Reference:

EJMECH 9864

To appear in:

European Journal of Medicinal Chemistry

Received Date: 24 July 2017 Revised Date:

6 October 2017

Accepted Date: 26 October 2017

Please cite this article as: B.M. Chougala, S. Samundeeswari, M. Holiyachi, N.S. Naik, L.A. Shastri, S. Dodamani, S. Jalalpure, S.R. Dixit, S.D. Joshi, V.A. Sunagar, Green, unexpected synthesis of bis-coumarin derivatives as potent anti-bacterial and anti-inflammatory agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.072. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Green, unexpected synthesis of bis-coumarin derivatives as potent antibacterial and anti-inflammatory agents Bahubali M. Chougalaa, Samundeeswari S.a, Megharaja Holiyachia, Nirmala S. Naika, Lokesh A. Shastri a,*,Suneel Dodamanib, Sunil Jalalpureb,c, Sheshagiri R. Dixitd, Shrinivas D. Joshid, Vinay

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A. Sunagare a

Department of Chemistry, Karnatak University, Dharwad, 580 003, Karnataka, India.

b

Dr. PrabhakarKore Basic Science Research Center, KLE University, Belagavi, 590010,

c

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Karnataka, India.

KLE University’s College of Pharmacy, Nehru Nagar, Belagavi, 590010, Karnataka, India.

d

Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,

e

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S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580002, Karnataka, India. Department of Chemistry, G.S.S. College, Belagavi, Karnataka, India.

N

O

HN

O

O

O

TE D

R

80

oC

O

X

O

120

o

C

O

HO

EP

HOOC

O O

O R

R

O

AC C

O

O

O

O H2N

O

O O

R' O R O

O

HCO2H

*Corresponding author: e-mail: [email protected] (Lokesh A. Shastri)

ACCEPTED MANUSCRIPT Green, unexpected synthesis of bis-coumarin derivatives as potent antibacterial and anti-inflammatory agents Bahubali M. Chougalaa,Samundeeswari S.a,Megharaja Holiyachia,Nirmala S. Naika,Lokesh A. Shastri a,*,SuneelDodamanib, Sunil Jalalpureb,c, Sheshagiri R. Dixitd,Shrinivas D. Joshid, Vinay A.Sunagare Department of Chemistry, Karnatak University, Dharwad, 580 003, Karnataka, India.

b

Dr. Prabhakar Kore Basic Science Research Center, KLE University, Belagavi, 590010,

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a

Karnataka, India.

KLE University’s College of Pharmacy, Nehru Nagar, Belagavi, 590010, Karnataka, India.

d

Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,

SC

c

S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580002, Karnataka, India. Department of Chemistry, G.S.S. College, Belagavi, Karnataka, India.

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e

*Corresponding author: e-mail: [email protected](Lokesh A. Shastri)

ABSTRACT:

A green and efficient protocol has been developed and a series of coumarin based

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pyrano[3,2-c]chromene derivatives (2) have been synthesized using multi-component reaction (MCR) approach. Unexpected 3-coumarinyl-3-pyrazolylpropanoic acids (3) and C4C4 chromenes (5) have been isolated instead of expected product 4 by the reaction of compound (2) in formic acid at 90 oC for about 4-5 h and at 130 oC for about 8-10 h

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respectively. Further, C4-C4chromenes (5) formation was confirmed by intramolecular cyclization of compounds (3). These compounds were screened for their biological activities

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and most of them exhibited promising antibacterial activity. The anti-inflammatory assay was evaluated against HRBC membrane stabilization method and the compounds exhibit excellent anti-inflammatory activity. Molecular docking study has been performed for all the synthesized compounds with Klebsiella pneumoni aeacetolactate synthase and results obtained are quite promising.

Key words: L-Proline, Pyrano[3,2-c]chromene, Protein denaturation, HRBC membrane

1

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1. Introduction In the past few decades, the overuse of antibiotics have dramatically increased due to the spread of multidrug-resistant bacteria[1,2]. The multidrug-resistant bacteria showing highest incidence rates worldwide include methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa (MRPA), vancomycin-resistant

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Enterococci (VRE) and more recently vancomycin-resistant Staphlyococcus aureus (VRSA)[3,4].Majority of these pathogens arise from the commensal bacteria in humans, which cause opportunistic infections when immune compromised or under certain medical conditions[5]. Because of the diminishing timeline between drug implementation in medical

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conditions and resistance, the current antibiotics continue to lose their efficacy[6]. Therefore, the investigation and development of new antibacterials that are not subject to the existing

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mechanisms of resistance offer a solution to this growing unmet medical need[7].

It has long been believed that small alterations in the structure of certain molecules can greatly affect their receptor binding affinity and biological activity[8]. Several studies have demonstrated that the introduction of various heterocyclic rings on parent moiety is effective in the production of variety of compounds with potential biological

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activities[9].Oftentimes, known heterocyclic moieties possessing antibiotic properties are modified to prolong their use for a known target[10].These may include the variations in the substitution pattern around the main heterocyclic nuclei, ring opening and ring closer[11-13].

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Meanwhile, inflammation is due to the reaction of living tissues towards injury and it comprises systemic and local responses[14].The main action of anti-inflammatory agents are

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the overproduction of leukotrienes and prostanoids (prostaglandins and thromboxane) in arachidonic acid pathway and inhibition of Cyclooxegenase enzymes which are responsible for the conversion of arachidonic acid to prostaglandins[15,16].Since, human red blood cell (HRBC) membranes are well matched to these lysosomal membrane constituents, the prevention of hypotonicity induced HRBC membrane lysis was taken as a measure in estimating the anti-inflammatory property. In the pathways of the anti-inflammatory process, the implication of non-steroidalanti-inflammatory drugs/ heterocyclic nucleus is particularly important[17]and they are widely used to treat the effects of inflammation through inhibition of cyclooxygenase enzymes (COX)[18].Among the immense figures of heterocycles, coumarins are one of the alluring heterocyclic aromatic organic compound which have important pharmacophore and privileged structure in the recent past[19,20].Substituted 2

ACCEPTED MANUSCRIPT coumarins have considerable interest as compounds with a wide spectrum of biological activity and low toxicity, allowing them to interact easily with the biopolymers of the living systems.

Against this background our efforts are extended on antibacterial and antiinflammatory drug discovery[11], we have designed a cogent approach for the synthesis of

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new biologically active scaffolds having chemotype featuring of chromene core at C4 position of coumarins via MCRs approach using L-prolineas a catalyst.

2. Results and Discussion

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2.1. Chemistry

The introduction of one more coumarin nucleus on parent coumarin molecule could

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improve their pharmaceutical activity, therefore an attempt has been made here to enhance the biological activity, a series of compounds (2a-j) were designed and synthesized (Scheme 1). The unexpected 3-coumarinyl-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)propanoic acids (3) and

4-coumarinyl 3,4-dihydropyrano[3,2-c]chromene-2,5-dione (5) (Scheme 3) were

isolated. R'

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NC

CHO R O

OH

O

O

O

H2N

75 oC, 2 h

O

R'

Catalyst, H2O

O R

O

O

1

O

EP

2

R= 6-Me, 6-OMe, 6-Cl, 7-Me, 7,8-Benzo; R'= CN, COOEt

AC C

Scheme 1. Synthesis of 2-amino-4,5-dihydro-5-oxo-4-(2-oxo-2H-chromen-4-yl)pyrano[3,2c]chromenes (2a-j)

The synthetic route for reaction leading to the desired 4-coumarinyl 2-amino-4,5-

dihydro-pyrano[3,2-c]chromene-3-carbonitriles(2)was

achieved

by

the

key

starting

compound 4-formyl coumarin (1)[21],4-hydroxy coumarin and malononitrile in one step multi component approach using green protocol.

Initially, the reaction was performed without catalyst using three component one step approach by the reaction of compound (1), (R= 6-CH3) malononitrile and 4-hydroxy 3

ACCEPTED MANUSCRIPT coumarin in water at 75 °C[22]. In this condition, it was noticed that the product formation around 30% (Table-1, Entry-1). While, there is no improvement in the yield with increase in temperature and time.

The above experimental results encouraged us to screen for ecofriendly catalyst using water as a solvent. Carbonates are the excellent mild bases, therefore sodium and potassium

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carbonates have been employed and achieved higher yield as compared to without catalyst (Table-1, Entry-2 and 3). Similarly, NaBr and NaCl afforded lower yield (Table-1, Entry-4 and 5). As we know that, L-proline is excellent organic green catalyst which has been used in organic synthesis, due to its dual nature, as proton abstracting and proton donor. Thus the L-

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proline catalyzed reaction afforded excellent yield (95%) (Table-1, Entry-6). In case of sodium benzoate (Table-1, Entry-8), sodium bisulphate (Table-1, Entry-9) afforded more

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than 75% and iodine catalyzed reaction gave around 86% yield.

The catalyst optimization results showed their efficiency for the formation of compound (2). Among all the catalyst, L-proline is more efficient and highly effective catalyst and it proved its excellency in the organic synthesis.

Entry

Catalyst

1

-

2

Na2CO3

3 4

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Table 1. Screening of catalysts in water for the synthesis of pyrano[2,3-c]chromenes. Temp. (°C)

Time (h)

Yield (%)**

Water

75

2.0

33

Water

75

2.0

76

K2CO3

Water

75

2.0

81

NaBr

Water

75

2.0

55

AC C

EP

Solvent

5

NaCl

Water

75

2.0

52

6

L-Proline

Water

75

2.0

95

7

L-Cysteine

Water

75

2.0

89

8

Sodium benzoate

Water

75

2.0

77

9

Sodium bisulfite

Water

75

2.0

78

10

Iodine

Water

75

2.0

86

** Isolated yield The mechanism (Scheme 2) of reaction is believed to involve the Knoevenagel condensation[23]of substituted 4-formylcoumarin and malononitrile or ethylcyanoacetate to 4

ACCEPTED MANUSCRIPT form an alkene intermediate (A). The intermediate undergoes Michael type of nucleophilic addition of 4-hydroxycoumarin followed by an intra-molecular cyclization to give polyfunctionalized pyrano[2,3-c]chromenes(2). All the synthesized compounds are listed inTable-2.

CHO Knoevenagel condensation

CN R O 1

O A

O

HN C O

R O

O

HO

R'

Mechanism

for

the

O

O

O

synthesis

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oxopyrano[3,2-c]chromenes.

O H

R

O

2

O

C

O

R O

O

N

HO

R'

O

O

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R'

Scheme2.

R

R'

O

OH

SC

H2N

H

R'

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N C

O

of

4-coumarinyl-2-amino-4,5-dihydro-5-

Entry

Compound Structure

Table 2.Pyrano[2,3-c]chromene derivatives (2a-j). Entry

Compound Structure

2a

O

O

EP

H2N NC

H3C

2f

O

AC C

O

H2N

2b

H2N

O

H2N

2g

O O

H2N O

2h

O O

O

O O O

O O

EtO2C Cl

O O

5

O

O

O

O

Cl

O

EtO2C H3CO

O

NC

O

O

O

H3CO

O

EtO2C H3C

O

NC

2c

H2N

O

ACCEPTED MANUSCRIPT H2N

2d

O

H2N

O

NC

2i

O O

EtO2C O

O H3C

O

H2N

H2N

O O

NC

O

O

O O

EtO2C

2j

O

O

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2e

H3C

O

O

O

O

O

SC

Based on the biological applications of pyrimidine nucleus it is worthy to synthesize pyrimidine fused with pyrano coumarins (4), which is outlined in Scheme 3 using compound (2). To the best of our knowledge amines and nitrile functional groups at 1 and 2 positions

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leads to pyrimidine nucleus in the presence of formic acid[24]. Unfortunately, the reported methods for the synthesis of (4) from coumarin-4-(2-amino-3-cyano) pyrane (2) was not successful. Moreover, the unexpected product (3) obtained was identified [13] by spectral analysis. The formation of product (3) was optimized at different temperature and time, results obtained are listed in the Table-3. Initially, heating of compound (2)at 80 °C for 3-4 h

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with formic acid leads to the desired product (3) in 70% yield (Table -3, Entry -1), whereas at 90 °C for about 4-5 h, exclusively compound (3) is the final product (Table-3, Entry-2). Gradual increase in temperature and time leads to two spots by TLC, one spot corresponds to the expected product (3) (60%) and the other corresponds to unexpected compound (5) (30%)

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(Table -3, Entry- 4), which was identified latter by the intramolecular cyclization of compound (3) using thionyl chloride (Scheme 5). Interestingly, the higher yield of compound

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(5) was noticed at 130 °C for about 8-10 h (Table-3, Entry-6). The plausible mechanism is given in Scheme 4 for the formation of compound (5). All the synthesized compounds (3) and (5) are listed in Table- 4 and 5.

Table 3. Optimization condition for the synthesis of compounds 3 and 5. Entry

Conditions

Temp. (°C)

Time (h)

Compound Yield (%)** 3a

5a

1

Compound 2a+HCOOH

80

3-4

70

--

2

Compound 2a+HCOOH

90

4-5

95

--

4

Compound 2a+HCOOH

110

6-8

60

30

6

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Compound 2a+HCOOH

110

8-10

50

40

6

Compound 2a+HCOOH

130

8-10

10

88

** Isolated yield HO O

HOOC HCOOH H2N

O

HN

X HCOOH

O

O

O

90 0C

O

R

O

R'

3

O

O

O

R

R O

O

O

O

O

O

O

O

HCOOH

2

4

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N

130 0C

O

SC

R

O 5

O

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R= 6-Me, 6-OMe, 6-Cl, 7-Me, 7,8-Benzo;R'= CN, COOEt.

Scheme 3. Synthesis of substituted 3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-3-(2-oxo-2Hchromen-4-yl)propanoic acids (3a-e) and Synthesis of substituted 3,4-dihydro-4-(2-oxo-2Hchromen-4-yl)pyrano[3,2-c]chromene-2,5-diones (5a-e).

Table

4.Substituted

3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-3-(2-oxo-2H-chromen-4-

Entry

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yl)propanoic acids (3a-e).

Compound Structure

Entry

Compound Structure HO

HO

O

HOOC H3C

3d

AC C

O

3b

3c

O

HOOC O

O

EP

3a

H3C

O

HO

HO

O

HOOC H3CO

O

O

3e

O

HOOC O

O O

O

O

HO O

HOOC Cl

O O

O

7

O

ACCEPTED MANUSCRIPT H HO

HO

O

O

O

HO

HO H

O

H O

O

O H

O 3

O

O

O

O

-H2O

R

O

O

O

O

R

R

O

O

O

O R O

O

O

5

chromen-4-yl)pyrano[3,2-c]chromene-2,5-diones.

O

HO

O

O

SOCl2, CH3CN

O

70oC, 2-3 h

R O

O

R

O 5

O

O

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3

SC

O

HOOC

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Scheme 4. Plausible mechanism for the synthesis of substituted 3,4-dihydro-4-(2-oxo-2H-

R= 6-Me, 6-OMe, 6-Cl, 7-Me, 7,8-Benzo.

Scheme 5. Synthesis of substituted 3,4-dihydro-4-(2-oxo-2H-chromen-4-yl)pyrano[3,2c]chromene-2,5-diones (5a-e).

Table

5.Substituted

3,4-dihydro-4-(2-oxo-2H-chromen-4-yl)pyrano[3,2-c]chromene-2,5-

Entry

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diones (5a-e).

Compound Structure O

Compound Structure O

O

O

5a

Entry

O O

5d O

O

EP

H3C

AC C

O

O

O

O O

O

O

O

O

O

5b

H3CO

O

H3C

5e

O

O O

O

O

O

O O

5c Cl

O O

O

Formation of products (2), (3) and (5) are well supported by spectroscopic analysis. In case of compound (2b), IR spectrum exhibited an intense stretching bands at 1670, 1701 and 8

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2192 cm due to lactone carbonyls and nitrile group respectively. The NH2 symmetric and asymmetric stretching bands are observed at 3397 and 3286 cm-1 respectively. Molecular ion peak of the compound (2b) observed at m/z 414 in mass spectrum confirms the desired product. Formation of product (2b) was further confirmed by 1H-NMR, wherein the C'10-H of chromene is resonated as doublet of doublet at 7.91 δppm (J = 8.0 and 1.6 Hz) and C'8-H of chromene is resonated at 7.75-7.74 δppm as multiplet. The NH2 protons of pyran appeared as

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singlet at 7.59 δppm. The C'9-H and C'7-H of chromene resonated around 7.54-7.48 δppm as multiplet. The C8-H of coumarin is appeared as a doublet at 7.41 δppm (J = 8.8 Hz), whereas C7-H of coumarin appeared as doublet of doublet at 7.27 δppm (J = 9.2 and 2.8 Hz). Two singlets at 6.44 and 5.23 δppm are due to C3-H of coumarin and C4-H of pyran respectively.

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Whereas, C6-OCH3 of coumarin is observed as a singlet at 3.82 δppm.

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Compound (3d), exhibited two intense bands in IR spectrum at 1748 and 1722 cm-1 due to lactone and carboxylic acid carbonyl respectively. The band at 3327 cm-1 is due to OH group. Molecular ion peak of the compound (3d) observed at m/z 392 in mass spectrum confirms the desired product. In 1H-NMR spectrum the propanoic acid OH and phenolic OH proton of chromene appeared as a broad singlet at 11.16 δppm. The C'10-H of chromene resonated at 7.91 δppm as doublet of doublet with coupling constants 8.0 and 1.6 Hz, whereas

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C'8-H of chromene is resonated at 7.75-7.74 δppm as multiplet. The C5-H of coumarin resonated as a doublet at 7.80 δppm (J= 7.2 Hz). The C'9-Hand C'7-H of chromene resonated as multiplet at 7.54-7.48 δppm. The C8-H of coumarin is appeared as a singlet at 7.21 δppm and C6-H of coumarin is observed as doublet at 7.13 δppm (J = 8.0 Hz). The singlet at 6.44

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δppm is due to the C3-H of coumarin and triplet at 5.10 δppm (J= 6.8 Hz) due to C3-H of propanoic acid. The two doublet of doublets at 3.25 δppm (J = 16.8 and 8.8 Hz) and 3.00

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δppm (J = 16.4 and 6.4 Hz) are due to CH2 of propanoic acid. The C7-CH3 of coumarin moiety is observed as singlet at 2.34 δppm

Compound (5d), shows two intense stretching bands in IR spectrum at 1733 and 1709

-1

cm due to lactone carbonyls. Molecular ion peak of the compound (5d) observed at m/z 374 in mass spectrum confirms the desired product. In 1H-NMR spectrum, the C'10-H of chromene resonated at 8.12 δppm as doublet with coupling constant 8.0 Hz, wherein the C5-H of coumarin is resonated as doublet at 7.92 δppm (J = 7.6 Hz). The C'9-H, C'8-H and C'7-H of chromene resonated around 7.57-7.42 δppm as multiplet and C6-H of coumarin observed as a doublet at 7.29 δppm (J = 10.4 Hz).The two singlets at 7.08 and 6.23 are due to C8-Hand C39

ACCEPTED MANUSCRIPT H of coumarin respectively. The C4-H of dihydro chromene observed as a triplet at 4.97 δppm (J= 8.4 Hz). The CH2 of dihydro chromene ring observed as two doublet of doublets at 2.97 δppm (J = 15.2 and 8.0 Hz) and 2.93 δppm (J = 16.4 and 7.6 Hz). The C7 methyl group of coumarin moiety observed as a singlet at 2.40 δppm.

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2.2. Single Crystal X-ray Studies For X-ray diffraction experiment, the single crystals of compound (2g) were developed by slow evaporation of methanol as a solvent. Suitable crystal was selected by using polarizing microscope and then mounted on a Mitegen Micromount. The data was

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collected from diffractometer at 296(2) K using graphite-monochromated MoKα radiation (λ = 0.71073 Å) and Bruker SAINT Software package with a narrow-frame algorithm. The crystal structure was solved by direct method using SHELXS-97 and refined by full-matrix

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least-squares refinement on F2 with anisotropic displacement parameters for non-hydrogen atoms using SHELXL-97[26]. Crystal information files (CIF) have been deposited at Cambridge Crystallographic Data Centre (CCDC), the CCDC number of compound(2g) is 1473917.

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X-ray study and molecular structure of ethyl 2-amino-4,5-dihydro-4-(6-methoxy-2oxo-2H-chromen-4-yl)-5-oxopyrano[3,2-c]chromene-3-carboxylate (2g) revealed that, it exists in the crystal phase as a one classical intramolecular hydrogen bonding between a carbonyl group of ester and a hydrogen atom of amine group. The crystal structure of

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compound (2g) is non-planar in nature with a significant dihedral angle 86.35° of coumarin and pyrano[3,2-c]chromene moiety. The ORTEP and molecular packing diagrams of

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compound (2g) are given in Fig. 1and 2. Table5 shows the crystal refinement parameters of compound (2g).

O

H N

O O

O

H O

O O

O

Figure 1.Structure and ORTEP diagram with labels showing 50% displacement of ellipsoids. 10

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ACCEPTED MANUSCRIPT

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Figure 2.Packing daigram and hydrogen bonding with π....π stacking interactions of (2g).

Table 5.The crystal data and structure refinement parameters of compound (2g). Identification code

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2g

Chemical formula

C25H19NO8

Formula weight

461.41

Temperature

296(2) K

MoKα radiation, λ

0.71073 Å

Space group

Triclinic

Z

AC C

Density

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Unit cell dimensions

Volume

P-1

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Crystal system

a=9.1505(3) Å

α=76.367(2)°

b=9.5489(3) Å

β=82.759(2)°

c=13.5841(4) Å

γ=67.437(2)°

1064.37(6) Å3 2 1.440 g/cm3

Absorption coefficient µ

0.109 mm−1

F(000)

480.0

Crystal size

0.21 × 0.14 × 0.10 mm3

2θ range for data collection/°

3.08 to 53.42°

Index ranges

-11 ≤ h ≤ 11, -12 ≤ k ≤ 12, -17 ≤ l ≤ 17

Reflections collected

16643

Independent reflections

4490 [Rint= 0.0671, Rsigma= 0.0544]

Data / restraints / parameters

4490/0/354

Goodness-of-fit on F2

0.919 11

ACCEPTED MANUSCRIPT Final R indices [I>=2σ(I)]

R1= 0.0471, wR2= 0.1227

R indices (all data)

R1= 0.0777, wR2= 0.1364

Largest diff. peak and hole/ e Å-3

0.39/-0.17 e Å-3

CCDC Number

1473917

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2.3. Pharmacological screening 2.3.1. Antibacterial activity

All the synthesized compounds (2a-j), (3a-e) and (5a-e) were evaluated for their antibacterial activity against three Gram-positive (S. aureus, E. faecalis and B. cereus) and

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three Gram-negative (E. coli, P. aeruginosa and P. intermedia) bacterial strains with ciprofloxacin as a standard by using Broth microdilution method[27].The minimum inhibitory concentration (MIC) of the synthesized compounds were compared with

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ciprofloxacin, it revealed that most of the newly synthesized compounds showed varying degrees of inhibition against the tested microorganisms.

Table 6. In vitro antibacterial activity data of compounds (2a-j), (3a-e) and (5a-e). Minimum inhibitory concentration in µg/mL (MIC) R

Gram +ve bacteria

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Entry

S.aureus E.faecalis 6-Me

2b

6-OMe

2c

6-Cl

2d 2e

B.cereus

E.coli

P.aeruginosa

P.intermedia

6.25

1.56

6.25

6.25

25

6.25

12.5

3.25

6.25

1.56

1.56

6.25

25

3.25

25

6.25

6.25

6.25

7-Me

1.56

6.25

1.56

1.56

1.56

6.25

7,8-Benzo

3.25

1.56

1.56

6.25

25

1.56

2i

AC C

EP

2a

Gram -ve bacteria

7-Me

12.5

6.25

25

6.25

6.25

6.25

2j

7,8-Benzo

6.25

6.25

6.25

1.56

1.56

6.25

3a

6-Me

1.56

1.56

25

3.25

25

6.25

3b

6-OMe

3.25

1.56

6.25

1.56

1.56

6.25

3c

6-Cl

3.25

1.56

25

1.56

25

3.25

3d

7-Me

3.25

3.25

6.25

3.25

6.25

25

2f 2g 2h

6-Me

3.25

6.25

6.25

1.56

6.25

6.25

6-OMe

6.25

6.25

6.25

6.25

25

1.56

6-Cl

12.5

1.56

25

6.25

1.56

1.56

12

ACCEPTED MANUSCRIPT 7,8-Benzo

6.25

1.56

6.25

3.25

6.25

25

5a

6-Me

12.5

6.25

50

1.56

25

3.25

5b

6-OMe

6.25

6.25

6.25

3.25

6.25

25

5c

6-Cl

6.25

3.25

25

6.25

25

6.25

5d

7-Me

6.25

3.25

6.25

1.56

25

3.25

5e

7,8-Benzo

25

6.25

25

6.25

1.56

25

Ciprofloxacin

-

6.25

6.25

6.25

6.25

6.25

6.25

RI PT

3e

The preliminary antibacterial activity revealed that, majority of the synthesized

SC

compounds showed excellent activity against both gram positive and gram negative bacterial stains. In general, more selective inhibitory activity against Gram positive E. faecalis and

obtained are summarized in Table 6.

M AN U

Gram negative E. coli bacterial strains. The minimum inhibitory concentrations (MIC) results

MIC results of series (2a-j), compound (2c) showed (Chloro substitution on coumarin) less activity against Gram positive S. aureus and B. cereus bacterial stains, whereas active against Gram negative bacterial stains. Moreover, other substitutions of these

substituted

TE D

series such as methyl, methoxy and benzo are found to be more active. Interestingly, 3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-3-(2-oxo-2H-chromen-4-yl)propanoic

acids (3a-e) are more active compared to parent molecules 4-coumarinyl-2-amino-4,5dihydro-5-oxopyrano[3,2-c]chromenes (2a-e). However, all the substitution on coumarin

EP

nucleus of compounds (3a-e) are highly active and more potent against Gram positive S. aureus and E. faecalis bacterial strains. In the series (5a-e), compounds 5c (chloro

AC C

substitution at C6 of coumarin nucleus) and 5d (methyl substitution at C7 of coumarin nucleus) are highly active against E. faecalis Gram positive bacteria. Whereas, compounds (5a-e) are more promising against E. coli bacteria in comparison with other two Gram negative bacteria P. aeruginosa and P. intermedia. The oral observation from Table 6, among all the series, compounds (3a-e) have exhibited excellent activity against Gram positive and Gram negative bacterial strains, it might be due to the hydrophilic functional groups such as carboxylic and hydroxy.

2.3.2. In vitro Anti-inflammatory activity

13

ACCEPTED MANUSCRIPT Human Red Blood Cell (HRBC) membrane stabilization method The anti-inflammatory activities of all newly synthesized compounds were also screened by using Human Red Blood Cell (HRBC) membrane stabilization method[28]. This method is more conventional, sensitive and well accepted for screening of newer antiinflammatory agents. The compounds activity was screened at the concentration of 100

RI PT

µg/mL oral dose and the same dose concentration of standard drug acetyl salicylic acid. The results obtained are presented in Table 7.

Table 7. In vitro anti-inflammatory activity in HRBC membrane stabilization method of

R

Control

-

% Inhibition of Erythrocyte in 100 µg/mL -

2a

6-Me

11.21±0.45*

3a

6-Me

14.10±0.2*

2b

6-OMe

15.27±0.34*

3b

6-OMe

23.31±0.1*

2c

6-Cl

43.34±0.24*

3c

6-Cl

61.19±0.34**

2d

7-Me

69.07±0.34**

3d

7-Me

76.40±0.3**

2e

7,8-Benzo

54.42±0.23**

3e

7,8-Benzo

59.09±0.2**

2f

6-Me

42.33±0.35**

5a

6-Me

31.37±0.1*

2g

6-OMe

52.10±0.34**

5b

6-OMe

50.54±0.4**

2h

6-Cl

62.25±0.24**

5c

6-Cl

70.73±0.3**

2i

7-Me

69.36±0.46**

5d

7-Me

36.82±0.2*

2j

7,8-Benzo

61.19±0.34**

5e

7,8-Benzo

37.61±0.1*

Acetyl

-

36.16 ± 0.11

Acetyl

-

36.16 ± 0.11

R

Control

AC C

EP

Entry

M AN U

Entry

-

% Inhibition of Erythrocyte in 100 µg/mL -

TE D

SC

compounds (2a-j), (3a-e) and (5a-e).

salicylic acid

salicylic acid

Level of significance * p<0.01**=p<0.05* percent inhibition of migration was calculated relative control.

The bio-screening results disclosed that all the compounds exhibited inhibitions of

HRBC membrane stabilization at the concentration of 100 µg/mL and most of the compounds showed more than half maximal (50%) inhibition except compounds (2a),(2b),(3a) and (3b).Compounds (3c), (5a), (5d) and (5e) showed erythrocyte hemolysis membrane stabilization inhibition close to the standard drug. Among all the synthesized compounds, (2d),(2i),(3d) and (5c)have shown excellent inhibition, almost twice compared with the standard acetyl salicylic acid inhibition 36.16%.To generalize the therapeutic properties of all 14

ACCEPTED MANUSCRIPT the synthesized compounds (2a-j), (3a-e) and (5a-e) for inflammation, compounds (2a-j) are more potent compared to other series of compounds.

2.4. Docking study To understand the efficiency of a biologically active drug molecule as a therapeutic agent, the knowledge of its binding location in the DNA environment is very essential and

RI PT

significant. A docking experiment was performed to obtain insight of selected binding site along with the preferred orientation of the ligand inside the active site of enzyme. All the synthesized compounds were docked into the active site of the enzyme and the docked view of the same has been depicted in the Fig. 3. The observed C-score values of all the

TE D

M AN U

SC

compounds are in the range of from 1.87 to 5.17, the score values are listed in Table 8.

Figure 3. Interaction of all the synthesized compounds at the active site of the enzyme (PDB

EP

ID: 1OZH).

AC C

Table 8.Surflex Docking score (kcal/mol) of the coumarinyl derivatives (2a–j), (3a-e) and (5a-e) with Klebsiella pneumoniae acetolactate synthase(PDB ID: 1OZH). Entry

C scorea

Crash

Polar

scoreb

scorec

D scored

PMF

G scoref

Chemscoreg

scoree

2a

2.72

-0.63

2.11

-183.62

0.28

-142.07

-27.51

2b

3.09

-1.99

2.31

-422.48

-38.78

-169.12

-27.74

2c

1.88

-0.42

1.66

-44.29

-27.97

-108.68

-17.78

2d

3.84

-0.95

1.02

-310.31

2.63

-166.57

-21.39

2e

2.71

-0.49

2.83

22.11

13.04

-82.46

-21.01

2f

2.50

-0.84

3.21

-178.81

2.34

-82.93

-16.87

15

ACCEPTED MANUSCRIPT 1.87

-0.50

0.00

-232.27

-9.46

-40.59

-10.90

2h

2.44

-0.45

0.32

-413.54

-32.64

-136.51

-16.36

2i

3.88

-1.12

0.00

-305.53

34.86

-186.93

-15.95

2j

3.69

-0.83

1.80

-285.45

14.87

-130.28

-24.63

3a

3.72

-1.56

2.17

-411.33

41.97

-115.12

-16.40

3b

4.54

-1.04

1.71

-491.67

21.63

-177.49

-21.07

3c*

5.17

-0.83

1.83

-364.53

31.89

-140.50

-17.78

3d

4.17

-1.19

0.96

-272.73

35.74

-156.67

-17.64

3e*

5.54

-1.63

2.96

-440.66

13.97

-161.15

-33.96

5a

3.58

-0.59

2.54

-174.96

24.09

-104.45

-20.44

5b

3.86

-0.71

2.39

-229.34

47.53

-138.42

-30.04

5c

2.83

-0.56

1.13

-154.72

5.54

-147.41

-24.62

5d

3.66

-1.62

2.09

-43.81

35.26

-133.90

-17.50

5e

4.19

-1.15

0.00

-208.04

-19.71

M AN U

SC

RI PT

2g

-240.65

57.71

As depicted in Fig. 4, compound (3c), makes three hydrogen bonding interaction at the active site of the enzyme (PDB ID: 1OZH), among them two bonding interactions raised from oxygen atom of carboxylic acid group with hydrogens of ARG228 (-COOH ---- H-

TE D

ARG228, 1.84 & 2.69 Å), hydrogen of hydroxyl group present at the C4 position of coumarin ring makes a bonding interaction with oxygen atom of ASN252 (-OH ---- O-ASN252, 2.19

AC C

EP

Å).

Figure 4. Interaction of compound (3c) at the active site of the enzyme (PDB: 1OZH). Fig.5 shows docking view of compound (3e)which makes three hydrogen bonding interaction with amino acid residues, among them two bonding interactions raised from 16

ACCEPTED MANUSCRIPT oxygen atom of carboxylic acid group with hydrogens of ARG228 (-COOH ---- H-ARG228, 1.96 & 2.07 Å), hydrogen of hydroxyl group present at the C4 position of coumarin ring

SC

RI PT

makes a bonding interaction with oxygen atom of GLY246 (-OH ---- O-GLY246, 1.96 Å).

M AN U

Figure 5. Interaction of compound (3e) at the active site of the enzyme (PDB: 1OZH).

3. Conclusion

A new coumarin based pyrano-coumarin (2) has been synthesized in ecofriendly condition and its structure was confirmed by X-ray crystallography. The unexpected pyran

TE D

ring opened product 3 and ring closer product 5 were obtained in single reaction at variable temperature. The in vitro antibacterial activity results confirmed the effectiveness of all the synthesized compounds. Specially, compounds (3a-e) exhibited higher antibacterial activity

EP

compared to other series (2a-j and 5a-e) of compounds. Noticeably, these series of compounds are more selective against S. aureus, E. faecalis and E. coli bacterial strains. This part of effectiveness may be due to the presence of hydrophilic groups such as

AC C

carboxylic and hydroxy. Moreover, these results are well supported by molecular docking study, which shows more number of interactions with amino acid residues and higher C score values. While, compounds (2a-j and 5a-e) are equally potent in comparison with standard ciprofloxacin. Further, the in vitro anti-inflammatory activity results display most of the compounds are equipotent activity in comparison with standard clinical drug molecule acetyl salicylic acid. Particularly, compounds 2d, 2i, 3d and 5c are found to be highly potent activity. Further, these results conclude that an ample scope for the small structural modifications for potential drug discovery against bacterial and inflammatory study.

4. Experimental protocol 4.1. Materials and methods 17

ACCEPTED MANUSCRIPT All the chemicals were purchased by commercial source and used without further purification unless otherwise stated. The reactions were monitored by thin-layer chromatography (TLC) analysis with Merck Kieselgel 60 F254 plates and visualized using UV light at 254 nm and KMnO4 staining or iodine vapour. The purity of compound was also checked by TLC. The

RI PT

melting points were determined by open capillary method and are uncorrected. The IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer in KBr disc method and mass spectra were recorded using Shimadzu GCMSQP2010S. 1H-NMR and

13

C-NMR spectra

SC

were measured on a Bruker 400 MHz and Jeol solution for innovation 500 MHz spectrometer (400 MHz or 100 MHz, respectively) using DMSO-d6as solvent and TMS as an internal

M AN U

standard. Chemical shifts are reported in δppm relative to internal tetramethylsilane standard (TMS, δppm 0.00). The peak patterns are indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; q, quartet; dd, doublet of doublets; br, broad. The coupling constant, J, is reported in Hertz (Hz). The X-ray diffraction study was performed on a BRUKER AXS

TE D

SMART APEX CCD diffractometer. The CHN elemental analysis of all the products were recorded by LECO TRUSPEC CHN analyzer.

4.1.1. General procedure for the synthesis of 2-amino-4,5-dihydro-4-(2-oxo-2H-chromen-

EP

4-yl)-5-oxopyrano[3,2-c]chromene (2a-j) A solution of 4-formylcoumarin (1 mmol), malononitrile or ethylcyanoacetate (1

AC C

mmol) and catalyst (5 mmol %) was prepared in water. The resulting suspension was stirred at room temperature for 10 minutes to this 4-hydroxycoumarin (1 mmol) was added and the reaction mixture left to heat on oil bath for about 2 h at 75 °C equipped with chilled water circulating condenser (monitored by TLC). The reaction mixture was allowed to cool and poured into ice cold water with stirring. The separated solid was filtered and washed with water to obtain a pure desired product (2).

4.1.2. General procedure for the synthesis of 3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-3-(2oxo-2H-chromen-4-yl)propanoic acid (3a-e)

18

ACCEPTED MANUSCRIPT A mixture of (2)(1 mmol) and formic acid (10 mL) was prepared and the reaction mixture was heated on oil bath for about 4-5 h at 90 °C. The completion of the reaction was confirmed by TLC. The reaction mixture was allowed to cool and poured into ice cold water with stirring. The separated solid was filtered, repeatedly washed with water and dried to obtain pure white product (3).

RI PT

4.1.3. Synthesis of 3,4-dihydro-4-(2-oxo-2H-chromen-4-yl)pyrano[3,2-c]chromene-2,5dione (5a-e)

A mixture of (2)(1 mmol) and formic acid (10 mL) was prepared and the reaction mixture was heated on oil bath for about 8-10 h at 130 °C. The completion of the reaction

SC

was confirmed by TLC. The reaction mixture was allowed to cool and poured into ice cold water with stirring. The separated solid was filtered, washed twice with NaHCO3solution

M AN U

(20%) and repeatedly washed with water and dried to obtain pure product (5).

4.1.4. General procedure for the synthesis of 3,4-dihydro-4-(2-oxo-2H-chromen-4yl)pyrano[3,2-c]chromene-2,5-dione (5a-e)

A mixture of compound 3 (1 mmol) in acetonitrile was charged into round bottomed flask to this SOCl2 (5 mmol) was added in cold condition and the reaction mixture was reflux

TE D

on oil bath for about 3 h. The progress of the reaction was monitored by TLC. The reaction mixture was poured into ice cold water with stirring and neutralized by NaHCO3. The separated solid was filtered, washed with NaHCO3 solution (20%) and repeatedly washed

EP

with water to obtain pure product.

4.2. Spectral characterization of compounds (2a-j), (3a-e) and (5a-e)

AC C

2-Amino-4,5-dihydro-4-(6-methyl-2-oxo-2H-chromen-4-yl)-5-oxopyrano[3,2-c]chromene3-carbonitrile (2a)

White solid; Yield 95%; m.p: 188-190 °C; IR (KBr): 3330, 3152, 2196, 1721cm-1; 1H-

NMR (400 MHz, DMSO-d6) δppm: 7.97 (s, 1H, C5-H of coumarin), 7.91 (d, 1H, J= 8.4 Hz, C7-H of coumarin), 7.76-7.72 (m, 1H, C'10-H of chromene), 7.56 (s, 2H, NH2 of pyran), 7.537.48 (m, 3H, C'7-H, C'8-H and C'9-H of chromene), 7.34 (d, 1H, J= 8.4 Hz, C8-H of coumarin), 6.40 (s, 1H, C3-H of coumarin), 5.22 (s, 1H, C4-H of pyran), 2.38 (s, 3H, C6-CH3 of coumarin);

13

C-NMR (100 MHz, DMSO-d6) δppm: 160.33, 159.69, 158.63, 158.60,

155.00, 152.29, 151.31, 139.07, 133.25, 133.20, 124.99, 124.72, 124.69, 122.60, 118.67, 116.65, 116.54, 113.04, 106.93, 102.01, 55.44, 20.89; LCMS (m/z): 398 (M+); HPLC: 95.08 19

ACCEPTED MANUSCRIPT %; Anal. Calcd. for C23H14N2O5 (%): Calcd.: C, 69.34; H, 3.54; N, 7.03. Found: C, 69.35; H, 3.52; N, 7.05.

2-Amino-4,5-dihydro-4-(6-methoxy-2-oxo-2H-chromen-4-yl)-5-oxopyrano[3,2c]chromene-3-carbonitrile (2b) White solid; Yield 93%; m.p: 264-266 °C; IR (KBr): 3397, 3286, 2192, 1701, 1670

RI PT

cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 7.91 (dd, 1H, J= 8.0 and 1.6 Hz, C'10-H of chromene), 7.75-7.74 (m, 1H, C'8-H of chromene), 7.59 (s, 2H, NH2 of pyran), 7.59 (s, 1H, C5-H of coumarin), 7.54-7.48 (m, 2H, C'9-H and C'7-H of chromene), 7.41 (d, 1H, J= 8.8 Hz, C8-H of coumarin), 7.27 (dd, 1H, J= 9.2 and 2.8 Hz, C7-H of coumarin), 6.42 (s, 1H, C3-H of

SC

coumarin), 5.23 (s, 1H, C4-H of pyran), 3.82 (s, 3H, C6-OCH3 of coumarin);13C-NMR (100 MHz, DMSO-d6) δppm: 160.26, 159.63, 158.74, 155.44, 155.23, 152.34, 147.58, 133.26,

M AN U

124.75, 122.56, 119.58, 118.80, 118.66, 117.87, 117.87, 116.69, 114.76, 112.98, 108.15, 101.92, 55.77, 55.25, 38.99; MS (m/z): 414 (M+). Anal. Calcd. for C23H14N2O6 (%): Calcd.: C, 66.67; H, 3.41; N, 6.76. Found: C, 66.65; H, 3.46; N, 6.75.

2-Amino-4-(6-chloro-2-oxo-2H-chromen-4-yl)-4,5-dihydro-5-oxopyrano[3,2-c]chromene3-carbonitrile (2c)

TE D

White solid; Yield 92%; m.p: 278-280 °C; IR (KBr): 3339, 3311, 2201, 1717, 1707 -1 1

cm ; H-NMR (400 MHz, DMSO-d6) δppm: 8.30 (s, 1H, C5-H of coumarin), 7.91-7.89 (m, 1H, C'10-H of chromene), 7.83 (dd, 1H, J= 8.8 and 1.6 Hz, C7-H of coumarin), 7.77-7.76 (m, 1H, C'8-H of chromene), 7.58 (s, 2H, NH2 of pyran), 7.50-7.48 (m, 2H, C'7-H and C'9-H of

EP

chromene), 7.32 (d, 1H, J= 8.0 Hz, C8-H of coumarin), 6.56 (s, 1H, C3-H of coumarin), 5.30 (s, 1H, C4-H of pyran);

13

C-NMR (100 MHz, DMSO-d6) δppm: 160.35, 159.72, 158.58,

AC C

158.53, 155.02, 152.29, 151.73, 133.25, 132.16, 128.66, 124.80, 124.76, 122.62, 122.60, 121.41, 118.83, 118.70, 116.69, 113.06, 101.81, 55.32; LCMS (m/z): 418 (M+). Anal. Calcd. for C22H11ClN2O5 (%): Calcd.: C, 63.10; H, 2.65; N, 6.69. Found: C, 63.15; H, 2.66; N, 6.72.

2-Amino-4,5-dihydro-4-(7-methyl-2-oxo-2H-chromen-4-yl)-5-oxopyrano[3,2-c]chromene3-carbonitrile (2d) White solid; Yield 94%; m.p: 286-288 °C; IR (KBr): 3387, 3293, 2192, 1696, 1672 -1 1

cm ; H-NMR (400 MHz, DMSO-d6) δppm: 7.93 (s, 1H, C5-H of coumarin), 7.84 (d, 1H, J= 8.4 Hz, C6-H of coumarin), 7.76-7.75 (m, 1H, C'10-H of chromene), 7.56 (s, 2H, NH2 of pyran), 7.53-7.44 (m, 3H, C'7-H, C'8-H and C'9-H of chromene), 7.36 (d, 1H, J= 8.0 Hz, C8-H 20

ACCEPTED MANUSCRIPT of coumarin), 6.40 (s, 1H, C3-H of coumarin), 5.20 (s, 1H, C4-H of pyran), 2.32 (s, 3H, C7CH3 of coumarin);

13

C-NMR (100 MHz, DMSO-d6) δppm: 162.08, 160.92, 159.78, 155.41,

150.83, 150.79, 136.97, 134.11, 129.06, 128.10, 127.66, 124.40, 124.10, 122.02, 121.55, 119.95, 119.84, 116.16, 115.11, 113.92, 112.49, 101.85, 55.84, 24.10; LCMS (m/z): 398 (M+). Anal. Calcd. for C23H14N2O5 (%): Calcd.: C, 69.34; H, 3.54; N, 7.03. Found: C, 69.38;

RI PT

H, 3.57; N, 7.05.

2-Amino-4,5-dihydro-5-oxo-4-(2-oxo-2H-benzo[h]chromen-4-yl)pyrano[3,2-c]chromene-3carbonitrile (2e)

Light brown solid; Yield 91%; m.p: 282-285 °C; IR (KBr): 3440, 3308, 3205, 2218,

SC

1725, 1643 cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 8.40 (d, 1H, J= 8.8 Hz, C'10-H of coumarin), 8.07 (d, 1H, J= 8.4 Hz, C7-H of coumarin), 7.72-7.68 (m, 2H, C8-H and C9-H of

M AN U

coumarin), 7.63 (d, 1H, J= 8.4 Hz, C5-H of coumarin), 7.58 (d, 1H, J= 8 Hz, C6-H of coumarin), 7.56 (s, 2H, NH2 of pyran), 7.55-7.48 (m, 4H, C'7-H, C'8-H, C'9-H and C'10-H of chromene), 6.45 (s, 1H, C3-H of coumarin), 5.21 (s, 1H, C4-H of pyran);

13

C-NMR (100

MHz, DMSO-d6) δppm: 160.38, 159.12, 155.09, 151.49, 141.92, 138.62, 137.88, 134.56, 130.08, 129.57, 129.37, 128.59, 128.12, 124.47, 123.18, 122.55, 122.17, 121.70, 120.38, 116.73, 116.25, 114.51, 112.66, 103.38, 55.66; LCMS (m/z): 434 (M+). Anal. Calcd. for

TE D

C26H14N2O5 (%): Calcd.: C, 69.34; H, 3.54; N, 7.03. Found: C, 69.39; H, 3.58; N, 7.04.

Ethyl

2-amino-4,5-dihydro-4-(6-methyl-2-oxo-2H-chromen-4-yl)-5-oxopyrano[3,2-

c]chromene-3-carboxylate (2f)

EP

White solid; Yield 94%; m.p: 230-232°C; IR (KBr): 3378, 3272, 1719, 1710, 1644 cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 7.93 (s, 1H, C5-H of coumarin), 7.88 (d, 1H, J=

AC C

8 Hz, C7-H of coumarin), 7.76-7.72 (m, 2H, C'8-H and C'10-H of chromene), 7.62 (s, 2H, NH2 of pyran), 7.53-7.46 (m, 2H, C'7-H and C'9-H of chromene), 7.38 (d, 1H, J= 8.4 Hz, C8-H of coumarin), 6.42 (s, 1H, C3-H of coumarin), 5.22 (s, 1H, C4-H of pyran), 4.12-4.06 (m, 2H, CH2 of ethyl group), 2.38 (s, 3H, C6-CH3 of coumarin); 1.66-1.58 (m, 3H, CH3 of ethyl group);

13

C-NMR (100 MHz, DMSO-d6) δppm: 167.85, 162.44, 161.69, 155.73, 150.11,

145.71, 142.69, 136.02, 134.44, 129.28, 128.65, 128.11, 126.85, 126.76, 120.69, 118.69, 117.94, 117.61, 111.09, 108.38, 79.15, 56.20, 21.01, 13.92; LCMS (m/z): 445 (M+). Anal. Calcd. for C25H19NO7 (%): Calcd.: C, 67.41; H, 4.30; N, 3.10. Found: C, 67.43; H, 4.34; N, 3.12.

21

ACCEPTED MANUSCRIPT Ethyl

2-amino-4,5-dihydro-4-(6-methoxy-2-oxo-2H-chromen-4-yl)-5-oxopyrano[3,2-

c]chromene-3-carboxylate (2g) White solid; Yield 92%; m.p: 194-196°C; IR (KBr): 3397, 3286, 1701, 1670 cm-1; 1HNMR (400 MHz, DMSO-d6) δppm: 7.95 (s, 1H, C5-H of coumarin), 7.81 (d, 1H, J= 8.4 Hz, C7-H of coumarin), 7.75-7.70 (m, 2H, C'8-H and C'10-H of chromene), 7.53 (s, 2H, NH2 of pyran), 7.52-7.44 (m, 2H, C'7-H and C'9-H of chromene), 7.30 (d, 1H, J= 8.0 Hz, C8-H of

RI PT

coumarin), 6.48 (s, 1H, C3-H of coumarin), 5.39 (s, 1H, C4-H of pyran), 4.18-4.04 (m, 2H, CH2 of ethyl group), 3.86 (s, 3H, C6-OCH3 of coumarin) 1.46-1.38 (m, 3H, CH3 of ethyl group);

13

C-NMR (100 MHz, DMSO-d6) δppm: 167.12, 162.25, 162.94, 162.62, 157.16,

155.08, 153.29, 150.01, 145.01, 142.22, 135.94, 135.30, 134.43, 129.36, 129.12, 126.73,

SC

120.62, 118.22, 117.56, 111.01, 108.43, 79.62, 58.22, 56.16, 16.36; LCMS (m/z): 461 (M+). Anal. Calcd. for C25H19NO8 (%): Calcd.: C, 65.07; H, 4.15; N, 3.04. Found: C, 65.12; H,

Ethyl

M AN U

4.14; N, 3.07.

2-amino-4-(6-chloro-2-oxo-2H-chromen-4-yl)-4,5-dihydro-5-oxopyrano[3,2-

c]chromene-3-carboxylate (2h)

White solid; Yield 91%; m.p: 256-258 °C; IR (KBr): 3433, 3315, 1719, 1712 cm-1; 1

H-NMR (400 MHz, DMSO-d6) δppm: 8.32 (s, 1H, C5-H of coumarin), 7.97-7.93 (m, 1H,

TE D

C'10-H of chromene), 7.85 (dd, 1H, J= 8.8 and 1.6 Hz, C7-H of coumarin), 7.75-7.73 (m, 1H, C'8-H of chromene), 7.60 (s, 2H, NH2 of pyran), 7.53-7.50 (m, 2H, C'7-H and C'9-H of chromene), 7.46 (d, 1H, J= 8.0 Hz, C8-H of coumarin), 6.55 (s, 1H, C3-H of coumarin), 5.38 (s, 1H, C4-H of pyran), 4.11-4.08 (m, 2H, CH2 of ethyl group),1.36-1.32 (m, 3H, CH3 of ethyl 13

C-NMR (100 MHz, DMSO-d6) δppm: 168.00, 164.08, 162.61, 159.02, 152.89,

EP

group);

152.08, 146.50, 138.00, 132.62, 131.54, 128.72, 126.18, 124.99, 124.60, 120.93, 119.21,

AC C

119.13, 115.85, 114.08, 111.24, 79.21, 54.50, 14.61; LCMS (m/z): 465 (M+). Anal. Calcd. for C24H16ClNO7 (%): Calcd.: C, 61.88; H, 3.46; N, 3.01. Found: C, 61.90; H, 3.44; N, 3.04.

Ethyl

2-amino-4,5-dihydro-4-(7-methyl-2-oxo-2H-chromen-4-yl)-5-oxopyrano[3,2-

c]chromene-3-carboxylate (2i) White solid; Yield 93%; m.p: 212-214 °C; IR (KBr): 3385, 3275, 1716, 1683 cm-1; 1

H-NMR (400 MHz, DMSO-d6) δppm: 8.15 (d, 1H, J= 8.4 Hz C5-H of coumarin), 7.99-7.96

(m, 1H, C'10-H of chromene), 7.88 (s, 2H, NH2 of pyran), 7.73-7.69 (m, 1H, C'8-H of chromene), 7.52-7.43 (m, 2H, C'9-H and C'7-H of chromene), 7.24 (dd, 1H, J= 8.4 and 1.2 Hz, C6-H of coumarin), 7.20 (s, 1H, C8-H of coumarin), 6.14 (s, 1H, C3-H of coumarin), 5.25 22

ACCEPTED MANUSCRIPT (s, 1H, C4-H of pyran), 3.88-3.77 (m, 2H, CH2 of ethyl group), 2.42 (s, 3H, C7-CH3 of coumarin) 0.72 (t, 3H, J= 6.8 Hz CH3 of ethyl group);

13

C-NMR (100 MHz, DMSO-d6)

δppm: 167.32, 160.58, 160.00, 158.53, 153.77, 152.68, 152.04, 142.52, 138.61, 133.03, 125.11, 124.87, 124.67, 122.69, 116.56, 116.24, 111.98, 111.54, 108.63, 75.83, 59.07, 36.00, 21.00, 13.67; MS (m/z): 445 (M+). Anal. Calcd. for C25H19NO7 (%): Calcd.: C, 67.41; H,

Ethyl

RI PT

4.30; N, 3.14. Found: C, 67.45; H, 4.34; N, 3.16.

2-amino-4,5-dihydro-5-oxo-4-(2-oxo-2H-benzo[h]chromen-4-yl)pyrano[3,2-

c]chromene-3-carboxylate (2j)

White solid; Yield 90%; m.p: 247-249 °C; IR (KBr): 3397, 3286, 1703, 1672 cm-1; H-NMR (400 MHz, DMSO-d6) δppm: 8.45 (d, 1H, J= 8.4 Hz, C10-H of coumarin), 8.17 (d,

SC

1

1H, J= 8.8 Hz, C7-H of coumarin), 7.82-7.78 (m, 2H, C8-H and C9-H of coumarin), 7.73 (d,

M AN U

1H, J= 8.0 Hz, C5-H of coumarin), 7.70 (d, 1H, J= 8.4 Hz, C6-H of coumarin), 7.54 (s, 2H, NH2 of pyran), 7.50-7.45 (m, 4H, C'7-H, C'8-H, C'9-H and C'10-H of chromene), 6.34 (s, 1H, C3-H of coumarin), 5.21 (s, 1H, C4-H of pyran), 4.02 (m, 2H, CH2 of ethyl group), 1.49 (m, 13

C-NMR (100 MHz, DMSO-d6) δppm: 165.45, 162.25, 161.42,

3H, CH3 of ethyl group);

160.13, 156. 71, 150.61, 143.11, 137.51, 136.11, 130.26, 129.51, 128.57, 127.97, 126.50, 125.97, 125.45, 122.28, 121.42, 118.56, 117.89, 111.24, 77.42, 55.08, 17.13; LCMS (m/z):

69.88; H, 3.99; N, 2.93.

TE D

481 (M+). Anal. Calcd. for C28H19NO7 (%): Calcd.: C, 69.85; H, 3.98; N, 2.91. Found: C,

acid (3a)

EP

3-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-3-(6-methyl-2-oxo-2H-chromen-4-yl)propanoic White solid; Yield 85%; m.p: 211-212 °C; IR (KBr): 3412, 3302, 1739, 1712 cm-1; H-NMR (400 MHz, DMSO-d6) δppm: 11.12 (br, 2H, OH of propanoic acid and phenolic

AC C

1

OH of chromene), 8.06 (d, 1H, J= 8.4 Hz, C'10-H of chromene), 7.82 (s, 1H, C5-H of coumarin), 7.38-7.24 (m, 3H, C'7-H, C'8-H and C'9-H of chromene), 7.11 (d, 1H, J= 8.0 Hz, C7-H of coumarin), 7.09 (d, 1H, J= 7.6 Hz, C8-H of coumarin), 6.48 (s, 1H, C3-H of coumarin), 5.12 (t, 1H, J= 7.2 Hz, C3-H of propanoic acid), 3.29 (dd, 1H, J= 16.4 and 8.4 Hz, C2-H of propanoic acid), 3.06 (dd, 1H, J= 16.0 and 6.8 Hz, C2-H of propanoic acid), 2.32 (s, 3H, C6-CH3 of coumarin);

13

C-NMR (100 MHz, DMSO-d6) δppm: 177.10, 163.15, 163.82,

161.13, 156.15, 155.50, 150.08, 148.87, 137.15, 136.10, 126.78, 125.61, 121.68, 120.22, 119.97, 118.23, 115.93, 112.57, 109.47, 42.57, 32.50, 19.26; LCMS (m/z): 392 (M+).

23

ACCEPTED MANUSCRIPT HPLC:97.96%; Anal. Calcd. for C22H16O7 (%): Calcd.: C, 67.35; H, 4.11; Found: C, 67.38; H, 4.09.

3-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-3-(6-methoxy-2-oxo-2H-chromen-4-yl)propanoic acid (3b) White solid; Yield 82%; m.p: 219-221 °C; IR (KBr): 3251, 1747, 1701 cm-1; 1H-NMR

RI PT

(400 MHz, DMSO-d6) δppm: 11.02 (br, 2H, OH of propanoic acid and phenolic OH of chromene), 7.46 (d, 1H, J= 8.4 Hz, C'10-H of chromene), 7.32 (s, 1H, C5-H of coumarin), 7.28-7.17 (m, 3H, C'7-H, C'8-H and C'9-H of chromene), 7.11 (d, 1H, J= 8.0 Hz, C7-H of coumarin), 7.06 (d, 1H, J= 7.6 Hz, C8-H of coumarin), 6.40 (s, 1H, C3-H of coumarin), 5.15

SC

(t, 1H, J= 7.2 Hz, C3-H of propanoic acid), 3.82 (s, 3H, C6-OCH3 of coumarin), 3.23 (dd, 1H, J= 16.4 and 8.4 Hz, C2-H of propanoic acid), 3.00 (dd, 1H, J= 16.0 and 6.8 Hz, C2-H of 13

C-NMR (100 MHz, DMSO-d6) δppm: 172.58, 163.73, 161.50, 159.75,

M AN U

propanoic acid);

154.58, 150.2689, 144.58, 236.38, 134.08, 129.06, 127.86, 127.55, 123.73, 122.58, 121.75, 120.32, 119.93, 114.73, 112.75, 53.83, 36.38, 33.72; LCMS (m/z): 408 (M+). Anal. Calcd. for C22H16O8 (%): Calcd.: C, 64.71; H, 3.95; Found: C, 64.68; H, 3.99.

acid (3c)

TE D

3-(6-Chloro-2-oxo-2H-chromen-4-yl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)propanoic White solid; Yield 80%; m.p: 224-226 °C; IR (KBr): 3434, 1772, 1722 cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 11.16 (br, 2H, OH of propanoic acid and phenolic OH of chromene), 8.12 (d, 1H, , J=8.4 Hz, C'10-H of chromene), 7.84 (s, 1H, C5-H of coumarin),

EP

7.36-7.21 (m, 3H, C'7-H, C'8-H and C'9-H of chromene), 7.10 (d, 1H, J= 8.0 Hz, C7-H of coumarin), 7.05 (d, 1H, J= 7.6 Hz, C8-H of coumarin), 6.41 (s, 1H, C3-H of coumarin), 5.11

AC C

(t, 1H, J= 7.6 Hz, C3-H of propanoic acid), 3.31 (dd, 1H, J= 16.0 and 8.0 Hz, C2-H of propanoic acid), 3.04 (dd, 1H, J= 16.4 and 7.2 Hz, C2-H of propanoic acid); 13C-NMR (100 MHz, DMSO-d6) δppm: 172.04, 164.04, 160.33, 159.69, 158.63, 155.00, 152.29, 146.56, 141.97, 133.25, 133.20, 124.99, 124.72, 122.60, 118.67, 118.05, 116.65, 116.54, 113.04, 34.00, 31.60; LCMS (m/z): 412 (M+). Anal. Calcd. for C21H13ClO7 (%): Calcd.: C, 61.10; H, 3.17; Found: C, 61.08; H, 3.19.

3-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-3-(7-methyl-2-oxo-2H-chromen-4-yl)propanoic acid (3d)

24

ACCEPTED MANUSCRIPT White solid; Yield 83%; m.p: 260-262 °C; IR (KBr): 3327, 1748, 1722 cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 11.16 (br, 2H, OH of propanoic acid and phenolic OH of chromene), 7.91 (dd, 1H, J= 8.0 and 1.6 Hz, C'10-H of chromene), 7.75-7.74 (m, 1H, C'8-H of chromene), 7.80 (d, 1H, J= 7.2 Hz, C5-H of coumarin), 7.54-7.48 (m, 3H, C'9-H and C'7-H of chromene), 7.21 (s, 1H, C8-H of coumarin), 7.13 (d, 1H, J= 8.0 Hz, C6-H of coumarin), 6.44 (s, 1H, C3-H of coumarin), 5.10 (t, 1H, J= 7.6 Hz, C3-H of propanoic acid), 3.25 (dd, 1H, J= propanoic acid), 2.34(s, 3H, C7-CH3 of coumarin);

13

RI PT

16.8 and 8.8 Hz, C2-H of propanoic acid), 3.00 (dd, 1H, J= 16.4 and 6.4 Hz, C2-H of C-NMR (100 MHz, DMSO-d6) δppm:

172.52, 162.52, 161.68, 160.10, 155.03, 153.07, 152.17, 142.54, 132.50, 125.51, 123.99, 123.94, 122.05, 116.92, 116.43, 116.39, 115.95, 113.14, 104.03, 34.72, 32.74, 20.83; MS

SC

(m/z): 392 (M+). Anal. Calcd. for C22H16O7 (%): Calcd.: C, 67.35; H, 4.11; Found: C, 67.38;

M AN U

H, 4.13.

3-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-3-(2-oxo-2H-benzo[h]chromen-4-yl)propanoic acid (3e)

Light brown solid; Yield 81%; m.p: 203-205°C; IR (KBr): 3426, 1725, 1708 cm-1; 1HNMR (400 MHz, DMSO-d6) δppm: 11.14 (br, 2H, OH of propanoic acid and phenolic OH of chromene), 8.26 (d, 1H, J= 8.4 Hz, C10-H of coumarin), 8.03 (d, 1H, J= 8.4 Hz, C7-H of

TE D

coumarin), 7.94-7.75 (m, 4H, C'7-H, C'8-H, C'9-H and C'10-H of chromene), 7.68-7.57 (m, 2H, C8-H and C9-H of coumarin), 7.54 (d, 1H, J= 8.4 Hz, C6-H of coumarin), 7.47 (d, 1H, J= 8.0 Hz, C5-H of coumarin), 6.57 (s, 1H, C3-H of coumarin), 5.14 (t, 1H, J= 7.6 Hz, C3-H of propanoic acid), 3.24 (dd, 1H, J= 16.8 and 8.8 Hz, C2-H of propanoic acid), 3.03 (dd, 1H, J=

EP

16.4 and 6.4 Hz, C2-H of propanoic acid);

13

C-NMR (100 MHz, DMSO-d6) δppm: 177.42,

163.81, 160.49, 154.41, 148.38, 144.20, 136.23, 134.28, 133.87, 129.36, 129.31, 129.05,

AC C

128.95, 128.37, 126.26, 126.11, 123.80, 121.52, 120.96, 117.46, 113.56, 112.16, 42.11, 31.05; MS (m/z): 428 (M+). Anal. Calcd. for C25H16O7 (%): Calcd.: C, 70.09; H, 3.76; Found: C, 69.99; H, 3.74.

3,4-Dihydro-4-(6-methyl-2-oxo-2H-chromen-4-yl)pyrano[3,2-c]chromene-2,5-dione (5a) White solid; Yield 80%; m.p: 236-238 °C; IR (KBr): 1731 cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 8.02 (d, 1H, , J= 8.4 Hz, C'10-H of coumarin), 7.85 (s, 1H, C5-H of coumarin), 7.69 (d, 1H, J= 10.4 Hz, C7-H of coumarin), 7.57-7.42 (m, 3H, C'7-H, C'8-H and C'9-H of chromene), 7.08 (d, 1H, J= 8.0 Hz, C8-H of coumarin), 6.22 (s, 1H, C3-H of coumarin), 4.88 (t, 1H, J= 7.6 Hz, C4-H of pyran), 3.01 (dd, 1H, J= 15.6 and 8.0 Hz, C3-H of 25

ACCEPTED MANUSCRIPT pyran), 2.96 (dd, 1H, J= 16.0 and 7.6 Hz, C3-H of pyran), 2.38 (s, 3H, C6-CH3 of coumarin); 13

C-NMR (100 MHz, DMSO-d6) δppm: 170.05, 164.02, 161.59, 160.05, 159.99, 154.88,

154.66, 154.61, 148.17, 136.50, 136.43, 119.93, 119.17, 118.28, 115.62, 115.47, 107.56, 107.50, 34.11, 31.59, 24.17; LCMS (m/z): 374 (M+); HPLC: 98.55%; Anal. Calcd. for C22H14O6 (%): Calcd.: C, 70.59; H, 3.77; Found: C, 70.54; H, 3.75.

RI PT

3,4-Dihydro-4-(6-methoxy-2-oxo-2H-chromen-4-yl)pyrano[3,2-c]chromene-2,5-dione (5b) Light green solid; Yield 77%; m.p: 244-226 °C; IR (KBr): 1726 cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 7.57 (d, 1H, J= 8.4 Hz, C8-H of coumarin), 7.52-7.44 (m, 4H, C'7-H, C'8-H, C'9-H and C'10-H of chromene), 7.41 (dd, 1H, J= 8.8 and 2.8 Hz, C7-H of coumarin),

SC

7.35 (s, 1H, C5-H of coumarin), 6.49 (s, 1H, C3-H of coumarin), 4.62 (t, 1H, J= 7.2 Hz, C4-H of pyran), 3.69 (s, 3H, C6-OCH3 of coumarin), 3.03 (dd, 1H, J= 16.0 and 7.8 Hz, C3-H of 13

C-NMR (100 MHz, DMSO-d6)

M AN U

pyran), 2.98 (dd, 1H, J= 16.4 and 7.6 Hz, C3-H of pyran);

δppm: 167.79, 163.05, 159.95, 155.31, 149.42, 148.30, 140.88, 137.93, 136.93, 134.13, 129.57, 128.76, 121.88, 120.53, 119.77, 119.01, 118.64, 117.79, 117.06, 115.64, 112.05,110.07, 55.47, 34.88, 32.05; LCMS (m/z): 390 (M+). Anal. Calcd. for C22H14O7 (%): Calcd.: C, 67.69; H, 3.62; Found: C, 67.72; H, 3.60.

TE D

4-(6-Chloro-2-oxo-2H-chromen-4-yl)-3,4-dihydropyrano[3,2-c]chromene-2,5-dione (5c) White solid; Yield 75%; m.p: 228-230 °C; IR (KBr): 1711 cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 8.22 (s, 1H, C5-H of coumarin), 7.95-7.91 (m, 1H, C'10-H of chromene), 7.77 (dd, 1H, J= 8.4 and 1.6 Hz, C7-H of coumarin), 7.70-7.63 (m, 1H, C'8-H of chromene),

EP

7.58-7.45 (m, 2H, C'7-H and C'9-H of chromene), 7.36 (d, 1H, J= 8.4 Hz, C8-H of coumarin), 6.51 (s, 1H, C3-H of coumarin), 5.37 (s, 1H, C4-H of pyran), 3.02 (dd, 1H, J= 14.8 and 8.0

AC C

Hz, C3-H of pyran), 2.95 (dd, 1H, J= 16.4 and 6.8 Hz, C3-H of pyran); 13C-NMR (100 MHz, DMSO-d6) δppm: 166.54, 162.05, 160.92, 157.72, 152.36, 150.29, 148.37, 131.05, 128.56, 128.49, 126.93, 126.84, 125.50, 122.92, 122.54, 121.53, 117.25, 112.42, 111.17, 34.83, 33.07; LCMS (m/z): 394 (M+). Anal. Calcd. for C22H11ClO6 (%): Calcd.: C, 63.89; H, 2.81; Found: C, 63.92; H, 2.80.

3,4-Dihydro-4-(7-methyl-2-oxo-2H-chromen-4-yl)pyrano[3,2-c]chromene-2,5-dione (5d) White solid; Yield 78%; m.p: 217-219°C; IR (KBr): 1733, 1709 cm-1; 1H-NMR (400 MHz, DMSO-d6) δppm: 8.12 (d, 1H, , J=8.0 Hz, C'10-H of coumarin), 7.92 (d, 1H, J= 7.6 Hz, C5-H of coumarin), 7.57-7.42 (m, 3H, C'7-H, C'8-H and C'9-H of chromene), 7.29 (d, 1H, J= 26

ACCEPTED MANUSCRIPT 10.4 Hz, C6-H of coumarin), 7.08 (s, 1H, C8-H of coumarin), 6.23 (s, 1H, C3-H of coumarin), 4.97 (t, 1H, J= 8.4 Hz, C4-H of pyran), 2.97 (dd, 1H, J= 15.2 and 8.0 Hz, C3-H of pyran), 2.93 (dd, 1H, J= 16.4 and 7.6 Hz, C3-H of pyran), 2.40 (s, 3H, C7-CH3 of coumarin);

13

C-

NMR (100 MHz, DMSO-d6) δppm: 168.62, 162.52, 161.63, 160.10, 155.03, 153.07, 152.17, 142.54, 132.50, 125.51, 123.99, 123.94, 123.49, 116.92, 116.43, 116.39, 115.95, 113.14, 104.03, 34.72, 32.74, 20.68 ; MS (m/z): 374 (M+). Anal. Calcd. for C22H14O6 (%): Calcd.: C,

RI PT

70.59; H, 3.77; Found: C, 70.62; H, 3.80.

3,4-Dihydro-4-(2-oxo-2H-benzo[h]chromen-4-yl)pyrano[3,2-c]chromene-2,5-dione (5e) White solid; Yield 76%; m.p: 220-221 °C; IR (KBr): 1709 cm-1; 1H-NMR (400 MHz,

SC

DMSO-d6) δppm: 8.22 (d, 1H, J= 8.8 Hz, C10-H of coumarin), 8.03 (d, 1H, J= 8.4 Hz, C7-H of coumarin), 7.96-7.84 (m, 4H, C'7-H, C'8-H, C'9-H and C'10-H of chromene), 7.79-7.70 (m,

M AN U

2H, C8-H and C9-H of coumarin), 7.64 (d, 1H, J= 8.0 Hz, C6-H of coumarin), 7.59 (d, 1H, J= 8.4 Hz, C5-H of coumarin), 6.55 (s, 1H, C3-H of coumarin), 4.60 (t, 1H, , J= 7.6 Hz, C4-H of pyran), 3.19 (dd, 1H, J= 16.4 and 7.6 Hz, C3-H of pyran), 2.95 (dd, 1H, J= 16.0 and 7.2 Hz, C3-H of pyran);

13

C-NMR (100 MHz, DMSO-d6) δppm: 169.45, 160.12, 154.08, 150.11,

143.09, 140.90, 138.01, 137.21, 134.11, 129.45, 128.78, 126.87, 125.49, 121.96, 120.61, 119.80, 116.84, 115.81, 115.62, 112.12, 34.08, 31.96; LCMS (m/z): 410 (M+). Anal. Calcd.

4.3. Biological evaluation

TE D

for C25H14O6 (%): Calcd.: C, 73.17; H, 3.44; Found: C, 73.18; H, 3.46.

EP

4.3.1. Antibacterial screening

The susceptibility of the test organisms to synthetic compounds were assessed using

AC C

broth dilution assay, as minimum inhibitory concentration (MIC) Triplicates were performed for each of the standard strains. Culture media: Brain Heart Infusion (BHI) broth Test organisms: Four microorganisms were selected for the study: S. aureus microbial type culture collection MTCC 12598, E. faecalis MTCC 35550, E. coli MTCC 443 and P. aeruginosa MTCC 25668. All microorganisms were previously sub cultured in appropriate media and under gaseous conditions to confirm their purity at 35 °C for 48 h prior to testing of the vehicles. Inoculum preparation: The growth method or the log phase method was performed as follows. At least three to five well isolated colonies of the same morphological type were selected from an agar culture plate. Top of each colony was scooped with a loop, and the 27

ACCEPTED MANUSCRIPT growth was transferred into a tube containing 4–5 mL of BHI broth. The broth culture was incubated at 35°C for 2–6 h until it achieved the turbidity of the 0.5 McFarland standards. The turbidity of actively growing broth culture was adjusted with broth to obtain a final turbidity optically comparable to that of the 0.5 McFarland standards, done visually by comparing the inoculum tube and the standard against a white card with contrasting black

RI PT

lines. Broth dilution method: A total of 10 tubes were taken and nine dilutions of the vehicle were done with BHI for MIC. In the initial tube, only 200 µL of vehicle was added. For further dilutions, 200 µL of BHI broth was added to the next nine tubes separately. In the second

SC

tube, 200 µL of vehicle was added which already contained 200 µL of BHI broth. This was considered as 10 dilution. From the 10 diluted tube, 200 µL was transferred to the second tube to make 10 dilution. The serial dilution was repeated up to 10 dilution for each vehicle.

M AN U

From the maintained stock cultures of the required microorganisms, 5 µL was taken and added to 2 mL of BHI broth. In each serially diluted tube, 200 µL of the above culture suspension was added. The last tube contained only the media and the culture suspension, i.e. the negative control. The tubes were kept for incubation for 24 h at 37 °C in bacteriological

TE D

incubator and observed for turbidity.

4.3.2. Anti-inflammatory activity

Human Red Blood Cell (HRBC) membrane stabilization method Collection of blood samples: Human RBCs were collected for the study. 7 mL of blood was

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collected from healthy male human volunteers (aged 24-26 years) without a history of oral contraceptive or anticoagulant therapy and free from diseases using protocol issued by

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Institutional Ethics Committee. The collected RBCs were kept in a test tube with an anticoagulant EDTA under standard conditions of temperature 23±2 °C and relative humidity 55±10%.

Assay of membrane stabilization Erythrocyte suspension: The blood was washed three times using isotonic solution (0.9% saline). The volume of saline was measured and reconstituted as a 40% (v/v) suspension with isotonic buffer solution (pH 7.4) which contained in 1 L of distilled water: NaH2PO4. 2H2O, 0.26 g; Na2HPO4, 1.15 g; NaCl, 9 g (10 mM sodium phosphate buffer). Thus the suspension finally collected was the stock erythrocyte (RBC) suspension.

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ACCEPTED MANUSCRIPT Heat-induced hemolysis: Aliquots (5 mL) of the isotonic buffer, containing 0.1 mg/mL of synthesized compounds were put into two duplicate sets of centrifuge tubes (35).The vehicle, in the same amount, was added to another tube as control. Erythrocyte suspension (30 µL) was added to each tube and mixed gently by inversion. One pair of the tubes was incubated at 54 °C for 20 minutes in a water bath. The other pair was maintained at 0-5 °C in an ice bath. The reaction mixture was centrifuged for 3 minutes at 1300 rpm and the absorbance of the

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supernatant was measured at 540 nm using UV-VIS spectrometer (SHIMADZU, UV-1800). The percentage inhibition or acceleration of hemolysis in tests were calculated using the following equation.

Where, OD1=Test sample unheated,

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% Inhibition of Hemolysis=100 x 1- [(OD2-OD1)/(OD3-OD1)]

OD2=Test sample heated and

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OD3=Control sample heated.

4.4. Docking simulations

The crystal structures used was Klebsiella pneumoniaeacetolactate synthase with enzyme-bound cofactor and with an unusual intermediate (PDB ID: 1OZH) [29] for the

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docking studies was obtained from the Protein Data Bank. The proteins were prepared for docking by adding polar hydrogen atom with Gasteiger-Huckel charges and water molecules were removed. The 3D structure of the ligands was generated by the SKETCH module implemented in the SYBYL program (Tripos Inc., St. Louis, USA) and its energy-minimized

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conformation was obtained with the help of the Tripos force field using Gasteiger-Huckel[30] charges and molecular docking was performed with Surflex-Dock program that is interfaced

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with Sybyl-X 2.0. [31] And other miscellaneous parameters were assigned with the default values given by the software.

Acknowledgement The authors acknowledge the University Scientific Instrumentation Centre (USIC), Karnatak University, Dharwad for IR, Mass and SC-XRD data. We are also grateful the Department of Science and Technology (DST, Grant No. SR/FT.CS-19/2010 Dated: 29.10.2010)for financial support and also UGC-UPE and UGC-DSA New Delhi, for the 29

ACCEPTED MANUSCRIPT Departmental support. Authors also thank to NMR Research Centre, Indian Institute of Science (IISc), Bengaluru, India for carrying out the spectral analyses.

Appendix A. Supplementary data

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Supplementary data associated with this article is attached with manuscript.

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Highlights: The synthesis of substituted coumarin based pyrano[3,2-c]chromene derivatives were

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achieved in good to excellent yields underone-pot three component green method.

The unexpected 3-coumarinyl-3-pyrazolylpropanoic acid derivatives (3) and C4-C4 chromene derivatives (5) have been isolated by the reaction of compound (2) in acidic conditions.

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All the newly synthesized polyfunctionalized coumarin based pyrano[3,2-c]chromene derivatives are potent antibacterial and anti-inflammatory agents compared to standard.

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The molecular docking study reveals that, all newly synthesized polyfunctionalized coumarin based pyrano[2,3-c]pyrazole derivatives are well supported for invitro

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antibacterial activity.