Synthesis of 3-indolylmethyl substituted (pyrazolo/benzo)triazinone derivatives under Pd/Cu-catalysis: Identification of potent inhibitors of chorismate mutase (CM)

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Synthesis of 3-indolylmethyl substituted (pyrazolo / benzo)triazinone deriva-

tives under Pd/Cu-catalysis: Identification of potent inhibitors of chorismate

mutase (CM)

Gangireddy Sujeevan Reddy, Ampalam Venkata Snehalatha, Rebecca Kristina

Edwin, Kazi Amirul Hossain, Varadaraj Bhat Giliyaru, Raghu Chandrashekhar

Hariharapura, G. Gautham Shenoy, Parimal Misra, Manojit Pal

PII:

S0045-2068(19)30659-5

DOI:

https://doi.org/10.1016/j.bioorg.2019.103155

YBIOO 103155

Reference:

Bioorganic Chemistry

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Received Date:

Revised Date:

Accepted Date:

26 April 2019

4 July 2019

24 July 2019

Please cite this article as: G. Sujeevan Reddy, A. Venkata Snehalatha, R. Kristina Edwin, K. Amirul Hossain, V.

Bhat Giliyaru, R. Chandrashekhar Hariharapura, G. Gautham Shenoy, P. Misra, M. Pal, Synthesis of 3-indolylmethyl

substituted (pyrazolo / benzo)triazinone derivatives under Pd/Cu-catalysis: Identification of potent inhibitors of

chorismate mutase (CM), Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103155

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Synthesis of 3-indolylmethyl substituted (pyrazolo /

benzo)triazinone derivatives under Pd/Cu-catalysis:

identification of potent inhibitors of chorismate

mutase (CM)

Leave this area blank for abstract info.

Gangireddy Sujeevan Reddy, Ampalam Venkata Snehalatha, Rebecca Kristina Edwin, Kazi Amirul Hossain,

Varadaraj Bhat Giliyaru, Raghu Chandrashekhar Hariharapura, G. Gautham Shenoy, Parimal Misra and Manojit

Pal*

N

Q

N

O

N

Pd/Cu-cat

Q

O

+

N

Z

Et₃N, EtOH

60 ^oC

I

HN

Z

(Q = benzene / substituted pyrazole ring)

Design, synthesis and evaluation of 3-indolylmethyl substituted fused triazinone derivatives were undertaken to

identify potent inhibitors of chorismate mutase.

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Bioorganic Chemistry

journal homepage: www.elsevier.com

Synthesis of 3-indolylmethyl substituted (pyrazolo / benzo)triazinone derivatives

under Pd/Cu-catalysis: identification of potent inhibitors of chorismate mutase (CM)

Gangireddy Sujeevan Reddy,^a,bAmpalam Venkata Snehalatha,^aRebecca Kristina Edwin,^aKazi Amirul

Hossain,^aVaradaraj Bhat Giliyaru,^bRaghu Chandrashekhar Hariharapura,^bG. Gautham Shenoy,^bParimal

Misra^aand Manojit Pal^a,*

^aDr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India.

^bManipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Madhav Nagar, Manipal, 576 104, Karnataka, India.

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

The chorismate mutase (CM) is considered as an attractive target for the identification of potential

antitubercular agents due to its absence in animals but not in bacteria. A series of 3-indolylmethyl

substituted pyrazolotriazinone derivatives were designed and docked into CM in silico as potential

inhibitors. These compounds were efficiently synthesized using the Pd/Cu-catalyzed coupling-

cyclization in a single pot involving the construction of indole ring. The methodology was later

extended to the preparation of corresponding benzo analogs of pyrazolotriazinones i.e. 3-

indolylmethyl substituted benzotriazinone derivatives. Several of these novel compounds showed

significant inhibition of CM when tested in vitro at 30 µM. The SAR (Structure-Activity-

Relationship) studies suggested that benzotriazinone moiety was more favorable over the

pyrazolotriazinone ring. The two best active compounds showed IC₅₀~ 0.4-0.9 µM (better than

the reference/known compounds used) and no toxicity till 30 µM in vitro.

Available online

Keywords:

Triazinone

Indole

Pyrazole

Palladium

Chorismate mutase

1. Introduction

E-mail address: manojitpal@rediffmail.com (MP)

Tuberculosis or TB is a common and infectious lung disease

generally caused by the bacteria called Mycobacterium

tuberculosis (MTB) [1]. Roughly, 25% of the world population is

assumed to be infected by TB [2]. Indeed, TB remained one of the

leading causes of death worldwide due to (i) the extended duration

(6–9 months) of treatment, (ii) higher prevalence of (multi or

extensive) drug resistance, (iii) co-morbidity with HIV-AIDS and

(iv) decreased interest / effort in anti-infective drug research. The

chorismate mutase or CM (EC 5.4.99.5) that catalyzes the Claisen

rearrangement of chorismate to prephenate is considered as an

attractive target for the identification of effective antibacterial

agents due to its absence in animals but not in bacteria [3,4,5].

While several inhibitors of CM have been reported earlier [3] the

potent inhibitors possessing IC₅₀values in the submicromolar

range or less are not common in the literature. In our effort for the

discovery of novel inhibitors of CM we have reported a series of

N-aryl substituted fused triazinone derivatives as potential

inhibitors of CM [6]. One of them i.e. compound A (Fig. 1)

showed weak inhibition of CM (IC₅₀~ 15.13 ± 0.95 µM). In further

continuation of this research we became interested in

N

O

N

O

N

L

Ar

N

O

S

Me

S

O

Me

O

C

B

A

Fig. 1. Design of new triazinone derivative B based on known inhibitors

A and C.

the identification of fused triazinone based more potent inhibitors

B (Fig. 2). Our design mainly involved the introduction of a linker

‘‘L” at N-3 of the triazinone ring of A and replacing the naphthyl

ring by an indole moiety of the known inhibitor C [7]. We

anticipated that apart from introducing the structural flexibility

through the linker “L” assembling the structural features of two

different inhibitors e.g. A and C in a single molecular entity i.e. B

could enhance the overall potency. While a number of compounds

can be derived from C by varying ‘‘L” we focused on simpler

moiety like “CH₂” group as “L” at the initial stage.

To verify the merit and potential of our current designing

approach, the docking study of three representative molecules e.g.

B-1, B-2 and B-3 was performed using AutoDock Vina and the

binding affinities are presented in Chart 1. All molecules showed

----------------

* Corresponding author: Tel.: +91 40 6657; fax: +91 40 6657 1581

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Bioorganic Chemistry

better binding affinities than the reference compound A [6] and

[8] [i.e. 4-(3,4-dimethoxyphenethylamino)-3-nitro-5-

including B-1, B-2 and B-3. Herein we report the preliminary

results of our study that allowed us to discover a new class of CM

inhibitors which to our knowledge has not been explored earlier.

D

sulfamoylbenzoic acid] suggesting their possible superior

inhibitory properties against CM [the binding affinity of the most

active compound of series represented by C is -6.8 Kcal/mol (see

Fig. S-7 of suppl data file)]. Notably, the docking results showed

that the binding energy of these ligands at interface site was lower

than the other possible sites of MtbCM (indicating that these

molecules were preferably stabilized at the interface site of

MtbCM). The compound B-1 (3a) was potentially stabilized (Fig.

2(i), see also suppl data file) by several hydrophobic interactions

with the hydrophobic residues (TRP61, LEU65, PRO66, ILE67,

TYR110, PHE113 of A chain and LEU65, PRO66, ILE67 of B

chain) and hydrophobic regions of polar and charged residues

(GLN64, SER 70 of A chain and GLN64, GLU68 of B chain). In

addition, residue GLU68 of chain A played vital role to stabilize

the docked complex through H-bonding (formed between its

nitrogen atom of GLU68 and the sulfonyl oxygen of B-1) in the

distance of 2.093 Å.

(i)

Chart 1. Binding affinities of molecules with MtbCM

Molecules

Binding affinity (Kcal/mol)

Br

N

O

N

-10.2

S

O

N

O

Me

B-1

Cl

(ii)

N

O

N

-10.1

S

N

O

Me

B-2

N

O

N

S

-8.9

Me

N

O

Me

B-3

N

-6.9

-7.3

N

O

Me

A

NO₂

H

N

MeO

H₂NO₂S

OMe

COOH

D

*A and D are known / reference compounds

Like B-1 the molecule B-2 also formed H-bond through its

sulfonyl oxygen with the nitrogen atom of the GLU68 of A chain

in the distance of 2.108Å (Fig. 2(ii)). However, the positive

charged residue LYS60 of A chain made a hydrophobic contact

here that was not observed in case of B-1. While the similar H-

bonding was observed for B-3 (see suppl data file) too (involving

its sulfonyl oxygen and the nitrogen atom of the GLU68A of A

chain in the distance of 2.107Å) some of the hydrophobic contacts

with residues like ILE67A, SER70A, GLN64B, and PHE113B

were missing in this case perhaps due to the less hydrophobicity

of B-3 (compared to the previous molecules) thereby accounting

for its marginally low binding affinity (Chart 1). Nevertheless, the

overall outcome of in silico study appeared to be promising and

hence prompted us to undertake the actual synthesis as well as in

vitro evaluation of compounds based on framework B (Fig. 1)

Fig. 2. The 3D interaction diagram of (i) molecule B-1 and (ii) molecule

B-2 with interface residues of chorismate mutase (PDB code: 2FP2) (the A

chain is in blue color, B chain is red, and the H-bond is in cyan color) that

were prepared in UCSF Chimera.

2. Results and discussion

2.1. Chemistry

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The designed compounds were conveniently prepared

^C10 mol% of each catalyst used.

following a coupling-cyclization strategy under Pd/Cu-catalysis.

Indeed, the transition metal mediated coupling / cyclization in the

same pot [7,9-11] has become a common strategy for the synthesis

of 2-substituted indoles [12]. This one-pot reaction typically

involves the use of terminal alkynes as key reactants that are

coupled with a 2-iodoanilide. The terminal alkyne i.e. 5-methyl-3-

(prop-2-ynyl)-7-propyl-3H-pyrazolo[4,3-d][1,2,3] triazin-4(5H)-

one (1a) required in the current study was prepared following a

known method and were coupled with appropriate 2-

iodosulphanilides (2) under Pd/Cu-catalysis (Scheme 1).

^d1 mol% of each catalyst used.

Indeed, further improvement in yield was observed when K₂CO₃

was replaced by Et₃N (entry 7, Table 1). Interestingly, a substantial

increase in yield was observed when the combination of

(PPh₃)₂PdCl₂-CuI was used as a catalyst system along with Et₃N

as a base (entry 8, Table 1). Notably 10 mol% of each catalyst was

used in all the cases earlier (entries 1-7, Table 1) whereas only 1

mol% of (PPh₃)₂PdCl₂and CuI each was necessary in the case of

entry 8 of Table 1 (indeed, the increase of catalysts quantity did

not improve the product yield whereas use of < 1 mol% of catalysts

decreased the yield. For further details, see: Table S-1, suppl data

file). Though the use of water as a cheap and non-hazardous

solvent was not successful in this case (entry 9, Table 1) we were

satisfied with the %yield of 3a obtained in case of entry 8 of Table

1. Thus the use of (PPh₃)₂PdCl₂-CuI and Et₃N in EtOH was found

to be optimal for the preparation of 3a and was used to prepare

other related analogues (Scheme 1, Table 2, see also Table S-2,

suppl data file). A number of 2-iodosulphanilides (2a-k, Z =

mesyl, tosyl, phenylsulfonyl, 2-thienosulfonyl) were reacted with

alkyne 1a under the optimized conditions. The reaction proceeded

well irrespective of the presence of various substituents [e.g. F

(3f), Cl (3b, 3d, 3h), Br (3a), NO₂(3j) and Me (3k)] affording the

R²

I

R²

Br

NH

Z

N

O

N

O

R¹

N

2

N

NH

N

R¹

N

Z

K₂CO₃

MeCN

RT, 3h

(PPh₃)₂PdCl₂

CuI, Et₃N

EtOH, 60^oC

2-3 h

Me

O

1aa

1a

3

Scheme 1. Synthesis of 3-indolylmethyl substituted

pyrazolotriazinone derivatives 3

Initially, the alkyne 1a was reacted with N-(4-bromo-2-

iodophenyl)-4-methylbenzenesulfonamide 2a in the presence of

CuI and K₂CO₃in PEG-400 at 60°C for 8 h (Table 1). While the

expected product 3a was isolated in this case the yield was poor

(entry 1, Table 1). The change of base to Et₃N (entry 2, Table 1)

or solvent to EtOH (entry 3, Table 1) did not improve the yield.

The use of other catalyst e.g. InCl₃(entry 4, Table 1) or

combination of CuI and InCl₃(entry 5, Table 1) was also found to

be not effective. The product yield was improved to some extent

when combination of 10%Pd/C-CuI-PPh₃was used as a catalyst

system along with K₂CO₃as a base (entry 6, Table 1).

desired 3-indolylmethyl substituted

derivatives in good yields.

pyrazolotriazinone

Table 2. Preparation^aof compound 3 (Scheme 1) and their in

vitro evaluation against CM.

Entry R¹= ; R²=; Z =

3^b

%Yield^c% inhibition^d

@30 µM

(2)

1

2

3

4

5

6

7

8

H, Br, Ts (2a)

H, Cl, Ms (2b)

H, H, SO₂Ph (2c)

H, Cl, Ts (2d)

H, H, Ts (2e)

H, F, Ts (2f)

3a

3b

3c

3d

3e

3f

82

78

72

78

80

74

76

78

ND

5

25

70

20

7

Table 1. Effect of reaction conditions on coupling of terminal

alkyne 1a with 2a.^a

Br

Catalyst

N

I

Br

Base

N

O

N

H, H, Ms (2g)

H, Cl,

3g

3h

37

ND

+

N

HN

Ts

N

Solvent

Time

N

O

Me

Ts

Me

2-Thienosulfonyl (2h)

H, H,

60 ^o

C

1a

2a

3a

(Ts = SO₂C₆H₄Me-p)

9

3i

78

24

2-Thienosulfonyl (2i)

H, NO₂, Ms (2j)

H, Me, Ts (2k)

Entry

Catalyst

Base

Solvent

Time

%Yield^b

10

11

3j

80

72

30

19

(10 mol%)

CuI

(h)

8

3k

1

2

K₂CO₃

Et₃N

PEG-400

EtOH

15

0

^aAll the reactions were carried out using alkyne 1a (1 mmol), 2 (1.2 mmol),

(PPh₃)₂PdCl₂(1 mol%), CuI (1 mol%), and Et₃N (2 mmol) in EtOH (10 mL) at

60 °C for 2-3 h.

CuI

8

3

CuI

K₂CO₃

8

20

0

4

InCl₃

EtOH

8

^bIdentified by ¹H and ¹³C NMR, IR, and MS.

^cIsolated yields.

5^c

6^c

CuI / InCl₃

CuI/ PPh₃

10%Pd/C

CuI/ PPh₃

10%Pd/C

CuI

EtOH

8

20

40

EtOH

8

^dCompound A and D showed 38% and 57% inhibition at 30 µM.

*Ms = mesyl, Ts = p-tosyl, ND = Not determined.

7^c

8^d

Et₃N

EtOH

H₂O

8

2

8

65

82

0

Having prepared a range of pyrazolotriazinone containing

novel indole derivatives (3) we focused on assessing generality of

the present Pd/Cu-catalyzed coupling-cyclization reaction by

using simpler alkyne (1b) derived from 1a e.g. replacing the

pyrazole moiety of 1a by a benzene ring (i.e. the benzo analogue

to 1a). Accordingly, 1b was prepared from benzo[d][1,2,3]triazin-

4(3H)-one (1bb) and employed in the Pd/Cu-catalyzed coupling-

cyclization with a number of 2-iodoanilides (2) under the

optimized conditions (entry 8, Table 1). The corresponding indoles

i.e. 3-indolylmethyl substituted benzotriazinone derivatives (4a-l)

Pd(PPh₃)₂Cl₂

CuI

9^d

K₂CO₃

Pd(PPh₃)₂Cl₂

CuI/ PPh₃

10%Pd/C

10^c

H₂O

0

^aReactions were carried out using alkyne 1a (1 mmol), 2a (1.2 mmol), base

(2.0 mmol) and catalyst in a solvent (10 mL) under nitrogen at 60 ^oC.

^bIsolated yield.

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Bioorganic Chemistry

were obtained in good yields in all these cases (Table 3, see also

 1.0 (t)

R²

Table S-2, suppl data file). Indeed, the use of three additional

anilides (2l-n) was also successful when the desired products were

isolated in good yields (entries 10-12, Table 3). Finally, the use of

6-bromo-3-(prop-2-yn-1-yl)benzo[d][1,2,3]triazin-4(3H)-one (1c)

[prepared from 6-bromobenzo[d][1,2,3]triazin-4(3H)-one (1cc),

see suppl data file] was examined that afforded the corresponding

indole derivative (4m) when coupled with the 2d (entry 13, Table

3).

 1.9-1.8 (m)

 3.0 (t)

N

 6.0

N

R¹

R²

N

Z

 6.1

Me

 4.3

O

150 ppm

3

N

 6.0

N

Table 3. Preparation^aof compound 4 and their in vitro

evaluation against CM.

R¹

N

 6.1

Z

O

155 ppm

4

R²

Br

2

Fig. 3. Partial ¹H and ¹³C NMR data of compound 3 and 4.

N

O

(PPh₃)₂PdCl₂

N

R¹

NH

N

K₂CO₃

MeCN

RT, 3h

CuI, Et₃N

EtOH, 60^oC

2-3 h

Z

O

Mechanistically (Scheme 2) [9,10,11], the present coupling-

cyclization method proceeded via (i) a first catalytic cycle i.e. the

known Sonogashira coupling [13] involving in situ generated

Pd(0) catalysed C-C bond forming reaction to give the alkyne E-1

followed by (ii) the second catalytic cycle i.e. Cu(I)-catalysed

intramolecular cyclization of E-1 to give the desired fused

triazinone containing indole derivatives 3 or 4. The second

catalytic cycle proceeded via interaction of Cu(I) with the alkyne

moiety of E-1 that seemed to be favoured by the coordination with

the proximate carbonyl group (of E-2) leading to E-3. Protonolysis

of the organo-copper species E-3 afforded the desired product via

E-4 with the regeneration of CuI to complete the second catalytic

cycle (the cyclization step).

O

1bb

1b

4

Entry

R¹=; R²=; Z =

(2)

4^b

Yield^c

% inhibition^d

@30 µM

38

1

2

3

4

5

6

7

8

H, Br, Ts (2a)

H, Cl, Ms (2b)

H, H, SO₂Ph (2c)

H, Cl, Ts (2d)

H, H, Ts (2e)

H, F, Ts (2f)

H, H, Ms (2g)

H, Cl,

4a

4b

4c

4d

4e

4f

80

78

74

81

77

71

80

26

42

78

56

41

4g

4h

20

N

BHI

N

57

Q

N

Q

CuI

Q

2-Thienosulfonyl (2h)

H, H,

O

(B = Et₃N)

9

4i

78

45

O

Z

N

Cu

2-Thienosulfonyl (2i)

H, F, Ms (2l)

I

N

E-1

E-3

Z

N

Z

10

11

12

13

4j

4k

4l

72

68

29

37

27

34

E-2

BH

Me, Me, Ms (2m)

Me, Me, SO₂Ph (2n)

H, Cl, Ts (2d)

BHI

66

Coupling

Cyclization

N

4m

79^e

Pd(0)

Pd(II)

Q

N

CuI

^aAll the reactions were carried out using alkyne 1b (1 mmol), 2 (1.2 mmol),

(PPh₃)₂PdCl₂(1 mol%), CuI (1 mol%), and Et₃N (2 mmol) in EtOH (10 mL) at

60 °C for 2-3 h.

1

2

+

O

H

3

4

or

Z

N

Cu

I

^bIdentified by ¹H and ¹³C NMR, IR, and MS.

^cIsolated yields.

E-4

Scheme 2. The plausible reaction mechanism for coupling-

cyclization of fused triazinone alkyne (1) with 2-iodoanilides

(2) under Pd/Cu-catalysis.

^dCompound A and D showed 38% and 57% inhibition at 30 µM

^e6-bromo-3-(prop-2-yn-1-yl)benzo[d][1,2,3]triazin-4(3H)-one (1c) was

used in place of 1b and the corresponding product obtained was 6-bromo-3-

{(5-chloro-1-tosyl-1H-indol-2-yl)methyl}benzo[d][1,2,3]triazin-

4(3H)-one (4m).

2.2. Biology

We then tested all the synthesized compounds i.e. 3 and 4 for

their CM inhibitory properties in vitro [14] using an assay that

involved measurement of catalytic activity of enzyme (CM) in the

conversion of chorismate (substrate) to prephenate. The known

inhibitor [8] D (Chart 1) was used as a reference compound. All

compounds were tested at an initial concentration of 30 µM and

compounds showed inhibition > 50% were identified as most

active inhibitors of CM. In case of pyrazolotriazinone series (3)

the compound 3d showed significant inhibition (Table 2) whereas

compound 4d, 4e and 4h from the benzotriazinone series were

identified as most active inhibitors (Table 3). Several other

compounds showed moderate inhibition (~35-40%) from both

series e.g. 3g (Table 2), 4a, 4c, 4f, 4i and 4k (Table 3). Notably,

greater number of compounds from benzotriazinone series was

found to be active compared to that of pyrazolotriazinone series

partially due to the fact that compounds from the later series

*Ms = mesyl, Ts = p-tosyl, ND = Not determined.

All the synthesized 3-indolylmethyl substituted pyrazolo and

benzotriazinone derivatives (3 and 4) were characterized by (¹H

and ¹³C NMR, and MS) spectral data. Briefly (Fig 3), a singlet near

1

 6.1 and 6.0 in the H NMR spectra accounted for the linker

“CH₂” moiety and C-3 indole proton, respectively. The compound

3 was further characterized by the presence of NCH₃protons near

 4.3 and the n-propyl moiety near  3.0 (t, CH₂), 1.9-1.8 (m, CH₂)

and 1.0 (t, CH₃). The “C=O” group of the fused triazinone moiety

appeared in the region 150-155 ppm in ¹³C NMR spectra and

showed a strong IR absorption in the region 1700-1670 cm^-1.

JOURNAL PRE-PROOF

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showed some solubility issue in the assay medium used. For

100

80

60

40

20

0

example compound 3a and 3h could not be tested because of their

precipitation in the assay medium. Indeed, compound 3 was

expected to be more lipophilic than 4 due to the presence of more

number of nitrogen atoms and alkyl side chains. Nevertheless, an

overview of SAR (Structure-Activity-Relationship) observations

noted from the current series of compounds is summarized in Fig

4. In general the “Cl” group at C-5 of indole ring was beneficial

for CM inhibition whereas other substituents like F, Br or Me or

no substituent at this position decreased the activity. No

substituent at C-7 position was generally favorable whereas a

“Me” group at the same position lowered the activity. Among the

sulfonyl substituents at indole N-1 position the p-tosyl group

contributed towards high activity though low activity was also

observed in few cases. The other groups like mesyl,

phenylsulfonyl and 2-thienosulfonyl groups were found to be

inferior. The aryl/heteroaryl ring fused with the triazinone moiety

also influenced the activity. This is

0.001

0.01

0.1

1

10

100

Log Concentration(M)

Fig. 5. Concentration dependent study of compound 3d against CM

To understand the interaction of these molecules with CM

through in silico studies it was desirable to analyze and compared

the docking results. Since the docking results of compound 3d

H - low to moderate activity

F - low to moderate activity

Cl - generally high activity

Br - moderate activity

NO₂- moderate activity

Me - low activity

R²

N

Q

N

R¹

N

Z

O

H - low to high activity

Me - low activity

Z = mesyl; low activity

Z = phenylsulfonyl; low to moderate activity

Z = tosyl; low to high activity

Z = 2-thienosulfonyl; low to moderate activity

Q = benzene ring; good activity

Q = pyrazole ring; low to good activity

Fig. 4. Summary of SAR for CM inhibitory activities of compound 3 and

4.

exemplified by the fact that three compounds (e.g. 4d, 4e and

4h) showed inhibition > 50% contain benzene ring fused with the

triazinone moiety whereas only one compound (3d) showed high

activity that contains pyrazole ring at the same place.

Fig. 6. The 3D interaction diagram of compound 4d with interface

residues of chorismate mutase (PDB code: 2FP2).

Nonetheless, the best active compounds i.e. 3d from the

pyrazolotriazinone series and 4d from the benzotriazinone series

were taken forward for concentration dependent study to

determine their IC₅₀values. Accordingly, the IC₅₀values were

found to be 0.40±0.05 µM (Fig.5) and 0.85±0.10 µM for

compound 3d and 4d, respectively whereas that of known

compound D was 5.3 ± 0.2 µM. It was therefore evident that both

the compounds were more potent than D in this assay. Moreover,

the current efforts afforded better active fused triazinone

derivatives than the previously reported inhibitor A (Fig. 1) [6].

These compounds were also evaluated for their potential toxicity

by MTT assay on RAW 264.7 cell line (a commonly used model

of mouse macrophages for the study of cellular responses to

microbes and their products) at different concentrations. None of

them showed significant toxicities till 30 µM of their concentration

tested. Indeed, % cell viability was found to be 100, 100, 86, 91,

68 and 42% at the concentration of 1, 3, 10, 30, 60 and 100 µM of

3d, respectively suggesting potential / acceptable therapeutic

window for this compound.

was already available (molecule B-2, Fig 2(ii)) hence docking of

4d was performed. According to the docking results (Fig. 6)

compound 4d (binding affinity -9.5 Kcal/mol) was potentially

stabilized by several hydrophobic interactions with the

hydrophobic residues (TRP61, LEU65, PRO66, ILE67, TYR110,

PHE113 of A chain and PRO66, ILE67, TYR110 of B chain) and

hydrophobic regions of polar and charged residues (GLN64 of A

chain and GLU68 of B chain). Unlike 3d the compound 4d (being

a benzotriazinone derivative rather than pyrazolotriazinone) was

stabilized by a slightly different binding mode at the interface site,

and formed H-bond at a distance of 2.036 Å with the residue

GLU68 of chain B instead of chain A.

Since the early detection of ADME (absorption, distribution,

metabolism, and excretion) or pharmacokinetic properties of

NCEs (new chemical entities) is an useful strategy for further

progress of molecules hence computational ADME prediction of

compounds 3d and 4d along with the known inhibitor D was

performed using SwissADME web-tool [15] and results are

presented in Table 4 (among the various descriptors only notable

Table 4. Computational ADME prediction of 3d, 4d and D.

Properties

Molecules

4d

3d

D

JOURNAL PRE-PROOF

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Bioorganic Chemistry

(i) Physicochemical

an useful template for the design, synthesis and identification of

Molecular Weight (g/mol)

511.00

4.41

-6.41

(poorly

soluble)

464.92

4.55

425.41

0.90

potent inhibitors of CM.

4. Experimental section

4.1. Chemistry

Consensus Log P^a

Log S (ESOL)^b

-6.44

-3.63

(poorly

soluble)

(soluble)

(ii) Pharmacokinetics

GI^cabsorption

4.1.1. General methods

High

No

High

No

Low

No

Yes

BBB^dpermeation

P-gp^esubstrate

Unless stated otherwise, reactions were performed under

nitrogen atmosphere using oven dried glassware. Reactions were

monitored by thin layer chromatography (TLC) on silica gel plates

(60 F254), visualizing with ultraviolet light or iodine spray. Flash

chromatography was performed on silica gel (230-400 mesh)

using distilled hexane and EtOAc. ¹H and ¹³C NMR spectra were

recorded in CDCl₃or DMSO-d₆solution by using a 400 and 100

MHz spectrometer. Proton chemical shifts (δ) are relative to

tetramethylsilane (TMS, δ = 0.00) as internal standard and

expressed in ppm. Spin multiplicities are given as s (singlet), d

(doublet), dd (doublet of doublet), td (triplet of doublet), t (triplet)

and m (multiplet) as well as bs (broad singlet). Coupling constants

(J) are given in hertz. Melting points were determined using

melting point apparatus and are uncorrected. MS spectra were

obtained on Agilent 6430 series Triple Quard LC-MS / MS

spectrometer. Chromatographic purity by HPLC (Agilent 1200

series ChemStation software) was determined by using area

normalization method and the condition Specified in each case:

column, mobile phase (range used), flow rate, detection

wavelength, and retention times. The starting compound 1aa and

1bb was prepared following a reported method [6, 16a].

(iii) Druglikenss

Lipinski rule

1 violation: No

No

MW^f>500

No

violation

No

violation

1 violation

(TPSA^g

>140; i.e.

182.15Å²)

0.11

Veber rule

violation

Bioavailability score

^aLog P: Lipophilicity.

0.17

0.55

^bLog S (ESOL): water solubility, calculated by ESOL method which

Quantitative Structure-Property Relationship (QSPR) based model.

^cGI: Gastrointestinal.

^dBBB: Blood Brain Barrier.

^eP-gp: permeability glycoprotein.

^fMW: molecular weight,

^gTPSA: Topological polar surface area.

one are listed in the table). In spite of one violation of Lipinski’s

criteria (molecular weight > 500) the desirable ADME was

predicted for compound 3d. The desirable ADME was predicted

for 4d too with no such violation. Indeed, high GI absorption, no

BBB (Blood Brain Barrier) penetration and no P-gp substrate

potential have been predicted for both molecules with superior

bioavailability score over the reference molecule D. Overall, the

compound 3d and 4d could be better inhibitors than D and

appeared to have medicinal value especially from the viewpoint of

developing potential antitubercular agents. Notably, favorable

biological properties of 1,2,3-benzotriazin-4-one derivatives have

been documented well in the literature [16]. The compound 3d and

4d therefore deserves further pharmacological profiling.

4.1.2. General procedure for the preparation of compound 1a-

c

Propargyl bromide (1.2 mmol) was added to a solution of 1aa

or 1bb or 1cc (1.0 mmol) and K₂CO₃(3.0 mmol) in CH₃CN (25

mL) under a nitrogen atmosphere. The mixture was stirred at room

temperature for 5h. After completion of the reaction (confirmed by

TLC), the mixture was diluted with ice-water (60 mL) and

extracted with EtOAc (3 x 15 mL). The organic layers were

collected, combined, dried over anhydrous Na₂SO₄, filtered and

concentrated under low vacuum. The residue was purified by

column chromatography using hexane- EtOAc as eluent to afford

the title compound.

4.1.3. 5-Methyl-3-(prop-2-yn-1-yl)-7-propyl-3H-pyrazolo[4,3-

d][1,2,3]triazin-4(5H)-one (1a)

3. Conclusion

White solid (78% yield); mp 55-57 °C; R_f(20% EtOAc/n-

hexane) 0.3; ¹H NMR (400 MHz, CDCl₃) δ: 5.19 (d, J = 2.4 Hz,

2H, NCH₂), 4.29 (s, 3H, NCH₃), 3.01(t, J = 7.6 Hz, 2H, CH₂), 2.37

(t, J = 2.4 Hz, 1H, HC≡C), 1.93-1.82 (m, 2H, MeCH₂), 1.01 (t, J

= 7.6 Hz, 3H, CH₃);¹³C NMR (100 MHz, CDCl₃) δ: 149.8 (C=O),

148.6, 136.1, 124.7, 77.0 (-C≡), 73.2 (HC≡), 38.8 (CH₂), 38.6

(NMe), 27.8 (CH₂), 22.3 (CH₂), 13.9 (Me); IR (KBr) _max3238,

2120, 1697, 1531 cm^-1; MS (ES mass): m/z 232.2 (M+1, 100%).

In conclusion, a series of novel 3-indolylmethyl substituted

pyrazolotriazinone derivatives were initially designed and docked

into CM in silico as potential inhibitors of CM. Synthesis of these

compounds were carried out efficiently using the Pd/Cu-catalyzed

coupling-cyclization in a single pot involving the construction of

indole ring. The methodology involved reaction of 5-methyl-3-

(prop-2-ynyl)-7-propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-

one with a number of 2-iodosulphanilides in the presence of

(PPh₃)₂PdCl₂, CuI and Et₃N in EtOH at 60 °C for 2-3 h to afford

the desired compounds in good yields. The methodology was later

extended to the preparation of corresponding benzene analogs of

4.1.4.

(1b)

3-(Prop-2-yn-1-yl)benzo[d][1,2,3]triazin-4(3H)-one

White solid (80% yield); mp 103-105 °C; R_f(20% EtOAc/n-

hexane) 0.35; ¹H NMR (400 MHz, CDCl₃) δ: 8.38 (dd, J = 8.0, 1.2

Hz, 1H, ArH ), 8.18 (d, J = 8.4 Hz, 1H, ArH), 8.01-7.94 (m, 1H,

ArH), 7.85-7.80 (m, 1H, ArH),5.23 (d, J = 2.8 Hz, 2H, NCH₂),

2.38 (t, J = 2.8 Hz, 1H, HC≡C); ¹³C NMR (100 MHz, CDCl₃) δ:

154.9(C=O), 144.3, 135.1, 132.7, 128.5, 125.2, 119.9, 76.9 (-C≡),

73.3 (HC≡), 39.4 (CH₂); MS (ES mass): m/z 186.20 (M+1, 100%).

pyrazolotriazinones

i.e.

3-indolylmethyl

substituted

benzotriazinone derivatives. Overall, more than 20 new

compounds were prepared and tested for their CM inhibitory

potentials in vitro. Several of these compounds showed significant

inhibition of CM when tested at 30 µM. The SAR (Structure-

Activity-Relationship) studies suggested that benzotriazinone

moiety was more favorable over the pyrazolotriazinone ring. The

two best active compounds showed IC₅₀values ~ 0.4-0.9 µM and

were found to be more potent than the reference / known

compounds used. They were also non-toxic towards RAW 264.7

cell line till 30 µM in an MTT assay. The present 3-indolylmethyl

substituted (pyrazolo / benzo)triazinone framework appeared to be

4.1.5.

6-Bromo-3-(prop-2-yn-1-yl)benzo[d][1,2,3]triazin-

4(3H)-one (1c)

White solid (75% yield); mp 92-94 °C; R_f(20% EtOAc/n-

1

hexane) 0.4; H NMR (400 MHz, CDCl₃) δ: 8.12-7.95 (m, 2H,

ArH), 8.51 (s, 1H, ArH), 5.21 (d, J = 2.8 Hz, 2H, NCH₂), 2.38 (t,

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7

J = 2.8 Hz, 1H, HC≡C); ¹³C NMR (100 MHz, CDCl₃) δ: 153.6

(C=O), 142.9, 138.5, 130.2, 128.0, 127.4, 121.2, 76.6 (-C≡), 73.5

(HC≡), 39.5 (CH₂); HPLC: 99.5%, column: X Bridge C-18

150*4.6 mm 5μm, mobile phase A: 5mM Ammonium Acetate in

water, mobile phase B: CH₃CN, (gradient) T/B% : 0/10, 23/90,

30/90, 31/90, 35/10; flow rate: 1 mL/min; Max plot, retention time

13.6 min; MS (ES mass): m/z 265.9 (M+2, 100%).

22.3 (CH₂), 14.0 (Me); HPLC: 98.9%, column: Eclips XDB C-

18 150*4.6 mm 5μm, mobile phase A: 0.05% TFA in water,

mobile phase B: 0.05% TFA in CH₃CN, (gradient) T/B% : 0/10,

5/10, 25/90, 30/90, 31/10, 35/10; flow rate: 1 mL/min; UV 218.0

nm, retention time 24.8 min; IR (KBr) _max2953, 1707, 1529, 1475

cm^-1; MS (ES mass): m/z 463.1 (M+1, 100%).

4.2.1. General procedure for the preparation of compound 3

and 4

4.2.5. 3-{(5-Chloro-1-tosyl-1H-indol-2-yl)methyl}-5-methyl-7-

propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one (3d)

A

mixture of compound 1a or 1b (1.0 mmol), 2-

White solid (78% yield); mp: 151-153 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 7.98 (d, J = 8.8 Hz,

1H, ArH), 7.78 (d, J = 8 Hz, 2H, ArH), 7.27-7.22 (m, 3H, ArH),

7.22-7.17 (m, 1H, ArH), 6.04 (s, 2H, NCH₂), 6.01 (s, 1H, indole

(C-3) H), 4.28 (s, 3H, NCH₃), 3.03 (t, J = 7.6 Hz, 2H, CH₂), 2.33

(s, 3H, ArCH₃), 1.96-1.85 (m, 2H, MeCH₂), 1.04 (t, J = 7.6 Hz,

3H, CH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 150.2 (C=O), 148.6,

145.5, 137.2, 136.1, 135.3, 135.0, 130.4, 130.1 (2C), 129.5, 126.6

(2C), 124.9, 124.6, 120.3, 115.5, 108.9, 46.8 (NCH₂), 38.8 (NMe),

27.9 (CH₂), 22.3 (CH₂), 21.6 (ArMe), 14.0 (Me); HPLC: 99.9%,

column: Cosmicsil Aura ODS C-18 150*4.6 mm 5μm, mobile

phase A: 0.1% TFA in water, mobile phase B: CH₃CN, (gradient)

T/B% : 0/10, 20/95, 27/95, 31/10, 35/10; flow rate: 1 mL/min; UV

220.0 nm, retention time 21.2 min; IR (KBr) _max2960, 1705,

1532, 1403 cm^-1; LCMS : m/z 511.1 (M+1, 100%).

iodosulphanilides (2, 1.2 mmol), (PPh₃)₂PdCl₂(1 mol%), CuI (1

mol%) and Et₃N (2.0 mmol) in EtOH (10 mL) was stirred at 60°C

for 2-3 h. After completion of the reaction (indicated by TLC) the

reaction mixture is diluted with EtOAc (50 mL) and filtered

through celite bed. The organic layer was collected, washed with

water (3 × 30 mL), dried over anhydrous Na₂SO₄, filtered and

concentrated under low vacuum. The crude residue was purified

by column chromatography on silica gel using hexane-EtOAc to

afford the desired product.

4.2.2. 3-{(5-Bromo-1-tosyl-1H-indol-2-yl)methyl}-5-methyl-7-

propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one (3a)

White solid (82% yield); mp: 170-172 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 7.95 (d, J = 8.8 Hz,

1H, ArH), 7.79 (d, J = 8.4 Hz, 2H, ArH), 7.44 (d, J = 1.7 Hz, 1H,

ArH), 7.36 (dd, J = 8.8, 2 Hz, 1H, ArH), 7.27 (d, J = 8.4 Hz, 2H,

ArH), 6.06 (s, 2H, NCH₂), 6.01 (s, 1H, indole (C-3) H), 4.30 (s,

3H, NCH₃), 3.05 (t, J = 7.6 Hz, 2H, CH₂), 2.36 (s, 3H, ArCH₃),

4.2.6. 5-Methyl-7-propyl-3-{(1-tosyl-1H-indol-2-yl)methyl}-

3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one (3e)

White solid (80% yield); mp: 151-153 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 8.07 (d, J = 8.4 Hz,

1H, ArH), 7.81 (d, J = 8.4 Hz, 2H, ArH), 7.34-7.22 (m, 4H, ArH),

7.18-7.14 (m, 1H, ArH), 6.08 (bs, 3H, NCH₂, indole (C-3) H),

4.30 (s, 3H, NCH₃), 3.05 (t, J = 7.6 Hz, 2H, CH₂), 2.33 (s, 3H,

ArCH₃), 1.97-1.86 (m, 2H, MeCH₂), 1.05 (t, J = 7.6 Hz, 3H, CH₃);

¹³C NMR (100 MHz, CDCl₃) δ: 150.2 (C=O), 148.6, 145.1, 137.1,

136.1, 135.6, 135.3, 130.0 (2C), 129.2, 126.6 (2C), 124.8, 124.7,

123.8, 120.8, 114.5, 109.8, 47.0 (NCH₂), 38.8 (NMe), 27.9 (CH₂),

22.3 (CH₂), 21.6 (ArMe), 14.0 (Me); HPLC: 99.9%, column:

Cosmicsil Aura ODS C-18 150*4.6 mm 5μm, mobile phase A:

0.1% TFA in water, mobile phase B: CH₃CN, (gradient) T/B% :

0/10, 20/95, 27/95, 31/10, 35/10; flow rate: 1 mL/min; UV 215.0

nm, retention time 18.8 min; IR (KBr) _max2962, 1697, 1529, 1453

cm^-1; MS (ES mass): m/z 477.1 (M+1, 100%).

1.97-1.86 (m, 2H, MeCH₂), 1.05 (t, J = 7.6 Hz, 3H, CH₃); ¹³

C

NMR (100 MHz, CDCl₃) δ: 150.2 (C=O), 148.6, 145.5, 136.9,

136.1, 135.7, 135.0, 130.9, 130.1 (2C), 127.6, 126.6 (2C), 124.6,

123.4, 117.2, 115.9, 108.8, 46.8 (NCH₂), 38.8 (NMe), 27.9 (CH₂),

22.3 (CH₂), 21.6 (ArMe), 14.0 (Me); HPLC: 98.9%, column: X

Bridge C-18 150*4.6 mm 3.5μm, mobile phase A: 0.1% TFA in

water, mobile phase B: CH₃CN:THF (80:20), (gradient) T/B% :

0/10, 20/95, 40/95, 41/10, 45/10; flow rate: 1 mL/min; UV 220.0

nm, retention time 18.6 min; IR (KBr) _max2965, 1706, 1533, 1402

cm^-1; MS (ES mass): m/z 557.1 (M+2, 100%), 555.1 (M⁺, 98%).

4.2.3. 3-[{5-Chloro-1-(methylsulfonyl)-1H-indol-2-yl}methyl]-

5-methyl-7-propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one

(3b)

White solid (78% yield); mp: 141-143 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 7.95 (d, J = 8.8 Hz,

1H, ArH), 7.42 (d, J = 2 Hz, 1H, ArH), 7.29 (dd, J = 8.8, 2 Hz,

1H, ArH), 6.27 (s, 1H, indole (C-3) H), 5.96 (s, 2H, NCH₂), 4.28

(s, 3H, NCH₃), 3.31 (s, 3H, SCH₃), 3.04 (t, J = 7.6 Hz, 2H, CH₂),

4.2.7. 3-{(5-Fluoro-1-tosyl-1H-indol-2-yl)methyl}-5-methyl-7-

propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one (3f)

White solid (74% yield); mp: 140-142 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 8.03 (dd, J = 8.8, 4.4

Hz, 1H, ArH), 7.79 (d, J = 8.4 Hz, 2H, ArH), 7.27 (d, J = 6.4 Hz,

2H, ArH), 7.02-6.96 (m, 2H, ArH), 6.06 (s, 2H, NCH₂), 6.03 (s,

1H, indole (C-3) H), 4.30 (s, 3H, NCH₃), 3.05 (t, J = 7.6 Hz, 2H,

CH₂), 2.36 (s, 3H, ArCH₃), 1.97-1.86 (m, 2H, MeCH₂), 1.05 (t, J

= 7.6 Hz, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 161.0 (C-F J

= 239.3 Hz), 150.3 (C=O), 148.7, 145.4, 137.4, 136.2, 135.1,

133.4, 130.3(C-F J = 10.3 Hz), 130.1 (2C), 126.6 (2C), 118.0,

115.7 (C-F J = 9.3 Hz), 112.8 (C-F J = 25.3 Hz), 109.5 (C-F J = 4

Hz), 106.4 (C-F J = 24 Hz), 46.9 (NCH₂), 38.8 (NMe), 27.9 (CH₂),

22.3 (CH₂), 21.6 (ArMe), 14.0 (Me); HPLC: 99.5%, column:

Eclips XDB C-18 150*4.6 mm 5μm, mobile phase A: 0.05% TFA

in water, mobile phase B: 0.05% TFA in CH₃CN, (gradient) T/B%

: 0/10, 5/10, 25/90, 30/90, 31/10, 35/10; flow rate: 1 mL/min; UV

215.0 nm, retention time 25.8 min; MS (ES mass): m/z 495.1 (M-

+1, 100%).

1.93-1.87 (m, 2H, MeCH₂), 1.04 (t, J = 7.6 Hz, 3H, CH₃); ¹³

C

NMR (100 MHz, CDCl₃) δ: 150.1 (C=O), 148.6, 136.9, 136.2,

135.0, 130.2, 129.7, 125.2, 124.6, 120.5, 115.0, 109.2, 45.9

(NCH₂), 41.2 (SMe), 38.8 (NMe), 27.9 (CH₂), 22.3 (CH₂), 14.0

(Me); HPLC: 99.9%, column: X Bridge C-18 150*4.6 mm 5μm,

mobile phase A: 0.1% TFA in water, mobile phase B: CH₃CN,

(gradient) T/B% : 0/30, 20/95, 30/95, 31/30, 35/30; flow rate: 1

mL/min; max plot, retention time 14.8 min; IR (KBr) _max3006,

1697, 1533, 1446 cm^-1; MS (ES mass): m/z 435.1 (M+1, 100%).

4.2.4. 5-Methyl-3-{(1-(phenylsulfonyl)-1H-indol-2-yl)methyl}-

7-propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one (3c)

White solid (72% yield); mp: 154-156 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 8.07 (d, J = 8.4 Hz,

1H, ArH), 7.97-7.88 (m, 2H, ArH), 7.57-7.52 (m, 1H, ArH), 7.46

(t, J = 8 Hz, 2H, ArH), 7.33 (d, J = 8 Hz, 1H, ArH), 7.30-7.24 (m,

1H, ArH), 7.20-7.14 (m, 1H, ArH), 6.10 (s, 1H, indole (C-3) H),

6.08 (s, 2H, NCH₂), 4.30 (s, 3H, NCH₃), 3.05 (t, J = 7.6 Hz, 2H,

CH₂), 1.97-1.87 (m, 2H, MeCH₂), 1.05 (t, J = 7.6 Hz, 3H, CH₃);

¹³C NMR (100 MHz, CDCl₃) δ: 150.2 (C=O), 148.6, 138.3, 137.0,

135.5, 133.9, 129.4 (2C), 129.2, 126.6 (2C), 124.8, 124.7, 123.8,

120.8, 116.7, 114.4, 110.0, 46.8 (NCH₂), 38.8 (NMe), 27.9 (CH₂),

4.2.8. 5-Methyl-3-[{1-(methylsulfonyl)-1H-indol-2-yl}methyl]-

7-propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one (3g)

White solid (76% yield); mp: 78-80 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 8.01 (d, J = 8.4 Hz,

1H, ArH), 7.45 (d, J = 7.6 Hz, 1H, ArH), 7.37-7.30 (m, 1H, ArH),

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Bioorganic Chemistry

7.27-7.23 (m, 1H, ArH), 6.32 (s, 1H, indole (C-3) H), 5.97 (s,

4.2.12. 5-Methyl-3-{(5-methyl-1-tosyl-1H-indol-2-yl)methyl}-

2H, NCH₂), 4.28 (s, 3H, NCH₃), 3.29 (s, 3H, SCH₃), 3.04 (t, J =

7.6 Hz, 2H, CH₂), 1.96-1.85 (m, 2H, MeCH₂), 1.04 (t, J = 7.6 Hz,

3H, CH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 150.1 (C=O), 148.6,

136.8, 136.3, 135.4, 129.0, 125.0, 124.7, 123.9, 121.0, 114.0,

110.0, 46.0 (NCH₂), 40.9 (SMe), 38.8 (NMe), 27.9 (CH₂), 22.3

(CH₂), 14.0 (Me); HPLC: 99.9%, column: Eclips XDB C-18

150*4.6 mm 5μm, mobile phase A: 5mM Ammonium Acetate in

water, mobile phase B: CH₃CN, (gradient) T/B% : 0/10, 20/95,

37/95, 40/90, 45/10; flow rate: 1 mL/min; max plot, retention time

16.9 min; IR (KBr) _max2960, 1697, 1532, 1405 cm^-1; MS (ES

mass): m/z 401.1 (M+1, 100%).

7-propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one (3k)

White solid (72% yield); mp: 111-113 °C; R_f(20% EtOAc/n-

hexane) 0.25; ¹H NMR (400 MHz, CDCl₃) δ: 7.94 (d, J = 8.4 Hz,

1H, ArH), 7.79 (d, J = 8.4 Hz, 2H, ArH), 7.08 (d, J = 8.0 Hz, 2H,

ArH), 7.23 (d, J = 8.8, 2H, ArH), 6.05 (s, 2H, NCH₂), 6.02 (s, 1H,

indole (C-3) H), 4.30 (s, 3H, NCH₃), 3.05 (t, J = 7.6 Hz, 2H, CH₂),

2.34 (2s, 6H, 2ArCH₃), 1.97-1.86 (m, 2H, MeCH₂), 1.04 (t, J = 7.6

Hz, 3H, CH₃);¹³C NMR (100 MHz, CDCl₃) δ: 150.2 (C=O), 148.5,

144.9, 136.1, 135.5, 135.4, 135.3, 133.3, 129.9 (2C), 129.4, 126.6

(2C), 126.1, 124.7, 120.6, 114.1, 109.7, 46.9 (NCH₂), 38.8 (NMe),

27.9 (CH₂), 22.3 (CH₂), 21.5 (ArMe), 21.2 (ArMe), 14.0 (Me);

HPLC: 97.4%, column: X Bridge C-18 150*4.6 mm 5μm, mobile

phase A: 5 mM Ammonium acetate in water, mobile phase B:

CH₃CN, (gradient) T/B% : 0/5, 20/90, 30/90, 31/5, 35/5; flow rate:

1 mL/min; Max plot, retention time 20.9 min; MS (ES mass): m/z

491.1 (M+1, 100%).

4.2.9.

3-[{5-Chloro-1-(thiophen-2-ylsulfonyl)-1H-indol-2-

yl}methyl]-5-methyl-7-propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-

4(5H)-one (3h)

White solid (78% yield); mp: 115-117 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 8.05 (d, J = 8.8 Hz,

1H, ArH), 7.76 (dd, J = 4.0, 1.6 Hz, 1H, ArH), 7.59 (dd, J = 4.8,

1.2 Hz, 1H, ArH), 7.32-7.26 (m, 2H, ArH), 7.05 (dd, J = 5.2, 4.0

Hz, 1H, ArH), 6.04 (bs, 3H, indole (C-3) H, NCH₂), 4.31 (s, 3H,

NCH₃), 3.04 (t, J = 7.6 Hz, 2H, CH₂), 1.97-1.86 (m, 2H, MeCH₂),

1.05 (t, J = 7.6 Hz 3H, CH₃);¹³C NMR (100 MHz, CDCl₃) δ: 150.2

(C=O), 148.7, 137.7, 136.9, 136.1, 135.2, 133.9, 133.2, 130.6,

129.9, 127.8, 125.1, 124.6, 120.5, 115.7, 109.7, 46.8 (NCH₂), 38.8

(NMe), 27.9 (CH₂), 22.3 (CH₂), 14.0 (Me); HPLC: 99.8%,

column: Symmetry C-18 75*4.6 mm 3.5μm, mobile phase A:

10mM Ammonium Formate in water, mobile phase B: CH₃CN

(gradient) T/B% : 0/5, 20/90, 30/90, 31/5, 25/20, 30/20; flow rate:

1 mL/min; UV: 225.0 nm, retention time 18.8 min; IR (KBr) _max

2956, 1705, 1531, 1447 cm^-1; MS (ES mass): m/z 503.10 (M+1,

100%).

4.2.13.

3-{(5-Bromo-1-tosyl-1H-indol-2-

yl)methyl}benzo[d][1,2,3]triazin-4(3H)-one (4a)

White solid (80% yield); mp: 145-147 °C; R_f(20% EtOAc/n-

1

hexane) 0.25; H NMR (400 MHz, CDCl₃) δ: 8.40 (d, J = 8 Hz,

1H, ArH), 8.23 (d, J = 8.4 Hz, 1H, ArH), 8.02 (t, J = 7.6 Hz, 1H,

ArH), 7.98 (d, J = 8.8 Hz, 1H, ArH), 7.86 (t, J = 7.6 Hz, 1H, ArH),

7.80 (d, J = 8.4 Hz, 2H, ArH), 7.43 (d, J = 1.2 Hz, 1H, ArH),

7.36(dd, J = 8.8 Hz,1.6 Hz, 1H, ArH), 7.27 (d, J = 8.4 Hz, 2H,

ArH), 6.10 (s, 2H, NCH₂), 6.07 (s, 1H, indole (C-3) H), 2.36 (s,

3H, ArCH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 155.3 (C=O), 145.5,

144.3, 136.8, 135.7, 135.3, 135.0, 132.8, 130.9, 130.1 (2C), 128.6,

127.6, 126.6 (2C), 125.3, 123.4, 119.8, 117.1, 115.8, 108.9, 47.7

(NCH₂), 21.6 (ArMe); HPLC: 99.2%, column: X Bridge C-18

150*4.6 mm 5μm, mobile phase A: 0.1% TFA in water, mobile

phase B: CH₃CN, (gradient) T/B% : 0/10, 3/10, 12/95, 23/95,

25/10, 30/10; flow rate: 1 mL/min; UV 225.0 nm, retention time

18.6 min; IR (KBr) _max2958, 1708, 1533, 1448 cm^-1; MS (ES

mass): m/z 510.9 (M+2, 100%), 508.9 (M⁺, 98%).

4.2.10. 5-Methyl-7-propyl-3-[{1-(Thiophen-2-ylsulfonyl)-1H-

indol-2-yl}methyl]-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one

(3i)

White solid (78% yield); mp: 152-154 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 8.11 (dd, J = 8.4, 0.8

Hz, 1H, ArH), 7.76 (dd, J = 4.0, 1.6 Hz, 1H, ArH), 7.54 (dd, J =

4.8, 1.6 Hz, 1H, ArH), 7.34-7.29 (m, 2H, ArH), 7.23-7.17 (m, 1H,

ArH), 7.01 (dd, J = 4.8, 4.0 Hz, 1H, ArH), 6.09 (s, 1H, indole (C-

3) H), 6.06 (s, 2H, NCH₂), 4.30 (s, 3H, NCH₃), 3.04 (t, J = 7.6 Hz,

2H, CH₂), 1.97-1.86 (m, 2H, MeCH₂), 1.05 (t, J = 7.6 Hz 3H,

CH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 150.2 (C=O), 148.6, 137.9,

136.8, 136.1, 135.3, 133.5, 133.0, 129.4, 127.7, 124.9, 124.6,

124.1, 120.9, 114.6, 110.5, 46.9 (NCH₂), 38.8 (NMe), 27.9 (CH₂),

22.3 (CH₂), 13.9 (Me); HPLC: 99.7%, column: Eclips XDB C-18

150*4.6 mm 5μm, mobile phase A: 0.05% TFA in water, mobile

phase B: 0.05% TFA in CH₃CN (gradient) T/B% : 0/10, 5/10,

25/90, 30/90, 31/10, 35/10; flow rate: 1 mL/min; UV 254.0 nm,

retention time 24.5 min; IR (KBr) _max2956, 1697, 1530, 1452 cm^-

¹; MS (ES mass): m/z 469.10 (M+1, 100%).

4.2.14.

3-[{5-Chloro-1-(methylsulfonyl)-1H-indol-2-

yl}methyl]benzo[d][1,2,3]triazin-4(3H)-one (4b)

White solid (78% yield); mp: 154-156 °C; R_f(20% EtOAc/n-

hexane) 0.25; H NMR (400 MHz, CDCl₃) δ: 8.36 (d, J = 8 Hz,

1

1H, ArH), 8.23 (d, J = 8 Hz, 1H, ArH), 8.02 (t, J = 7.6 Hz, 1H,

ArH), 7.95 (d, J = 8.8 Hz, 1H, ArH), 7.86 (t, J = 7.6 Hz, 1H, ArH),

7.41 (s, 1H, ArH), 7.29 (d, J = 9.2 Hz, 1H, ArH), 6.32 (s, 1H,

indole (C-3) H), 6.00 (s, 2H, NCH₂), 3.33 (s, 3H, SCH₃);¹³C NMR

(100 MHz, CDCl₃) δ: 155.3 (C=O), 144.4, 136.8, 135.3, 135.1,

132.9, 130.2, 129.7, 128.6, 125.2, 125.2, 120.5, 119.8, 115.0,

109.2, 46.8 (NCH₂), 41.2 (SMe); HPLC: 99.1%, column:

Cosmicsil Aura ODS 150*4.6 mm 5μm, mobile phase A: 5mM

Ammonium Acetate in water, mobile phase B: CH₃CN, (gradient)

T/B% : 0/10, 20/90, 28/90, 30/10, 35/10; flow rate: 1 mL/min; UV

225.0 nm, retention time 17.9 min; IR (KBr) _max1683, 1585,

1448, 1361 cm^-1; MS (ES mass): m/z 389.1 (M+1, 100%).

4.2.11. 5-Methyl-3-[{1-(methylsulfonyl)-5-nitro-1H-indol-2-

yl}methyl]-7-propyl-3H-pyrazolo[4,3-d][1,2,3]triazin-4(5H)-one

(3j)

4.2.15.

3-[{1-(Phenylsulfonyl)-1H-indol-2-

yl}methyl]benzo[d][1,2,3]triazin-4(3H)-one (4c)

White solid (80% yield); mp: 175-177 °C; R_f(20% EtOAc/n-

hexane) 0.2; H NMR (400 MHz, DMSO-d₆) δ: 8.47 (d, J = 2.0

White solid (74% yield); mp: 112-114 °C; R_f(20% EtOAc/n-

hexane) 0.25; ¹H NMR (400 MHz, CDCl₃) δ: 8.40 (m, J = 8.8 Hz,

1H, ArH), 8.23 (d, J = 8.4 Hz, 1H, ArH), 8.08 (d, J = 8 Hz, 1H,

ArH), 8.04-7.99 (m, 1H, ArH), 7.94(d, J = 8 Hz, 2H, ArH), 7.88-

7.83 (m, 1H, ArH), 7.55 (t, J = 7.6Hz, 1H, ArH), 7.48 (t, J = 7.6

Hz, 2H, ArH), 7.33-7.27 (m, 2H, ArH), 7.17 (t, J = 7.2 Hz, 1H,

ArH), 6.16 (s, 1H, indole (C-3) H), 6.12 (s, 2H, NCH₂); ¹³C NMR

(100 MHz, CDCl₃) δ: 155.3 (C=O), 144.3, 138.2, 137.0, 135.4,

135.2, 134.0, 132.7, 129.4 (2C), 129.2, 128.6, 126.6 (2C), 125.3,

124.8, 123.8, 120.8, 119.8, 114.4, 110.1, 47.8 (NCH₂); HPLC:

93.7%, column: Eclips plus 250*4.6 mm 5μm, mobile phase A:

0.1% TFA in water, mobile phase B: CH₃CN, (gradient) T/B% :

1

Hz, 1H, ArH), 8.23 (dd, J = 9.2, 2.4 Hz, 1H, ArH), 8.11 (d, J =

9.2 Hz, 1H, ArH), 6.71 (s, 1H, indole (C-3) H), 5.94 (s, 2H, NCH₂),

4.20 (s, 3H, NCH₃), 3.70 (s, 3H, SCH₃), 2.97 (t, J = 7.6 Hz, 2H,

CH₂), 1.93-1.87 (m, 2H, MeCH₂), 0.98 (t, J = 7.6 Hz, 3H, CH₃);

¹³C NMR (100 MHz, DMSO-d₆) δ: 149.6 (C=O), 147.0, 143.7,

139.1, 139.0, 135.5, 128.6, 124.6, 119.5, 117.0, 114.4, 109.2, 45.6

(NCH₂), 41.8 (SMe), 38.5 (NMe), 27.2 (CH₂), 21.6 (CH₂), 13.7

(Me); HPLC: 98.7%, column: X Bridge Phenyle 150*4.6 mm

3.5μm, mobile phase A: 0.05% TFA in water, mobile phase B:

CH₃CN:water (90:10), (gradient) T/B% : 0/2, 5/2, 20/90, 25/90,

26/2, 30/2; flow rate: 1 mL/min; UV: 250.0 nm, retention time 20.5

min; MS (ES mass): m/z 446.1 (M+1, 100%).

JOURNAL PRE-PROOF

9

0/5, 20/90, 30/90, 3/5, 35/5; flow rate: 1 mL/min; UV 215.0 nm,

Eclips XDB C-18 150*4.6 mm 5μm, mobile phase A: 0.05%

retention time 20.5 min; MS (ES mass): m/z 417.1 (M+1, 100%).

TFA in water, mobile phase B: 0.05% TFA in CH₃CN, (gradient)

T/B% : 0/10, 5/10, 25/90, 30/90, 31/10, 35/10; flow rate: 1

mL/min; UV 220.0 nm, retention time 20.0 min; IR (KBr) _max

3029, 1683, 1448, 1405 cm-1; MS (ES mass): m/z 355.1 (M+1,

20%), 208.1 (M-146, 100%).

4.2.16.

3-{(5-Chloro-1-tosyl-1H-indol-2-

yl)methyl}benzo[d][1,2,3]triazin-4(3H)-one (4d)

White solid (81% yield); mp: 154-156 °C; R_f(20% EtOAc/n-

hexane) 0.25; ¹H NMR (400 MHz, CDCl₃) δ: 8.39 (d, J = 8.0 Hz,

1H, ArH), 8.22 (d, J = 8.0 Hz, 1H, ArH), 8.03-7.99 (m, 2H, ArH),

7.85 (t, J = 7.6 Hz, 1H, ArH), 7.80 (t, J = 8.4 Hz, 2H, ArH), 7.28-

7.25 (m, 3H, ArH), 7.22 (dd, J = 8.8, 2.0 Hz, 1H, ArH), 6.09 (s,

2H, NCH₂), 6.08 (s, 1H, indole (C-3) H), 2.34 (s, 3H, ArCH₃); ¹³C

NMR (100 MHz, CDCl₃) δ: 155.3 (C=O), 145.5, 144.3, 136.9,

135.3, 135.2, 135.0, 132.8, 130.4, 130.1 (2C), 129.5, 128.6, 126.6

(2C), 125.3, 124.9, 120.3, 119.8, 115.5, 109.0, 47.7 (NCH₂), 21.6

(ArMe); HPLC: 99.9%, column: Symmetry C-18 4.6*75 mm

3.5μm, mobile phase A: 10mM Ammonium Formate in water,

mobile phase B: CH₃CN, (gradient) T/B% : 0/10, 3/10, 15/95,

20/95, 21/10, 25/10; flow rate: 1 mL/min; UV 225.0 nm, retention

time 14.8 min; IR (KBr) _max3088, 1672, 1596, 1442 cm^-1; MS

(ES mass): m/z 465.0 (M+1, 50%), 318.0 (M-146, 100%).

4.2.20.

3-{(5-Chloro-1-(thiophen-2-ylsulfonyl)-1H-indol-2-

yl}methyl)benzo[d][1,2,3]triazin-4(3H)-one (4h)

White solid (80% yield); mp: 213-215 °C; R_f(20% EtOAc/n-

hexane) 0.25; ¹H NMR (400 MHz, CDCl₃) δ: 8.37 (dd, J = 8.0, 0.8

Hz, 1H, ArH), 8.20 (d, J = 8.0 Hz, 1H, ArH), 8.05-7.96 (m, 2H,

ArH), 7.83 (t, J = 8.0 Hz, 1H, ArH), 7.76 (dd, J = 8.0, 1.2 Hz, 1H,

ArH), 7.57 (dd, J = 4.8, 1.2 Hz, 1H, ArH), 7.25 (d, J = 9.6 Hz, 2H,

ArH), 7.03 (dd, J = 4.8, 4.0 Hz, 1H, ArH), 6.07 (s, 1H, indole (C-

3) H), 6.06 (s, 2H, NCH₂); ¹³C NMR (100 MHz, CDCl₃) δ: 155.3

(C=O), 144.3, 137.7, 136.8, 135.3, 135.2, 133.9, 133.3, 132.8,

130.6, 129.9, 128.7, 127.9, 125.3, 125.1, 120.5, 119.8, 115.7,

109.8, 47.7 (NCH₂); HPLC: 99.9%, column: Symmetry C-18

75*4.6 mm 3.5μm, mobile phase A: 10mM Ammonium Formate

in water, mobile phase B: CH₃CN, (gradient) T/B% : 0/5, 20/90,

30/90, 31/5, 35/5; flow rate: 1 mL/min; UV 225.0 nm, retention

time 17.0 min; IR (KBr) _max3099, 1686, 1585, 1446 cm^-1; MS

(ES mass): m/z 457.0 (M+1, 69%), 309.9 (M-146, 100%).

4.2.17.

3-{(1-Tosyl-1H-indol-2-

yl)methyl}benzo[d][1,2,3]triazin-4(3H)-one (4e)

White solid (77% yield); mp: 173-175 °C; R_f(20% EtOAc/n-

hexane) 0.25; ¹H NMR (400 MHz, CDCl₃) δ: 8.40 (d, J = 7.6 Hz,

1H, ArH), 8.22 (d, J = 8.4 Hz, 1H, ArH), 8.09 (d, J = 8.40 Hz, 1H,

ArH), 8.00 (t, J = 7.6 Hz, 1H, ArH), 7.89-7.79 (m, 3H, ArH),

7.34-7.22 (m, 4H, ArH), 7.16 (t, J = 7.2 Hz, 1H, ArH), 6.14 (s, 1H,

indole (C-3) H), 6.12 (s, 2H, NCH₂), 2.34 (s, 3H, ArCH₃); ¹³C

NMR (100 MHz, CDCl₃) δ: 155.3 (C=O), 145.1, 144.3, 137.0,

135.4, 135.3, 135.1, 132.643, 130.0 (2C), 129.2, 128.5, 126.6

(2C), 125.3, 124.7, 123.7, 120.7, 119.8, 114.4, 109.8, 47.9

(NCH₂), 21.6 (ArMe); HPLC: 99.9%, column: X-Bridge C18

150*4.6 mm 5μm, mobile phase A: 0.1% TFA in water, mobile

phase B: CH₃CN, (gradient) T/B% : 0/10, 20/95, 31/10, 35/10;

flow rate: 1 mL/min; UV 220.0 nm, retention time 17.0 min; IR

(KBr) _max3084, 1679, 1595, 1450 cm^-1; MS (ES mass): m/z 431.1

(M+1, 25%), 284.0 (M-146, 100%).

4.2.21.

3-{(1-(Thiophen-2-ylsulfonyl)-1H-indol-2-

yl}methyl)benzo[d][1,2,3]triazin-4(3H)-one (4i)

White solid (78% yield); mp: 131-133 °C; R_f(20% EtOAc/n-

hexane) 0.25; ¹H NMR (400 MHz, CDCl₃) δ: 8.40 (dd, J = 8.0, 0.8

Hz, 1H, ArH), 8.23 (d, J = 8.0 Hz, 1H, ArH), 8.12 (d, J = 8.8 Hz,

1H, ArH), 8.05-7.97 (m, 1H, ArH), 7.89-7.81 (m, 1H, ArH), 7.78

(dd, J = 3.6, 1.2 Hz, 1H, ArH), 7.55 (dd, J = 4.8, 1.2 Hz, 1H, ArH),

7.31 (t, J = 7.6 Hz, 2H, ArH), 7.20 (t, J = 7.6 Hz, 1H, ArH), 7.02

(dd, J = 4.8, 4.0 Hz, 1H, ArH), 6.15 (s, 1H, indole (C-3) H), 6.08

(s, 2H, NCH₂); ¹³C NMR (100 MHz, CDCl₃) δ: 155.3 (C=O),

144.3, 138.0, 136.8, 135.2, 135.2, 133.6, 133.0, 132.7, 129.4,

128.6, 127.7, 125.3, 124.9, 124.1, 120.9, 119.8, 114.6, 110.6, 47.8

(NCH₂); HPLC: 99.8%, column: Eclips XDB C-18 150*4.6mm

5μm, mobile phase A: 0.05% TFA in water, mobile phase B:

0.05% TFA in CH₃CN, (gradient) T/B% : 0/10, 5/10, 25/90, 30/90,

31/10, 35/10; flow rate: 1 mL/min; UV 254.0 nm, retention time

22.3 min; IR (KBr) _max3090, 1684, 1606, 1451 cm^-1; MS (ES

mass): m/z 423.0 (M+1, 100%).

4.2.18.

3-{(5-Fluoro-1-tosyl-1H-indol-2-

yl)methyl}benzo[d][1,2,3]triazin-4(3H)-one (4f)

White solid (71% yield); mp: 176-178 °C; R_f(20% EtOAc/n-

hexane) 0.25; ¹H NMR (400 MHz, CDCl₃) δ: 8.40 (d, J = 7.6 Hz,

1H, ArH), 8.23 (d, J = 8.4 Hz, 1H, ArH), 8.08-7.98 (m, 2H, ArH),

7.86 (t, J = 7.2 Hz, 1H, ArH), 7.81 (d, J = 7.6 Hz, 2H, ArH), 7.27

(d, J = 7.2 Hz, 2H, ArH), 7.02-6.92 (m, 2H, ArH), 6.10 (bs, 3H,

NCH_2,indole (C-3) H), 2.36 (s, 3H, ArCH₃); ¹³C NMR (100 MHz,

CDCl₃) δ: 160.9 (C-F J = 245.6 Hz), 155.3 (C=O), 145.4, 144.291,

135.3, 135.2, 135.0, 132.8, 132.8, 130.1 (C-F J = 11.6 Hz), 130.1

(2C), 129.7, 128.6, 126.6 (2C), 125.3, 115.6 (C-F J = 10.7 Hz),

112.7 (C-F J = 25.6 Hz), 109.5 (C-F J = 2.8 Hz), 106.4 (C-F J =

24.7 Hz), 47.9 (NCH₂), 21.6 (ArMe); HPLC: 98.1%, column:

Cosmicsil Aura ODS C-18 150*4.6 mm 5μm, mobile phase A:

5mM Ammonium acetate in water, mobile phase B: CH₃CN,

(gradient) T/B% : 0/5, 20/90, 25/90, 26/3, 30/5; flow rate: 1

mL/min; UV 220.0 nm, retention time 19.3 min; IR (KBr) _max

3102, 1681, 1595, 1467 cm^-1; MS (ES mass): m/z 449.1 (M+1,

100%).

4.2.22.

3-[{5-Fluoro-1-(methylsulfonyl)-1H-indol-2-

yl}methyl]benzo[d][1,2,3]triazin-4(3H)-one (4j)

White solid (72% yield); mp: 154-156 °C; R_f(20% EtOAc/n-

1

hexane) 0.25; H NMR (400 MHz, CDCl₃) δ: 8.36 (d, J = 8 Hz,

1H, ArH), 8.23 (d, J = 8.4 Hz, 1H, ArH), 8.00 (m, 2H, ArH), 7.86

(t, J = 7.6 Hz, 1H, ArH), 7.07 (m, 2H, ArH), 6.33 (s, 1H, indole

(C-3) H), 6.00 (s, 2H, NCH₂), 3.32 (s, 3H, SCH₃); ¹³C NMR (100

MHz, CDCl₃) δ: 160.9 (C-F J = 239.6 Hz), 155.3 (C=O), 144. 4,

137.0, 135.3, 133.0, 132.8, 130.1(C-F J = 10.2 Hz), 128.6, 125.2,

119.8, 115.2 (C-F J = 9.2 Hz), 113.1 (C-F J = 25.1 Hz), 109.6 (C-F

J = 4 Hz), 106.6 (C-F J = 24 Hz), 46.9 (NCH₂), 41.0 (SMe); HPLC:

97.4 %, column: Eclipse plus C-18 250*4.6 mm 5μm, mobile

phase A: 0.1% TFA in water, mobile phase B: CH₃CN, (gradient)

T/B% : 0/5, 20/90, 28/90, 30/5, 35/5, flow rate: 1 mL/min; UV

225.0 nm, retention time 18.3 min; MS (ES mass): m/z 373.1 (M-

+1, 100%).

4.2.19.

3-{(1-(Methylsulfonyl)-1H-indol-2-

yl)methyl}benzo[d][1,2,3]triazin-4(3H)-one (4g)

4.2.23.

3-{(5,7-Dimethyl-1-(methylsulfonyl)-1H-indol-2-

White solid (80% yield); mp: 134-136 °C; R_f(20% EtOAc/n-

hexane) 0.25; ¹H NMR (400 MHz, CDCl₃) δ: 8.36 (d, J = 8.0 Hz,

1H, ArH), 8.21 (d, J = 8.0 Hz, 1H, ArH), 8.05-7.97 (m, 2H, ArH),

7.84 (t, J = 7.6 Hz, 1H, ArH), 7.44 (d, J = 7.6 Hz, 1H, ArH), 7.34

(t, J = 7.6 Hz, 1H, ArH), 7.25 (t, J = 7.2 Hz, 1H, ArH), 6.38 (s,

1H, indole (C-3) H), 6.01 (s, 2H, NCH₂), 3.31 (s, 3H, SCH₃); ¹³C

NMR (100 MHz, CDCl₃) δ: 155.3 (C=O), 144.4, 136.8, 135.3,

135.2, 132.8, 129.1, 128.6, 125.2, 125.1, 123.9, 121.0, 119.9,

114.0, 110.1, 47.0 (NCH₂), 41.0 (SMe); HPLC: 99.2%, column:

yl)methyl}benzo[d][1,2,3]triazin-4(3H)-one (4k)

White solid (68% yield); mp: 159-161 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 8.35 (d, J = 7.6 Hz,

1H, ArH), 8.19 (d, J = 8.0 Hz, 1H, ArH), 7.98 (t, J = 7.6 Hz, 1H,

ArH), 7.83 (t, J = 7.6 Hz, 1H, ArH), 7.26 (s, 1H, ArH), 6.94 (s,

1H, ArH), 6.43 (s, 1H, indole (C-3) H), 5.97 (s, 2H, NCH₂), 3.20

(s, 3H, SCH₃), 2.64 (s, 3H, ArCH₃), 2.32 (s, 3H, ArCH₃);¹³C NMR

(100 MHz, CDCl₃) δ: 155.2 (C=O), 144.3, 138.4, 137.1, 135.1,

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10

Bioorganic Chemistry

134.7, 132.7, 132.2, 130.5, 128.5, 127.1, 125.2, 119.9, 119.0,

charge calculation, deletion of co-crystal ligand, and addition of

hydrogen etc.) using AutoDock tool [20]. All ligands were docked

at the interface site of MtbCM (which is homodimer) using reliable

open-source tool AutoDock Vina [21]. The grid map was made up

of 20X20X20 points using AutoGrid with 0.54, 3.92, and 42.25 as

center_X, center_Y, and center_Z respectively. To search all

possible conformation of ligands thoroughly, exhaustiveness value

of 20 were used. The Maestro visualizer (Schrodinger, LLC) and

UCSF Chimera [22] was used to analyze the interactions of studied

ligands at defined binding site.

114.8, 48.0 (NCH₂), 40.0 (SMe), 21.8 (ArMe), 20.8 (ArMe);

HPLC: 99.4%, column: Eclips XDB C18 ODS 150*4.6 mm 5μm,

mobile phase A: 0.05% TFA in water, mobile phase B: 0.05% TFA

in CH₃CN, (gradient) T/B% : 0/10, 5/10, 25/90, 30/90, 31/10,

35/10; flow rate: 1 mL/min; UV 225.0 nm, retention time 22.1

min; IR (KBr) _max3111, 1685, 1461, 1444 cm^-1; MS (ES mass):

m/z 383.1 (M+1, 100%).

4.2.24.

3-{(5,7-Dimethyl-1-(phenylsulfonyl)-1H-indol-2-

yl)methyl}benzo[d][1,2,3]triazin-4(3H)-one (4l)

The molecular docking was performed to evaluate precise

binding of test molecules to the interface site of MtbCM. The

binding energy of best pose of each ligand is presented in Chart 1.

To check the reproducibility of docking result, the docking of each

individual ligand was performed at least ten times and maximum

difference found was ±0.2. The validation of docking protocol was

done by re-docking the co-crystal ligand (TSA) and calculating

RMSD difference between co-crystal one and docked one in

PyMOL [23], maximum RMSD was 1.713 (<2) which confirmed

the authenticity of current docking protocol.

White solid (66% yield); mp: 136-138 °C; R_f(20% EtOAc/n-

hexane) 0.24; ¹H NMR (400 MHz, CDCl₃) δ: 8.37 (dd, J = 8.0, 0.8

Hz, 1H, ArH), 8.20 (dd, J = 8.0, 0.4 Hz, 1H, ArH), 8.01-7.94 (m,

1H, ArH), 7.86-7.79 (m, 1H, ArH), 7.71-7.65 (m, 2H, ArH), 7.54-

7.47 (m, 1H, ArH), 7.41-7.35 (m, 2H, ArH), 6.88 (s, 1H, ArH),

6.81 (s, 1H, ArH), 6.12 (s, 1H, indole (C-3) H), 6.02 (s, 2H,

NCH₂), 2.60 (s, 3H, ArCH₃), 2.23 (s, 3H, ArCH₃); ¹³C NMR (100

MHz, CDCl₃) δ: 155.3 (C=O), 155.25, 144.3, 139.0, 137.2, 135.1,

134.7, 133.5, 132.8, 132.6, 130.4, 128.8 (2C), 128.5, 127.7, 126.6

(2C), 125.3, 119.9, 118.7, 115.0, 48.9 (NCH₂), 21.8 (ArMe), 20.8

(ArMe); HPLC: 99.9%, column: X-Bridge C18 150*4.6 mm 5μm,

mobile phase A: 0.1% TFA in water, mobile phase B: CH₃CN,

(gradient) T/B% : 0/10, 20/95, 31/10, 35/10; flow rate: 1 mL/min;

UV 220.0 nm, retention time 17.8 min; IR (KBr) _max3006, 1688,

1607, 1443 cm^-1; MS (ES mass): m/z 445.1 (M+1, 34%), 298.0 (M-

146, 100%).

6. Pharmacology

6.1. In vitro assay for CM inhibition

6.1.1. Enzyme and Reagents

Mycobacterium tuberculosis chorismate mutase (MtbCM) gene

was PCR amplified and cloned into expression vector pET22b.

MtbCM was purified from over expressed culture of BL21 (DE3)

harboring pET22b/ MtbCM by Ni-NTA affinity chromatography.

The substrate chorismic acid was obtained from Sigma (SIGMA

cat # 1701).

4.2.25.

6-Bromo-3-{(5-chloro-1-tosyl-1H-indol-2-

yl)methyl}benzo[d][1,2,3]triazin-4(3H)-one (4m)

White solid (79% yield); mp: 169-171 °C; R_f(20% EtOAc/n-

hexane) 0.3; H NMR (400 MHz, CDCl₃) δ: 8.54-8.51 (m, 1H,

1

ArH), 8.12-8.05 (m, 2H, ArH), 8.01 (d, J = 8.8 Hz, 1H, ArH), 7.79

(d, J = 8.4 Hz, 2H, ArH), 7.31-7.27 (m, 2H, ArH), 7.26-7.21 (m,

2H, ArH), 6.09 (s, 1H, indole (C-3) H), 6.07 (s, 2H, NCH₂), 2.36

(s, 3H, ArCH₃); ¹³C NMR (100 MHz, CDCl₃) δ: 154.1 (C=O),

145.5, 142.9, 138.6, 136.6, 135.4, 135.1, 130.3, 130.2, 130.1 (2C),

129.6, 128.1, 127.5, 126.6 (2C), 125.1, 121.0, 120.4, 115.6, 109.3,

47.8 (NCH₂), 21.6 (ArMe); HPLC: 99.7%, column: X-Bridge C-

18 4.6 mm 5μm, mobile phase A: 5 mM Ammonium acetate in

water, mobile phase B: CH₃CN, (gradient) T/B% : 0/10, 23/90,

30/90, 31/90, 35/10; flow rate: 1 mL/min; Max plot, retention time

23.4 min; MS (ES mass): m/z 544.9 (M+2, 40%), 317.9 (M-227,

100%).

6.1.2. CM enzymatic assay

Activity of chorismate mutase enzyme was based on the direct

observation of conversion of chorismate to prephenate

spectrophotometrically at OD₂₇₄. The reaction volume of the assay

was maintained at 100 µl. The substrate chorismic acid (2 mM)

was pre incubated at 37 ºC for 5 min in the buffer containing 50

mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.1 mg/ml bovine serum

albumin, and 10 mM β-Mercaptoethanol. The reaction was started

by adding 180 pmol of CM enzyme to the pre-warmed chorismic

acid solution. Inhibitory screening of the test compounds against

CM activity was measured at 30 µM (10 nM to 30 µM for

concentration dependent study) concentration of the effectors. The

reaction was allowed to proceed at 37 °C and was terminated after

5 min with 100 µl of 1 N HCl and absorbance was read at 274 nm.

Alternatively, the reaction was allowed to proceed for further 10

min and was then terminated with 180 µM of 2.5N NaOH and

absorbance was measured at 320 nm. A blank with no enzyme for

every reaction was kept as a control to account for the non

enzymatic conversion of chorismate to prephenate. The % of

enzyme inhibition caused by the test compound was calculated by

the following formula:

5. Docking study

5.1. Materials and methods

It is known that the secreted chorismate mutase of Mtb which

is encoded by Rv1885c gene is the major contributor in shikimate

pathway commonly occurred in cytoplasm of bacteria [17]. For

our study we searched for the available crystal structure of secreted

MtbCM and found four structures in Protein Data Bank (PDB)

[18], represented as follows

PDB ID

2AO2

2F6L

2FP1

2FP2

Resolution (Å)

Co-crystal ligand(s)

SO4 and Trp^a

Not available

PB

% inhibition = 100 – residual activity of CM

2.07

1.70

1.55

1.64

Residual activity of CM = [A₂₇₄{S + (E’ + C)} - A₂₇₄(S + C)] /

[A₂₇₄(S + E) – A₂₇₄(S)]

TSA^b

S = absorbance of the substrate (chorismic acid) at 274 nm; E’ =

absorbance of the enzyme (CM) at 274 nm with compound; E =

absorbance of the enzyme (CM) at 274 nm without compound; C

= test compound; A₂₇₄indicates absorbance at 274 nm (this is

replaced by A₃₂₀for absorbance at 320 nm).

^aTrp represents tryptophan.

^bTSA represents transition state analogue i.e. 8-hydroxy-2-oxa-

bicyclo[3.3.1]non-6-ene-3,5-dicarboxylic acid.

Among these four crystal structures, we have chosen 2FP2

(PDB ID), because of its low resolution value and presence of

substrate molecule as co-crystal structure.

6.2. MTT assay

20,000-25,000 cells per well were seeded in a 96-well plate and

incubated with the compound at different concentrations for 24

hours. After treatment, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-

All ligand structures were built in MarvinSketch [19]. Protein

(2FP2) as well as all ligands was prepared (means optimization,

JOURNAL PRE-PROOF

11

small molecules as potential anti-tuberculosis agents. Med.

diphenyltetrazolium bromide] reagent was added to a final

concentration of 0.25 mg/mL and incubated at 37°C for 2 hours.

After 2 hours, media was removed completely, and the

intracellular purple formazan crystals were dissolved in 100µL of

DMSO. The absorbance of this solution was measured at 570 nm.

Each concentration of compound was performed in triplicate and

the relative cell viability was expressed as a percentage relative to

the untreated control cells.

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Acknowledgements

GSR thanks DST, India for a INSPIRE fellowship (IF160590).

Authors thank the Management of DRILS, Hyderabad, India and

Manipal University, Manipal, India for encouragement and

support and DBT, New Delhi, India for financial assistance (Grant

No. BT/PR12817/COE/34/23/2015).

Supplementary data

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substructures. Chem. Rev. 103 (2003) 893-930.

Supplementary data associated with this article can be found, in

the on line version, at xxxxxxxxx

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Graphical Abstract

To create your abstract, type over the instructions in the template box below.

Fonts or abstract dimensions should not be changed or altered.

Synthesis of 3-indolylmethyl substituted (pyrazolo /

benzo)triazinone derivatives under Pd/Cu-catalysis:

identification of potent inhibitors of chorismate

mutase (CM)

Leave this area blank for abstract info.

Gangireddy Sujeevan Reddy, Ampalam Venkata Snehalatha, Rebeccakristina Edwin, Kazi Amirul Hossain,

Varadaraj Bhat Giliyaru, Raghu Chandrashekhar Hariharapura, G. Gautham Shenoy, Parimal Misra and Manojit

Pal*

N

Q

N

O

N

Pd/Cu-cat

Q

O

+

N

Z

Et₃N, EtOH

60 ^oC

I

HN

Z

(Q = benzene / substituted pyrazole ring)

Design, synthesis and evaluation of 3-indolylmethyl substituted fused triazinone derivatives were undertaken to

identify potent inhibitors of chorismate mutase.

.

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Tetrahedron Letters

Highlights

 3-Indolylmethyl substituted fused triazinones were explored as inhibitors of CM.

 Their synthesis involved coupling-cyclization under Pd/Cu-catalysis.

 Altogether 21 novel compounds were preapred in good yields.

 Several compound showed significant inhibition of CM in vitro.

 3d and 4d were potent (IC₅₀~ 0.4-0.9 µM) and non-toxic (till 30 µM in vitro).

Article Doi

Synthesis of 3-indolylmethyl substituted (pyrazolo/benzo)triazinone derivatives under Pd/Cu-catalysis: Identification of potent inhibitors of chorismate mutase (CM)

DOI: 10.1016/j.bioorg.2019.103155

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bioorg.2019.103155

Full text of DOI:10.1016/j.bioorg.2019.103155

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