Ligand-based virtual screening identifies a family of selective cannabinoid receptor 2 agonists

Accepted Manuscript

Ligand-based virtual screening identifies a family of selective cannabinoid re-

ceptor 2 agonists

Matteo Gianella-Borradori, Ivy Christou, Carole J.R. Bataille, Rebecca L. Cross,

Graham M. Wynne, David R. Greaves, Angela J. Russell

PII:

S0968-0896(14)00776-7

DOI:

http://dx.doi.org/10.1016/j.bmc.2014.11.002

BMC 11891

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

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Revised Date:

Accepted Date:

1 September 2014

31 October 2014

1 November 2014

Please cite this article as: Gianella-Borradori, M., Christou, I., Bataille, C.J.R., Cross, R.L., Wynne, G.M., Greaves,

D.R., Russell, A.J., Ligand-based virtual screening identifies a family of selective cannabinoid receptor 2 agonists,

Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.11.002

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Ligand-based virtual screening identifies a

family of selective cannabinoid receptor 2

agonists

Leave this area blank for abstract info.

Matteo Gianella-Borradori^a,†, Ivy Christou^b,†, Carole J. R. Bataille^a, Rebecca L. Cross^a, Graham M. Wynne^a,

David R. Greaves^b∗ and Angela J. Russell^a,c∗

^aDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford,

OX1 3TA, UK

^bSir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK

^cDepartment of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK

Bioorganic & Medicinal Chemistry

journal homepage: www.els evier.com

Ligand-based virtual screening identifies a family of selective cannabinoid receptor 2

agonists

Matteo Gianella-Borradori^a,†, Ivy Christou^b,†, Carole J. R. Bataille^a, Rebecca L. Cross^a, Graham M.

Wynne^a, David R. Greaves^b∗ and Angela J. Russell^a,c∗

^aDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK

^bSir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK

^cDepartment of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

The cannabinoid receptor 2 (CB₂R) has been linked with the regulation of inflammation, and

selective receptor activation has been proposed as a target for the treatment of a range of

inflammatory diseases such as atherosclerosis and arthritis. In order to identify selective CB₂R

agonists with appropriate physicochemical and ADME properties for future evaluation in vivo,

we first performed a ligand-based virtual screen. Subsequent medicinal chemistry optimisation

studies led to the identification of a new class of selective CB₂R agonists. Several examples

showed high levels of activity (EC₅₀< 200 nM) and binding affinity (K_i< 200 nM) for the

CB₂R, and no detectable activity at the CB₁R. The most promising example, DIAS2, also

showed favourable in vitro metabolic stability and absorption properties along with a clean

selectivity profile when evaluated against a panel of GPCRs and kinases.

Available online

Keywords:

Cannabinoid receptor 2

Cannabinoid receptor 1

Agonist

Inflammation

Ligand-based screening

1. Introduction

CB₂R has been proposed as a therapeutic target for the

treatment of pain and inflammatory conditions.¹¹The main

components of marijuana including ∆⁹-THC (1, Figure 1) and

other phytocannabinoids¹²have long been known to exhibit

analgesic activity, which has since been attributed to activation of

the CB₁R as well as the CB₂R.^8,13However the clinical use of

these agents is limited, in the most part due to their psychotropic

side effects which are thought to arise from activation of the

CB₁R.^14,15Thus, there have been extensive studies in recent years

to develop selective CB₂R modulators, particularly for the

management of pain.^11,16Indirect strategies to modulate the

cannabinoid receptors through inhibition of endocannabinoid

metabolism are also being explored.¹⁷

The endocannabinoid system (ECS) is an endogenous

signalling pathway that has a regulatory role in pain perception,

the immune response, metabolism and mood. Among the key

components of the ECS are the cannabinoid receptors 1 and 2

(CB₁R and CB₂R). While CB₁R is predominantly expressed in

the central nervous system, CB₂R is mainly expressed in

peripheral tissues, particularly immune cells,^1,2although studies

over the last decade have shown that CB₂R is also expressed in

the CNS in microglia³and brainstem neurons,⁴and that it is

upregulated in response to injury in dorsal root ganglia⁵and

sensory neurons.⁶

CB₂R belongs to the G-Protein Coupled Receptor (GPCR)

superfamily and mediates the downstream effects of endogenous

secreted cannabinoid receptor ligands (endocannabinoids) such

as anandamide and 2-arachidonoylglycerol (2-AG).⁷Activation

of the CB₂R has been shown to lead to a number of downstream

signalling events,⁸the most widely studied of which involve

inhibition of adenylyl cyclase and lowering of intracellular cyclic

AMP levels,⁹and mitogen-activated protein kinase (MAPK)

A variety of strategies have been explored to develop

alternative CB₂R agonists including structure activity relationship

(SAR) analyses around the structure of ∆⁹-THC (1) leading to the

discovery of compounds such as CP 55,940 (2, Figure 1)¹⁸and

the selective CB₂R agonist, HU-308 (5, Figure 2).¹⁹A homology

model of the CB₂R has been published using the crystal structure

of bovine rhodopsin,²⁰and this and more recent refine models

have been employed in virtual screening strategies to identify

new classes of CB₂R ligand.^21,22The most commonly employed

activation¹⁰via coupling through G_αi/oproteins.

———

∗ Corresponding authors. Tel.: +44-(0)1865-275643; fax: +44-(0)1865-285002; e-mail: angela.russell@chem.ox.ac.uk; tel.: +44-(0)1865-

289150; fax: +44-(0)1865-275515; e-mail: david.greaves@path.ox.ac.uk.

^†These authors contributed equally to this work.

strategies have been ligand-based virtual screening²³and high-

in vivo models. Herein we report our preliminary discovery

efforts towards this overarching goal.

2. Results and discussion

We opted to employ a virtual ligand-based screening strategy

in order to identify new chemical scaffolds which exhibited

agonist activity at the CB₂R to provide a starting point for initial

SAR analysis and optimisation. A set of reported CB₂R agonists

and their respective affinities for CB₁R and CB₂R were assessed

when making the final selection for the template ligand for the

virtual screen (See supplementary, Table S1). HU-308 (5,

Figure 2A) was chosen as the query molecule for the virtual

screen due to its high potency and degree of selectivity for CB₂R

over the CB₁R (> 400 fold).

2.1. Ligand-based virtual screening

Figure 1: Structures of cannabinoid receptor agonists, ∆⁹-

tetrahydrocannabinol (∆⁹-THC, 1), CP 55,940 (2), GW-842166X (3) and

Lilly’s recently disclosed CB₂R agonist (4).

Using the Omega (OpenEye) package, 40 possible low energy

conformers of HU-308 (5) were established based on the

minimised energy conformations of the molecule (kcal/mol)

(example in Figure 2B).⁴²Up to 100 conformers were also

generated of every molecule in our 25,000-member in-house

library.⁴³Finally the 40 conformers of HU-308 (5) were screened

against the virtual library using the programme rapid overlay of

chemical structures (ROCS, OpenEye).⁴⁴The screen was

performed under default parameters using full optimisation

mode. The compounds were ranked in order according to their

shape similarity compared to any of the 40 conformers of HU-

308 (5). The top 100 compounds that presented most similarity in

their overlay with HU-308 (5) were of interest, these overlay

results were confirmed through the use of an independent

alignment program, Forge (Cresset) (Figure 2C).⁴⁵The overall

score was based on a combination of two calculated parameters:

the colour score⁴⁶and shape Tanimoto score⁴⁷combined,

provided the combo score⁴⁷by which the compounds were

ranked (see supplementary, Table S2). On the basis of this

ranking the top 94 available compounds were selected for

screening for CB₂R activation.

throughput screening approaches,¹¹for example in the recent

discovery of 4 by Lilly (Figure 1).²⁴

Several CB₂R selective agonists show efficacy in in vivo

models of acute and chronic pain, and these effects are blocked

upon co-treatment with CB₂R antagonists such as SR144528²⁵

and AM630,²⁶or in CB₂R knockout animals, supporting a CB₂R-

mediated mechanism of action.¹¹Despite these promising

preclinical data, only a few candidates have entered clinical trials

to date for evaluation in the treatment of inflammatory pain, and

none has progressed beyond Phase 2. For instance

GW-842166X (3, Figure 1)²⁷was recently evaluated in a Phase

2a trial for acute inflammatory pain following third molar

extraction,²⁸but failed to demonstrate sufficient efficacy and has

been discontinued. In this case the lack of efficacy was attributed

to insufficient exposure and CB₂R occupancy.²⁸

Moreover, there is emerging in vivo data to support the role of

CB₂R as a therapeutic target in other disease indications

including osteoporosis,²⁹multiple sclerosis³⁰or in other diseases

with an inflammatory component³¹such as allergic contact

dermatitis,³²colitis^33,34and atherosclerosis.³⁵A non-psychoactive

cannabinoid derivative, ajulemic acid, has also been reported to

show anti-inflammatory effects.³⁶CB₂R activation has been

shown to induce monocyte chemotaxis,³⁷and reduce macrophage

chemotaxis to a range of chemoattractants including MCP-1,

MIP-1α, MCP-1, RANTES and fMLP.^35,38,39Support for CB₂R

being an physiologically relevant modulator of macrophage

inflammation in vivo came from analysis of endocannabinoid

levels in human atherosclerotic lesions in carotid arteries and

reduced MMP-2 secretion by human inflammatory cells treated

with the CB₂R-selective agonist JWH-133.⁴⁰There is therefore

still a real need for additional highly selective CB₂R agonists

with improved pharmacokinetic and pharmacodynamic profiles

to evaluate their clinical efficacy as anti-inflammatory agents. To

date most groups have focussed on CNS penetrant CB₂R ligands,

many of which have high lipophilicity.⁴¹

2.2. Biochemical screening and hit identification

The 94 compounds were screened using a commercially

available complementation immunoassay measuring levels of β-

galactosidase (β-gal) labelled cAMP.⁴⁸The assay was performed

using CB₂R-expressing Chinese Hamster Ovary (CHO) cells

stimulated with forskolin. In the absence of an agonist binding to

the CB₂R, intracellular cAMP competes with labelled cAMP for

binding to the anti-cAMP antibody increasing levels of free β-gal

labelled cAMP in the cell. The addition of a CB₂R agonist leads

to inhibition of adenylate cyclase activity by the G_αicomplex

formed upon receptor activation. This in turn leads to a lowering

of the level of intracellular cAMP, increased sequestration of

labelled cAMP by the anti-cAMP antibody and thus a decrease in

the level of free β-gal labelled cAMP.

From the compounds tested several CB₂R activators were

identified as having modest activity. Six different cores were

present in the top 16 hits. Interestingly the compound that ranked

highest in the virtual screen did not demonstrate the highest

activity in the in vitro assay. DIAS1 (6) was selected for further

evaluation (Figure 2D) on the basis of its activity, relatively low

molecular weight (258 Da), and predicted lower lipophilicity

compared to HU-308 (clogP HU-308, 6.9; clogP DIAS1, 4.2).

This compound exhibited good activity and in a dose dependent

manner at the CB₂R, 3 µM (57.2 %) and 1 µM (52.1 %). The

selected compound ranked 13^thin the virtual screen based on its

combo score (see supplementary, Table S2). An overlay of

DIAS1 (6) with HU-308 (5) using Forge⁴⁵illustrates good

Despite the range of current CB₂R agonists available in the

published literature, there still exists a scientific need for new

CB₂R-selective tool compounds that are less lipophilic and

peripherally restricted that can be used to specifically address the

role of CB₂R in modulating macrophage chemotaxis and

activation in preclinical models of inflammation.³⁴We therefore

sought to identify a new family of highly selective CB₂R agonists

with improved in vitro ADME properties to investigate their use

as peripherally-restricted anti-inflammatory agents in a range of

Scheme 1: Reagents and conditions: (i) thiosemicarbazide, K₂CO₃, H₂O,

reflux, 16 h; (ii) butyl iodide or 2-cyanobenzyl bromide, NEt₃, MeOH, rt,

16 h.

Figure 2. A. Structure and properties of HU-308 (5); B. 3-D representation of the lowest energy conformer of HU-308 (5) using Omega (OpenEye);⁴²C. overlay

of HU-308 (5) and DIAS1 (6) using Forge (Cresset);⁴⁵D. structure and properties of DIAS1 (6) and DIAS2 (7). ^aCalculated using the CLogP algorithm in

ChemBioDraw 14.0.

alignment of the hydrophobic alkyl chains and the aromatic core

in both molecules (Figure 2C).

11 and 12 – 24, which were isolated in 50% to quantitative yield

(Scheme 2).

Following the identification of 6 as an active hit, our

compound library was further mined using the 3-mercapto

triazinoindole core of 6 to identify available molecules with the

same substructure to allow screening of close analogues, and an

evaluation of preliminary SARs. This search identified a small

number of additional compounds with variations at C(3) and

N(5) positions of the tricyclic core. The compounds were

screened in the cAMP assay (Figure S1) and one, DIAS2 (7)

exhibited improved activity of (EC₅₀= 120 nM), in comparison to

DIAS1 (6) (EC₅₀= 296 nM) with a reduced clogP value (3.8).

Interestingly DIAS2 was not amongst the top 16 ranked

compounds in the virtual screen, nor did it appear in the top 400

compounds based on their combo score. This discrepancy may

reflect the accuracy of the algorithms used but may also just

reflect a limitation in this ligand-based screening method: for

instance hits are ranked on the basis of similarity to a probe

ligand (in this case HU-308) and not on the basis of predicted

best fit with the CB₂R.

The array of C(3)-substituted triazinoindoles were evaluated

for activity at the CB₂R using the CHO cell assay described

above. The preliminary SAR assessment had demonstrated that

the presence of linear thioalkyl groups C(3) larger than n-butyl

led to a reduction in activity at the CB₂R (Table S2), and it was

therefore of interest to determine whether a small thiomethyl (10)

substituent, or more bulky β-branched thiomethylcyclohexyl (11)

would show activity at the CB₂R. Compound 11 was also of

particular interest as a fully saturated counterpart for comparison

with the C(3)-thiobenzyl substituted analogues. Intermediate 9

was also tested and showed no detectable agonist activity at the

CB₂R,

nor

did

the

C(3)-thiomethyl-

or

C(3)-

thiomethylcyclohexyl-substituted derivatives 10 and 11 (EC₅₀

3000 nM, Table 1).

>

Having established a relatively restricted tolerance for

variations in C(3)-thioalkyl substituents, attention turned to

exploring systematic variations of C(3)-thiobenzyl substituents.

To this end a selection of compounds with varying aryl

substituents and regiochemistry were prepared and assayed.

(Table 1). Interestingly, removal of the cyano group (C(3)-

2.3. Hit validation of DIAS1 (6) and DIAS2 (7)

Verification of the activity of DIAS1 (6) and DIAS2 (7) was

carried out via the re-synthesis of an authentic sample.

Compounds 6 and 7 were synthesised via a cyclisation of isatin 8

with thiosemicarbazide to yield the tricycle 9,⁴⁹followed by

alkylation or benzylation of 9 to yield 6 and 7 in an overall yield

of 76 % and 79 % respectively (Scheme 1).^50,51

thiobenzyl, 12) led to a reduction in activity at the CB₂R (EC₅₀

=

10000 nM, Table 1). Regiochemistry was also found to influence

activity, with 3-cyano substituted derivative 13 showing

markedly reduced activity at the CB₂R compared to its 2-cyano

substituted counterpart DIAS2 7 (13: EC₅₀= 6450 nM; 7 EC₅₀

=

112 nM, Table 1). Introduction of fluoro or chloro substituents at

any site on the aryl ring showed no detectable activity (14 – 19;

EC₅₀> 3000 nM, Table 1). In contrast, the 2-nitro substituted

The resynthesised samples of DIAS1 (6) and DIAS2 (7) gave

EC₅₀values of 296 and 112 nM respectively in the cAMP assay.

Having confirmed the functional activity of DIAS1 (6) and

DIAS2 (7) as CB₂R agonists, attention next turned to exploring

preliminary structure-activity-relationships within this series in

order to optimize activity.

derivative 20 showed good levels of activity at the CB₂R (EC₅₀

=

33 nM, Table 1); regiochemistry was again found to influence

activity, with the 4-nitro substituted counterpart (21) showing

reduced activity at the CB₂R (EC₅₀= 617 nM, Table 1).

2.4. DIAS2 optimisation: variation at C(3)

The first region of the tricyclic moiety to be explored was the

C(3) benzyl thiol moiety. A synthetic route enabling a library to

be efficiently enumerated was optimised from the previous

synthesis of 7 via intermediate 9. This common intermediate was

used to prepare a series of compounds representing S-alkyl, S-

aryl and S-benzyl substituted derivatives. Intermediate 9 allowed

efficient substitution of a range of alkyl and arylmethyl halides in

the presence of Et₃N to yield the S-substituted compounds 10 –

Scheme 2: Reagents and conditions: (i) alkyl halide, Et₃N, MeOH, rt, 16 h; (ii) benzyl halide, Et₃N, MeOH, rt, 16 h; (iv) NaOH (aq.), rt, 16 h.

Table 1. Variation of sulfide substituent and effects on CBR activity and affinity. EC₅₀determined using CHO cells expressing hCB₂R/hCB₁R. Ki

determined using radioligand displacement assay using either [³H]WIN-55212-2 (CB₂R) or [³H]CP 55,940 (CB₁R). All assays were performed in duplicate.

N.D. = Not Determined.

Yield from 9

(%)

EC₅₀(CB₂R)/

nM

EC₅₀(CB₁R)/

nM

K_i(CB₂R)/

nM

K_i(CB₁R)/

nM

Entry

ID

10

R¹

Me

R²

-

1

2

95

80

>3000

296

N.D.

6

(DIAS1)

11

n-Bu

-

3

4

c-Hexylmethyl

-

Ph

67

75

>3000

10000

N.D.

>30000

N.D.

12

7

-

5

-

2-CN-C₆H₄-

quant.

112

>30000

355

>60000

(DIAS2)

13

14

6

7

8

9

10

11

12

13

14

15

16

17

18

-

3-CN-C₆H₄-

2-F-C₆H₄-

4-F-C₆H₄-

2,6-F₂-C₆H₃-

2-Cl-C₆H₄-

58

57

82

54

79

73

59

98

62

74

50

83

99

6450

>3000

33

>30000

N.D.

>30000

N.D.

155

N.D.

>60000

N.D.

15

16

17

18

19

20

3-Cl-C₆H₄-

4-Cl-C₆H₄-

2-NO₂-C₆H₄-

4-NO₂-C₆H₄-

2-CF₃-C₆H₄-

2-OCF₃-C₆H₄-

2-CO₂Et-C₆H₄-

2-CO₂H-C₆H₄-

21

22

617

>3000

2185

23

24

25

-

These initial SAR observations suggested that there was a

preference for electron withdrawing groups incorporating a

hydrogen bond acceptor in the ortho site of the C(3)-thiobenzyl

group. Previous studies have sought to rank and quantify

hydrogen bond acceptor ability of various functional groups,⁵²

and it was noted that examples incorporating a good hydrogen

bond acceptor in the ortho site of the S-benzyl group such as

nitro (20) and cyano (7) showed the highest levels of activity

compared to poorer hydrogen bond acceptors such as halogens

(14 and 17). Three further examples were therefore synthesized

in order to further probe any relationship between electron

withdrawing ability or hydrogen bond acceptor ability and

activity: o-trifluoromethoxy (23), o-ethoxycarbonyl (24) and o-

carboxlic acid (25, prepared through saponification of the

corresponding ester 24). Trifluoromethoxy substituted derivative

23 showed no detectable activity at the CB₂R (EC₅₀> 3000 nM,

Table 1), further suggesting that a bulky group cannot be

accommodated at this site. Interestingly, both ester 24 and

carboxylic acid 25 displayed little or no detectable activity at the

CB₂R (24: EC₅₀>3000 nM; 25 EC₅₀= 2185, Table 1) despite

both groups being good hydrogen bond acceptors.

DIAS2 so it was of further interest to determine the effect of S-

oxidation on bioactivity. However, S-oxidation led to a

significant reduction of activity at the CB₂R (26: EC₅₀= 3550

nM, Scheme 3).

Representative compounds showing activity at the CB₂R were

also assessed for activity at the CB₁R again using

a

complementation immunoassay measuring levels of β-

galactosidase (β-gal) labelled cAMP, analogous to the assay used

to measure CB₂R activity.⁴⁸The assay was performed using

CB₁R-expressing CHO cells stimulated with forskolin and ∆⁹-

THC, a non-specific CB₁R agonist, was used as a positive control

(data not shown). DIAS2 (7) and analogues 12, 13 and 20 were

all evaluated for activity at the CB₁R but none showed any

detectable agonist activity up to the highest concentrations tested

(EC₅₀> 60000 nM, Table 1).

In addition to measuring the functional activity of these

compounds at the CB₁R and CB₂R, the binding affinity of

representative examples at both receptors was determined, in

order to measure K_ivalues. Two representative compounds, 2-

nitro- and 2-cyano-substituted S-benzyl derivatives (20 and 7)

were thus assessed for CB₁R and CB₂R affinity using a

radioligand displacement assay. Compound binding was

measured by displacement of [³H]-CP 55,940 or [³H]-

WIN55212-2 in CHO cells expressing the human CB₁R or CB₂R

respectively.⁵³

The effect of varying the linker between C(3) and the aryl group

was also investigated. DIAS2 (7) was oxidised chemoselectively

to the corresponding sulfoxide 26 in 95% yield using a mixture

of trifluoroacetic acid and peroxytrifluoroacetic acid (Scheme 3).

Compound 26 was also considered as a potential metabolite of

These studies confirmed the binding of 20 and 7 to the CB₂R

(7: K_i= 155 nM; 20: K_i= 355 nM, Table 1). No binding was

detected at the CB₁R for either compound (K_i> 60000 nM,

Table 1) providing further support for the development of this

series of compounds as selective CB₂R agonists.

2.5. DIAS2 optimisation: variation at C(6)-C(9)

Having established preliminary structure-activity-relationships

through variation at C(3), attention next turned to exploring the

effect of introducing a substituent at C(6)-C(9) of the tricyclic

Scheme 3: Reagents and conditions: (i) CF₃CO₃H, CF₃CO₂H, rt, 16 h.

core. In an initial effort to establish which position could tolerate

further substitution, a bromo substituent was introduced at each

of the four available sites. Following the same synthetic

procedure developed for the preparation of DIAS2 (7),

bromoisatins 27 – 30 were first treated with thiosemicarbazide to

afford the corresponding tricyclic species 31 – 34 in 34 – 97 %

yield (Scheme 4). Intermediates 31 – 34 were subsequently

treated with 2-cyanobenzyl bromide to give the bromo-

substituted tricyclic derivatives 35 – 38 in 64 – 92 % yield. 9-

Bromo substituted tricycle 39 was also prepared in 66 % yield by

treating 35 with 2-nitrobenzyl bromide (Scheme 4).

Treatment of 4-bromoisatin 30 with either cyclopropyl

boronic acid or potassium cyclopropyl trifluoroborate under a

range of conditions gave no conversion to the desired 4-

cyclopropylisatin product. To circumvent this problem, isatin

was first treated with 4-methoxybenzyl chloride and sodium

hydride to give the corresponding N-PMB protected isatin 40.

Treatment of 40 with potassium cyclopropyl trifluoroborate and

Pd(dppf)₂Cl₂afforded 44 in 96 % yield. Cyclisation, S-

benzylation and deprotection using trifluoroacetic acid gave 9-

cyclopropyl substituted derivative 53 in 46 % yield over the three

steps (Scheme 5).

Scheme 4: Reagents and conditions: (i) thiosemicarbazide, K₂CO₃, H₂O,

rt, 16 h; (ii) benzyl halide, Et₃N, MeOH, rt, 16 h.

Table 2: Variation of the substitution pattern around the triazinoindole

core and effects on CBR activity and affinity. EC₅₀determined using

CHO cells expressing hCB₂R/hCB₁R. Ki determined using radioligand

displacement assay using either [3H]WIN-55212-2 (CB₂R) or [3H]CP

55,940 (CB₁R). All assays were performed in duplicate.

Scheme 5: Reagents and conditions: (i) NaH, 4-methoxybenzyl chloride,

DMF, 0 °C, 30 min then 40 °C, 3 h; (ii) Potassium aryl trifluoroborate,

K₃PO₄, Pd(PPh₃)₂Cl₂, THF/H₂O (3:1), 130 °C, MW, 4 h; (iii) Potassium

cyclopropyl trifluoroborate, K₃PO₄, Pd(dppf)₂Cl₂, THF/H₂O (3:1), 130

°C, MW, 4 h; (iv) thiosemicarbazide, K₂CO₃, H₂O, reflux, 16 h; (v) 2-

cyano benzyl bromide, Et₃N, MeOH, rt, 16 h; (vi) CF₃CO₂H, 90 °C, 16 h.

EC₅₀

(CB₂R)/

nM

EC₅₀

(CB₁R)/

nM

K_i(CB₂R)/

nM

K_i(CB₁R)/

nM

Entry

ID

Table 3: Variation of substituents at C9 of triazinoindole core and effects

on CB₂R activity and affinity. EC₅₀determined using CHO cells

expressing hCB₂R. All assays were performed in duplicate.

1

2

3

4

5

6

7

112

>30000

N.D.

≈20000

N.D.

355

N.D.

22.5

N.D.

>60000

N.D.

3200

N.D.

35

36

37

38

39

>3000

39

Yield (step

(i) or (ii),

Yield

(steps (iii)-

EC₅₀

(CB₂R)/

nM

Entry

ID

R

37

%)

-

42

68

59

43

(iv), %)

-

55

17

50

44

1

2

3

4

5

7

H

112

The bromo-substituted tricycles 36 – 40 were assessed for

CB₂R activity using the CHO cell assay. Introducing a bromo

substituent at C(6) (35), C(7) (36) or C(8) (37) was observed to

lead to a loss of activity at the CB₂R in all cases (EC₅₀>3000

nM, Table 2). Conversely, 38 and 39 both of which incorporated

a bromo substituent at C(9) maintained good levels of activity at

the CB₂R (EC₅₀= 39 and 37 nM respectively, Table 2). The 9-

bromo derivative 38 was selected for CB₁and CB₂receptor

affinity studies for comparison with DIAS2 (7). Compound 38

showed high binding affinity at the CB₂R and weak to moderate

affinity at the CB₁R (K_i= 22.5 and 3200 nM respectively, Table

2). In accordance with the affinity studies, 38 also showed very

weak agonist activity when evaluated in CB₁R-expressing CHO

cells (EC₅₀≈20000 nM, Table 2).

50

51

52

o-MeC₆H₄-

p-MeC₆H₄-

3-thienyl

4300

>3000

1760

231

53 cyclopropyl

The array of C(9)-substituted compounds were evaluated for

CB₂R activity using the CHO cell assay. While 9-cyclopropyl-

substituted derivative 53 showed good levels of activity at the

CB₂R (EC₅₀= 231 nM, Table 3), introducing a bulkier 2-thienyl

substituent at C(9) led to a reduction in activity at the CB₂R (52:

EC₅₀= 1760 nM, Table 3). Of the two C(9)-aryl substituted

derivatives tested (50 and 51) both showed markedly reduced

activity at the CB₂R (50: EC₅₀= 4300 nM; 51: EC₅₀> 3000 nM,

Table 3).

In an effort to rationalise the steep SAR observed upon

variation at C(9), lowest energy conformers were calculated for

representative examples using the Maestro modelling package

(Schrodinger): 51 (p-methylphenyl substituted), 52 (3-thienyl

substituted) and 53 (cyclopropyl substituted) (Figure 3). The

results suggest that 51 and 52 both preferentially adopt a non-

planar conformation about the exocyclic C(9)-C bond, consistent

with the conformational preferences of ortho-substituted biaryls.

A possible explanation for the reduced activity of 52 compared to

7 and 38, and the lack of detectable activity of 51, could be that

this non-planar conformation cannot be accommodated upon

binding to the CB₂R.

Having established that introducing a bromo substituent at

C(9) was tolerated, we sought to further explore the effect of

varying the C(9) group on activity and affinity at the CB₂R. It

was envisaged that a selection of groups could be introduced at

this position via a Suzuki cross-coupling reaction using 4-

bromoisatin 30 as starting material.⁵⁴4-(3’-Thienyl)isatin 41, 4-

(2’-methylphenyl)isatin 42 and 4-(4’-methylphenyl)isatin 43

were all prepared in moderate to good yield via a Pd(PPh₃)₄Cl₂-

mediated cross-coupling using 4-bromoisatin and the

corresponding

potassium

trifluoroborate

salts.⁵⁵

The

intermediates 41 – 43 were next treated stepwise with

thiosemicarbazide then 2-cyanobenzyl bromide to afford the

C(9)-substituted derivatives 50 – 52 in 17 – 50 % yield over the 2

steps (Scheme 5).

Compound 53 is predicted to have a number of low energy

conformers, one of which is depicted in Figure 3. The two

carbons at the base of the cyclopropyl ring can either be oriented

above or below the plane of the tricycle, and thus a possible

activity at the CB₁R up to the highest concentrations tested (K_i,

EC₅₀> 60000 nM, Table 4), providing additional useful insights

into the requirements for CB₁R/CB₂R selectivity in this series.

N-Sulfonyl and N-oxycarbonyl substituted derivatives were

also investigated. N-phenylsulfonyl derivative 62 showed

comparable levels of activity at the CB₂R to DIAS2 7 (EC₅₀= 76

nM, Table 4). However, introducing functional groups to the N-

phenylsulfonyl substituent (exemplified by N-4-methylphenyl

sulfonyl 63 and N-2-nitrophenyl sulfonyl 64) led to loss of

Figure 3: 3-D representations of calculated lowest energy conformers of

51, 52, and 53. Calculations were performed using Maestro

(Schrodinger).

explanation for the moderate CB₂R agonist activity of 53 is that

one of these conformers can be accommodated upon binding to

the receptor.

detectable activity up to the highest concentration tested (EC₅₀>

3000 nM, Table 4). The reasons for this unexpectedly steep SAR

are unclear at this time. N-Benzyloxycarbonyl derivative 65

showed no detectable activity at the CB₂R (EC₅₀> 3000 nM;

Table 4). Of more concern was the fact that the N-sulfonyl and

N-oxycarbonyl derivatives were also found to show limiting

solubility, even in the vehicle (DMSO), and therefore neither

series were pursued further.

2.6. DIAS2 optimisation: variation at N(5)

Having investigated the effects of varying substituents at C(3)

and C(6)-C(9) attention next turned to exploring the effect of

introducing a substituent at N(5). It was envisaged that a range of

N-substituted derivatives could be readily prepared from the

corresponding S-substituted tricycles. Several conditions were

trialled, but the optimal route was found to be treatment of

triazenoindoles 7, 14 and 20 with NaH (60 % dispersion in

mineral oil) in DMF followed by the requisite electrophile giving

a range of N-alkyl, N-benzyl, N-sulfonyl, N-oxycarbonyl and N-

acyl substituted analogues in 19 – 96 % yield (Scheme 6). Three

N-acyl substituted examples could not be accessed using this

method, and were therefore prepared using alternative conditions

by treating the triazenoindole with DMAP and the requisite acyl

chloride: this afforded 73 from 20 in 73% yield, and 78 – 79 from

7 in 55 % and 56 % yield respectively (Scheme 6).

A series of aliphatic N-acyl substituted derivatives were next

examined. While N-acetyl analogue 66 showed no detectable

activity at the CB₂R, all other aliphatic N-acyl substituted

analogues tested showed good levels of activity: N-

cyclopropylcarbonyl 67, N-cyclopentylcarbonyl 68 and N-

cyclohexylcarbonyl 69 all showed comparable activity with

DIAS2 7 (67: EC₅₀= 181 nM; 68: EC₅₀= 49 nM; 69: EC₅₀= 32

nM, Table 4). These observations were consistent with the trend

in activity for the N-alkyl substituted series – small N-

substituents show no or reduced activity at the CB₂R. It was also

of interest to determine whether polar groups could be

accommodated within the N(5) substituent as it was envisaged

these would aid with improving other important parameters such

as solubility, lipophilicity and potentially metabolism. Thus,

homologous esters 70 and 71 were prepared and evaluated for

agonist activity at the CB₂R: encouragingly both showed good

levels of activity (70: EC₅₀= 48 nM; 71: EC₅₀= 42 nM, Table 4).

N-Cyclopentyl 68, N-cyclohexyl 69 and ester 70 were also

selected as representative examples for CB₂R/CB₁R binding

affinity studies. All showed high affinity at the CB₂R, consistent

with the activity observed in CB₂R-expressing CHO cells (68: K_i

= 320 nM; 69: K_i= 30 nM; 70: K_i= 360 nM, Table 4), but no

detectable activity up to the highest concentrations tested at the

CB₁R (K_i> 60000 nM), Table 4).

The array of N(5)-substituted analogues were evaluated for

activity at the CB₂R in the CHO cell assay. A much broader

range of substituents appeared to be tolerated, with most of the

derivatives prepared and tested showing some agonist activity at

the CB₂R (Table 4). Of the N-alkyl substituted variants, N-

methyl derivative 54 showed no detectable activity at the CB₂R

(EC₅₀> 3000 nM, Table 4), consistent with the preliminary SAR

assessment (Table S2 and Figure S1). In contrast larger linear

N-alkyl groups, N-ethyl 55 and N-(n-butyl) 56, and β-branched

N-alkyl

groups,

N-cyclopropylmethyl

57

and

N-

cyclohexylmethyl 58, were all tolerated and showed similar

activities at the CB₂R (55: EC₅₀= 674 nM; 56: EC₅₀= 106 nM;

57: EC₅₀= 144 nM; 58: EC₅₀= 270 nM, Table 4).

The introduction of an N-benzoyl group 72 led to comparable

levels of activity at the CB₂R (EC₅₀= 113 nM, Table 4) to

DIAS2 7 and its N-benzyl substituted counterpart 59. Similarly,

N-benzoyl derivative 73 also showed comparable activity at the

CB₂R (EC₅₀= 75 nM, Table 4) to its N(5)-unsubstituted

counterpart 20. Both N-benzoyl substituted derivatives 72 and 73

were selected for CB₂R and CB₁R affinity studies: both showed

good affinity for the CB₂R (72: K_i= 300 nM; 73: K_i= 235 nM;

Table 4) and no detectable binding at the CB₁R (72: K_i> 10000

nM; 73: K_i> 60000 nM, Table 4). Interestingly the lack of CB₁R

binding observed for N-benzoyl substituted species 72 was in

contrast to the data obtained for its N-benzyl substituted

counterpart 59 (K_i= 61 nM, Table 4). This suggests that the

presence of the carbonyl functionality is not tolerated by the

N-Benzyl substituted derivatives 59 and 61 also showed

similar levels of activity at the CB₂R compared to their

unsubstituted counterpart DIAS2 7 (59: EC₅₀= 214 nM; 61: EC₅₀

= 53 nM; Table 4). Interestingly, N-benzyl S-(2-fluorobenzyl)

derivative 60 showed moderate activity at the CB₂R in contrast to

its N(5)-unsubstituted counterpart 14, which showed no activity

up to the highest concentrations tested (60: EC₅₀= 850 nM,

Table 4). In order to confirm the activity of the N-substituted

derivatives and determine K_ivalues, N-benzyl substituted

derivatives 59 and 60 and N-(2-nitro)benzyl 61 were selected as

representative examples for CB₂R and CB₁R affinity studies. All

were found to have moderate to high binding affinity for the

CB₂R (59: K_i= 11 nM; 60: K_i= 215 nM; 61: K_i= 290 nM; Table

4), and intriguingly both 59 and 60 also showed similar levels of

binding affinity for the CB₁R (59: K_i= 61 nM; 60: K_i= 275 nM;

Table 4). These binding affinity measurements were supported

by subsequent functional activity studies at the CB₁R: 59 showed

CB₁R, possibly as it introduces

preferentially adopts conformation that cannot be

accommodated at the receptor.

a polar interaction or

a

moderate agonist activity in CB₁R-expressing CHO cells (EC₅₀

=

1553 nM, Table 4). These results were in complete contrast to

the analogues with no substituent at N(5) which showed no

affinity or activity at the CB₁R up to the highest concentrations

tested. Furthermore N-(2-nitro)benzyl 61 showed no affinity or

Scheme 6: Reagents and conditions: (i) electrophile, NaH (60 % dispersion in mineral oil), DMF or THF, 0 °C to rt, 16 h; or DMAP, CH₂Cl₂, rt, 16 h.

Table 4: Variation of substituents at N5 of triazolyl-indole core and effects on CBR activity and affinity. IC₅₀determined using CHO cells expressing

hCB₂R/hCB₁R. Ki determined using radioligand displacement assay using either [3H]WIN-55212-2 (CB₂R) or [3H]CP 55,940 (CB₁R). All assays were

performed in duplicate. N.D. = Not Determined.

EC₅₀(CB₂R)/ EC₅₀(CB₁R)/

K_i(CB₂R)/

nM

K_i(CB₁R)/

nM

Entry

ID

R₁

R₃

Yield (%)

nM

112

>3000

33

nM

>30000

N.D.

>30000

N.D.

1553

N.D.

>30000

N.D.

1

2

3

4

5

6

7

8

7

2-CN-C₆H₄CH₂-

2-F-C₆H₄CH₂-

H

Me

Et

N/A

69

78

68

52

19

80

40

96

84

92

83

96

77

76

42

71

81

72

73

67

58

42

67

55

56

78

85

72

355

N.D.

155

N.D.

11

>60000

N.D.

>60000

N.D.

61

275

>60000

N.D.

>60000

N.D.

>10000

>60000

N.D.

>60000

N.D.

14

20

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

2-NO₂-C₆H₄CH₂-

2-CN-C₆H₄CH₂-

2-F-C₆H₄CH₂-

>3000

674

106

144

270

214

850

53

n-Bu

Cyclopropylmethyl

Cyclohexylmethyl

Benzyl

2-NO₂-C₆H₄-CH₂-

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

215

290

2-CN-C₆H₄CH₂-

2-NO₂-C₆H₄CH₂-

2-CN-C₆H₄CH₂-

PhSO₂-

76

N.D.

320

30

360

N.D.

300

4-Me-C₆H₄SO₂-

2-NO₂-C₆H₄SO₂-

BnOCO-

>3000

181

49

MeCO-

Cyclopropylcarbonyl

Cyclopentylcarbonyl

Cyclohexylcarbonyl

MeCO₂CH₂CH₂CO-

MeCO₂CH₂CH₂CH₂CO-

PhCO-

32

48

42

113

75

PhCO-

235

4-F-C₆H₄CO-

3-CF₃-C₆H₄CO-

2-CF₃-4-F-C₆H₃CO-

4-(CF₃O)-C₆H₄CO-

4-NMe₂-C₆H₄CO-

3,4-(MeO)₂-C₆H₃CO-

2-Furfurylcarbonyl

5-Isoxazolylcarbonyl

1-Morpholinocarbonyl

57

27

N.D.

380

N.D.

490

>3000

73

>3000

160

46

>10000

>60000

N.D.

19

92

450

N.D.

Having established that the introduction of a benzoyl

substituent at N(5) retained activity and affinity at the CB₂R, and

selectivity over the CB₁R, a number of substituted analogues

were next explored. All para- and meta-substituted analogues

showed similar levels of activity at the CB₂R compared to their

N-benzoyl substituted counterpart 72 (74, 4-fluoro: EC₅₀= 57

derivatives were prepared and, as anticipated, all showed good

activity at the CB₂R (80: EC₅₀= 46 nM; 81: EC₅₀= 19 nM; 82:

EC₅₀= 92 nM; Table 4). In subsequent affinity studies,

representative examples 80 and 81 also showed good affinity for

the CB₂R (80: K_i= 490 nM; 81: K_i= 450 nM, Table 4), and no

affinity at the CB₁R (80: K_i> 10000 nM; 81: K_i> 60000 nM,

Table 4).

nM; 75, m-trifluoromethyl: EC₅₀

=

27 nM; 77, p-

trifluoromethoxy: EC₅₀= 73 nM; 79, m-, p-dimethoxy: EC₅₀

=

Evaluation of physicochemical properties and metabolic stability

of DIAS2 and analogues

160 nM; Table 4), except for 4-dimethylamino substituted

derivative 78 which showed no detectable activity at the CB₂R

(EC₅₀> 3000 nM, Table 4). It was noted that 78 was extremely

poorly soluble under the assay conditions, and this may have led

to the lack of observable activity. Introducing a trifluoromethyl

group in the ortho site up to the highest concentrations tested

(EC₅₀>3000 nM, Table 4). Both of the examples investigated

had comparatively bulky ortho substituents (A-values: CF₃= 2.1

kcal/mol; NO₂= 1.13 kcal/mol⁵⁶) and it is therefore possible that

the reduced conformational freedom and the preferred

conformations cannot be accommodated at the CB₂R.

An analysis of commonly employed synthetic and semi-

synthetic cannabinoid receptor modulators revealed that the

majority are highly lipophilic, with high clogP and logD values

(HU-308: clogP = 8.0, Table S1). This is perhaps not surprising

given the preference of cannabinoid receptors for lipophilic

endogeneous ligands. Moreover, the majority of cannabinoid

receptor modulators to date have been developed and

investigated for neuroinflammation and neuropathic pain

indications and therefore require good distribution into the brain

and CNS, a property often associated with higher than average

logD values.^57,58To investigate the effects of CB₂R agonists in

peripheral pain and inflammation it was of interest to generate

compounds with reduced lipophilicity in the hope that this would

translate into reduced accumulation in the CNS and an improved

A selection of heterocyclic substituents were next investigated

as these were anticipated to confer more favourable

physicochemical properties. N-(2-Furfuryl)carbonyl (80), N-(5-

isoxazolyl)carbonyl (81) and N-(1-morpholino)carbonyl (82)

Table 5: Variation of substituents at N(5) of triazenoindole core and effects on CB₂R binding activity and affinity, solubility and mouse liver microsome

metabolic stability. N.D. = Not Determined. ^aCalculated using the CLogP algorithm in ChemBioDraw 14.0. ^bThe value determined is the mid-point

between the upper and lower solubility limits. *Not observable even at the earliest time point, suggesting stability is very low and t_1/2/was not

calculable.

Solubility

(µM)^b

EC₅₀(CB₂R)/

nM

MLM Stability

t_1/2(min)

Entry

ID

R¹

R²

R³

CLogP^a

LLE

1

2

3

6

7

n-Bu

H

296

112

33

4.2

3.8

3.9

2.3

3.2

3.7

2.0

3.75

N.D.

46

2-CN-C₂H₄CH₂-

2-NO₂-C₆H₄CH₂-

20

43

4

5

6

7

8

38

56

57

58

59

62

67

70

71

72

81

82

2-CN-C₂H₄CH₂-

Br

H

n-Bu

Cyclopropylmethyl

Cyclohexylmethyl

Benzyl

39

106

144

270

214

76

181

48

42

4.6

5.2

4.6

6.2

5.1

3.8

3.3

3.7

4.6

2.6

3.1

2.8

1.5

2.3

0.3

1.6

2.1

3.0

4.0

3.7

2.4

5.2

3.9

N.D.

3.75

2.0

5

<5

9

PhSO₂-

<5

10

11

13

14

15

16

Cyclopropylcarbonyl

MeCO₂CH₂CH₂CO-

MeCO₂CH₂CH₂CH₂CO-

PhCO-

5-Isoxylcarbonyl

1-Morpholinocarbonyl

N.D.

-*

N.D.

113

19

92

6.5

10.5

pharmacokinetic profile. Given the relative expression patterns of

CB₂R (predominantly peripheral tissue)^1,2and CB₁R

(predominantly CNS)^3,4it was also antipated that aiming for a

peripherally restricted molecule would lead to improved in vivo

efficacy and reduced side-effects.

within the N(5) substituent had lower clogP values and higher

LLE values than DIAS2 (esters 70 and 71: clogP = 3.3 and 3.7,

LLE = 4.0 and 3.7 respectively; 1-morpholinocarbonyl 82: clogP

=

3.1, LLE = 4.0). Of particular interest was N-(5-

isoxazolyl)carbonyl substituted derivative 81, which had a clogP

value of 2.6 and, due to its high activity at the CB₂R, a high value

for LLE (5.2).

Values for clogP were therefore calculated for representative

active compounds to allow comparisons to be made (Table 5).

Relative values for lipophilic ligand efficiency (LLE) were also

calculated based on the corresponding EC₅₀values for each

compound. LLE has been shown to be a useful parameter in

medicinal chemistry – optimal values for LLE have been shown

in many studies to correlate with higher chances of success in the

clinic.⁵⁹Moreover, a comparison of LLE within a series can give

a good indication whether increasing activity may be directly

related to increasing lipophilicity – i.e. it is likely to be a function

of the free energy of solvation and not an increase in the free

energy of binding to the receptor.⁶⁰While the value for clogP for

DIAS1 (6) was determined to be 4.2, clogP for DIAS2 (7) was

determined to be 3.2, with a correspondingly lower value for

LLE, and was therefore deemed to be a promising candidate for

optimisation and development.^59c,61The clogP value for 20 was

similar to DIAS2 7 (3.9), giving a proportionally higher value for

LLE (3.7) due to its modestly increased activity. Unsurprisingly

the clogP value for 9-bromo substituted derviative 38 was higher

than DIAS2 (4.6), leading to a correspondingly lower value for

LLE (2.8).

To further investigate any relationship between activity (in

this case pEC₅₀) and clogP within this hit series, a plot correlating

these two parameters for each active compound was constructed

(Figure 4). Supporting the analyses in Table 5, the plot showed

no correlation between clogP and activity (R²< 0.2).

Besides clogP and LLE it was of real interest to optimise

physicochemical properties within the series, particularly

It was of particular interest to compare the values for clogP

and LLE for a selection of the N-substituted variants. It was

observed that a range of groups were tolerated at this site, and it

was therefore anticipated that those examples incorporating polar

functional groups would give more favourable values for clogP

and thus proportionally higher values for LLE. As anticipated, N-

alkyl- and N-benzyl substituted derivatives 56 – 59 all had higher

clogP values (4.6 – 6.2), and correspondingly much lower values

for LLE (0.3 – 2.3). Similarly, N-phenylsulfonyl (62), N-

cyclopropylcarbonyl (67) and N-benzoyl (72) had similar or

higher values for clogP to DIAS2 (62 = 5.1; 67 = 3.8; 72 = 4.6),

and lower values for LLE (62 = 2.1; 67 = 3.0; 72 = 2.4).

Conversely, all those derivatives incorporating a polar group

Figure 4: pIC₅₀and cLogP correlation of triazinoindole series

aqueous solubility. The majority of cannabinoid ligands

described in the literature are relatively poorly soluble in aqueous

solution, presumably as a consequence of their high lipophilicity,

a property that limits their application as chemical tools. It was

anticipated that given that polar groups could be accommodated

within the N(5) substituent, aqueous solubility could also be

tuned by varying this substituent. Thus, the kinetic solubility of a

selection of active variants was measured at 37 °C, pH 7.0.⁶²

DIAS1 (6) showed low aqueous kinetic solubility (2.0 µM),

while DIAS2 (7) showed a slight improvement (3.75 µM, Table

5). A selection of derivatives incorporating N-acyl and/or polar

groups within the N(5) substituent were next evaluated. While the

introduction of an ester group within the N(5) substituent offered

no improvement in aqueous kinetic solubility (70 and 71: 3.75

µM, Table 5), N-benzoyl substituted derivative 72 showed

reduced solubility (2.0 µM) compared to its unsubstituted

counterpart, DIAS2 (7). However, the heterocycle-containing

analogues, N-(5-isoxazolyl)carbonyl 81 and N-(1-morpholino)

carbonyl 82 both showed modestly improved solubility compared

to DIAS2 (81: 6.5 µM; 82: 10.5 µM, Table 5) demonstrating that

through varying the N(5)-substituent solubility could be

improved within this series. Adequate solubility is a highly

desirable parameter for a range of drug properties, and whilst

some improvement was evident, further optimisation is still

required.⁶³

It was of interest to investigate absorption properties of

DIAS2 (7): ideally it was desired to generate an orally

bioavailable compound that would not accumulate within the

CNS. To give an indication of cellular permeability and efflux,

and in particular an indication of whether significant CNS

penetration was likely, Madin-Darby canine kidney cells

transfected with multidrug resistance protein 1 (MDCK-MDR1)

were used as an in vitro model. These cell lines were selected as

it can give an indication of p-glycoprotein-mediated efflux, a

major pathway through which compounds are effluxed within the

gut and at the blood brain barrier (BBB). DIAS2 (7) was

determined to have a permeability of 3.21 x 10^-6cm.s^-1predicting

that the compound would be readily absorbed within the gut. The

efflux ratio was determined to be < 1 indicating DIAS2 (7) is not

a p-glycoprotein substrate. This permeability data also predicts

that the compound would be on the boundary of CNS

penetration.⁶⁷

As the overall aim was to develop a selective CB₂R agonist

for evaluation in vivo, it was also of interest to investigate the

metabolic stability of representative examples. To give an

indication of in vivo metabolic stability, the compounds were

incubated in vitro with mouse liver microsomes (MLM) and the

concentration monitored by mass spectrometry over time

allowing a value for half-life (t_1/2) to be determined.⁶⁴Diazepam

(t_1/2= 5-10 min) and diphenhydramine (t_1/2= 30-40 min) were

used as controls. A representative selection of compounds were

assessed using this in vitro stability assay: S-benzyl derivatives 7

and 20, C(9)-bromo substituted derivative 38, N-alkyl substituted

derivatives 56 – 58, N-benzyl analogue 59, N-sulfonyl analogue

62, and N-acyl derivatives 67 and 72 (Table 5).

It had already been demonstrated that DIAS2 (7) showed

Figure 5: DIAS 2 (7) and its key pharmacological, physicochemical and

in vitro ADMR properties. ^aCalculated using ChemDraw v13.0.

^bDetermined using MDCK-MDR1 cells. ^c< 30 % activity when tested at

10 µM against a panel of 12 GPCRs (see Supporting Information). ^d< 30

% activity when tested at 10 µM against a panel of 96 kinases (see Tables

S3 and S4).

DIAS2 7 showed reasonable metabolic stability in the MLM

assay (t_1/2= 46 min): interestingly the S-oxidation product 26 was

not detected amongst the metabolites. Replacing the 2-

cyanobenzyl with a 2-nitrobenzyl group 20 did not significantly

alter the stability (20: t_1/2= 43 min), whereas incorporating a 9-

bromo substituent (38) led to a ten-fold reduction in half-life (t_1/2

= 5 min) compared to DIAS2 (7). The decreased stability of 38

may be due in part to its increased lipophilicity (clogP = 4.6): in

many instances more lipophilic compounds have been shown to

be better substrates for microsomal enzymes resulting in

increased turnover.⁶⁵All examples tested incorporating an N(5)

substituent showed a significant reduction in stability, regardless

of the nature of the substituent: N-alkyl substituted derivatives 56

– 58, N-benzyl analogue 59, N-sulfonyl analogue 62, and N-acyl

derivative 67 all gave values for t_1/2of less than 5 minutes. In the

case of N-benzoyl derivative 72, no parent compound could be

detected at the earliest time point (5 minutes) precluding the

determination of a value for t_1/2and suggesting extremely rapid

turnover. Importantly, the compounds were stable in the presence

of microsomes in the absence of added cofactor (NADPH),

suggesting a microsomal enzyme-mediated degradation and,

moreover, in all of the studies carried out on the N(5)-substituted

derivatives, DIAS2 (7) was observed as the principal metabolite.

This observation suggested that the main origin of metabolic

instability of the N(5)-substituted derivatives was the lability of

the exocyclic N-C bond, independent of the nature of the linkage.

selectivity for the CB₂R over the CB₁R (Table 1), but it was also

of interest to assess whether 7 also interacted with other targets,

particularly related GPCRs and also kinases. Thus, 7 was

screened for off-target activity against a selection of 24 GPCRs⁶⁸

(Table S3) and 97 kinases⁶⁹(Table S4). Kinases were of interest

to measure off target activity as they have been shown to bind

polycyclic heteroaromatic structures, and as the cell-based

activity screen measures intracellular cAMP levels it could in

principle respond to a compound interacting with other cellular

components. The GPCR screen used a radioligand displacement

assay platform analogous to the affinity measurements at he

CB₁R and CB₂R determined previously. Encouragingly at a

concentration of 1 µM, no significant binding was observed for 7

at any of the GPCRs tested (Table S3), nor with any of the

kinases when tested at a concentration of 10 µM (Table S4).⁶⁹

3. Conclusion

In summary, a novel series of CB₂R activating molecules have

been identified following a ligand-based screening approach

using a known CB₂R agonist HU-308 as a template.

Systematical SAR analyses were conducted around the

tricyclic core, varying substituents at C(3), C(6)-C(9) and N(5).

At C(3), thiobenzyl substituents incorporating small electron

withdrawing groups at the ortho site gave the highest levels of

activity and affinity at the CB₂R while maintaining selectivity

over the CB₁R. A small substituent was also found to be tolerated

at C(9), however this was also found to give rise to modest

affinity at the CB₁R. A range of substituents were found to be

tolerated at N(5), which allowed tuning of the selectivity (e.g. N-

benzyl 59: CB₁R K_i= 61 nM, CB₂R K_i= 11 nM; N-benzoyl 72:

CB₁R K_i> 10000 nM, CB₂R K_i= 300 nM) and, importantly,

2.7. DIAS2 (7) further evaluation

On the basis of the activity, affinity, selectivity, solubility and

in vitro metabolic stability analyses DIAS2 (7) and 20 were

deemed to possess the most promising overall profiles. The

nitrile functionality within 7 was favoured over the nitro group

within 20 due to the well-documented possibility of toxicophores

presented by the nitro group upon degradation,⁶⁶and thus DIAS2

(7) was selected for further evaluation.

other molecular properties. While the introduction of polar

groups within the substituent at N(5) was found to improve

solubility and reduce lipophilicity, all analogues examined were

rapidly metabolised in in vitro mouse liver microsome stability

studies: the major metabolite being DIAS2 (7) resulting from

cleavage of the exocyclic N(5)-C bond. The use of these

analogues as possible pro-drugs to liberate DIAS2 (7) in vivo is

currently being explored.

To a suspension of 5H-[1,2,4]triazino[5,6-b]indole-3-thiol (1

equiv) in methanol (5 mL) was added Et₃N (1.5 equiv). To the

resulting suspension was added the requisite electrophile (1

equiv) and the reaction mixture stirred at rt for 16 h. The

resulting precipitate was filtered and washed with a solution of

Et₃N in water (1:25, 5% Et₃N), which was dried in vacuo to yield

the S-substituted product which was purified as specified in each

example below.

DIAS2 (7) was deemed to possess the most promising overall

profile in terms of CB₂R activity, affinity, selectivity,

lipophilicity and in vitro metabolic stability and was therefore

advanced to further in vitro analyses. DIAS2 (7) was showed

good in vitro absorption properties and a low efflux ratio.

Moreover, no significant off-target activity was observed when

DIAS2 (7) was screened against a panel of GPCRs and kinases.

Overall we believe these properties make DIAS2 (7) a promising

candidate for further investigation and application in vivo to

assess the impact of CB₂R activity in inflammation (and other

pathologies). A detailed study of the formulation, in vivo

pharmacokinetic properties, in vitro and in vivo efficacy of

DIAS2 (7) in murine models of inflammation will be imminently

forthcoming.

4.2.2. General Procedure 2: N-substitution

A flask was flame dried and cooled under an atmosphere of

argon. To the flask was added the S-substituted-5H-

[1,2,4]triazino[5,6-b]indole-3-thiol (1 equiv) and anhydrous

DMF or THF (5 mL) under argon. The resulting suspension was

cooled to 0 °C using an ice bath. NaH (60 % dispersion in

mineral oil, 1.1 - 1.5 equiv) was added, resulting in a clear yellow

solution, which was stirred for 15 min at 0 °C. The corresponding

electrophile (1.5 equiv) was added gradually (dropwise or

portionwise)

at

0 °C. After addition was complete, the reaction mixture was

allowed to warm to rt and stirred for 16 h under argon. The

reaction was quenched with water and the product extracted as

specified below.

4. Experimental

4.3. Chemical synthesis

4.3.1. 5H-[1,2,4]Triazino[5,6-b]indole-3-thiol (9)⁵⁰

4.1. General chemical methods

To a stirred suspension of isatin 8 (5.00 g, 33.2 mmol) and

K₂CO₃(7.05 g, 51.0 mmol) in water (100 mL), was added

thiosemicarbizide (3.09 g, 33.2 mmol). The mixture was heated

under reflux for 16 h. Upon cooling the solution was acidified

with glacial acetic acid to afford a precipitate, which was filtered

and washed with a water/acetic acid (24:1, v/v) mixture. The

resulting solid was triturated with hot DMF, filtered and dried to

All reactions involving organometallic or moisture-sensitive

reagents were carried out under an argon atmosphere using

standard vacuum line techniques and glassware that was flame

dried and cooled under argon before use. Solvents were dried

according to the procedure outlined by Grubbs and co-workers.⁷⁰

Water was purified by an Elix® UV-10 system. All other

solvents and reagents were used as supplied (analytical or HPLC

grade) without prior purification. Organic layers were dried over

anhydrous MgSO₄. Pet ether refers to the fraction of petroleum

spirit boiling between 30 and 40 °C. Thin layer chromatography

was performed on aluminium plates coated with 60 F254 silica.

Plates were visualised using UV light (254 nm) or 1% aq

KMnO₄. Melting points were recorded either on a Gallenkamp

Hot Stage apparatus (Gallenkamp) or a EZ-Melt Automated

Melting Point Apparatus (EZ Melt) and are uncorrected. IR

yield the product (9) as

a

yellow solid (6.25 g,

92 %); mp (Gallenkamp) > 300 °C [lit.⁵⁰mp >300 °C]; δ_H(400

MHz, DMSO–d₆, 363 K) 7.34 (1 H, app t, J = 7.6 Hz, C(8)H),

7.44 (1 H, d, J = 8.1 Hz, C(6)H), 7.62 (1 H, app t, J = 7.6 Hz,

C(7)H), 8.00 (1 H, d, J = 7.6 Hz, C(9)H); δ_C(100 Hz, DMSO–d₆)

113.9, 118.6, 122.7, 123.9, 132.7, 136.5, 144.0, 150.0, 179.9; m/z

(ESI^–) 201 ([M–H]^–, 100%).

4.3.2. 3-(Methylthio)-5H-[1,2,4]triazino[5,6-b]indole (10)

spectra were recorded on

a

Bruker Tensor 27 FT−IR

Methyl iodide (308 uL, 4.95 mmol) was added to a stirred

solution on 5H-[1,2,4]triazino[5,6-b]indole-3-thiol 9 (1.0 g, 4.95

mmol) and K₂CO₃(683 mg, 4.95 mmol) and stirred at rt for 16 h

during which time the yellow solution turned green. The solvent

was removed in vacuo to yield the product (10) as a light green

solid (1.01 g, 95 %); mp (EZ Melt) >300 °C; ν_max(solid) 3056,

2799, 1600, 1576, 1460, 1415, 1232; HPLC (Method 2) >99 %,

tR = 11.31; δ_H(400 MHz, DMSO–d₆, 363 K) 2.66 (3H, s, CH₃),

7.43 (1H, app td, J = 8.2, 1.0 Hz, C(8)H), 7.58 (1H, app dd, J =

8.2, 1.0 Hz, C(6)H), 7.67 – 7.71 (1H, m, C(7)H), 8.31 (1H, app

dd, J = 8.2, 1.0 Hz, C(9)H), 12.60 (1H, br s, NH); δ_C(100 MHz,

DMSO–d₆) 13.4, 112.7, 117.7, 121.4, 122.4, 130.8, 140.3, 140.8,

146.7, 167.6; m/z (ESI^–) 215 ([M–H]^–, 100%); HRMS (ESI⁺)

C₁₀H₈N₄NaS ([M+Na]⁺) requires 239.0362; found 239.0360.

spectrometer with diamond ATR module. Selected

a

characteristic peaks are reported in cm^–1. NMR spectra were

recorded on Bruker Avance spectrometers in the deuterated

solvent stated. The field was locked by external referencing to the

relevant deuteron resonance. Chemical shifts (δ) are reported in

ppm and coupling constants (J) are reported in Hz and are

unaveraged. Low-resolution mass spectra were recorded on either

a VG MassLab 20-250 or a Micromass Platform 1 spectrometer.

Accurate mass measurements were run on either a Bruker

MicroTOF internally calibrated with polyalanine, or a Micromass

GCT instrument fitted with a Scientific Glass Instruments BPX5

column (15 m × 0.25 mm) using amyl acetate as a lock mass, by

the mass spectrometry service of the Chemistry Research

Laboratory, University of Oxford, UK. HPLC analysis was

carried out on a Waters Xterra reverse phase C₁₈with gradients

of water (0.1 % TFA) and acetonitrile (0.1 % TFA).

4.3.3.

DIAS 1)

3-(Butylthio)-5H-[1,2,4]triazino[5,6-b]indole (6,

To a suspension of 5H-[1,2,4]triazino[5,6-b]indole-3-thiol 9

(80 mg, 0.40 mmol) and Cs₂CO₃(194 mg, 0.94 mmol) in

methanol (10 mL) was added iodobutane (45 µL, 0.4 mmol) and

the resulting mixture stirred for 16 h at 80 °C to afford a yellow

solution. Upon cooling, the solvents were removed in vacuo and

resulting solid was washed with water/methanol to yield the title

4.2. General synthesis procedures

4.2.1. General Procedure 1: S-substitution

compound (6, DIAS1) as a yellow solid (82 mg, 80 %); mp

(Gallenkamp) 249 – 252 °C; ν_max(solid) 3058, 2957, 2802, 1607,

1461, 1186; HPLC (Method 2) >99 %, tR = 10.73 min; δ_H(400

MHz, DMSO–d₆, 363 K) 0.94 (3H, t, J = 7.3 Hz, C(4’)H), 1.42 –

1.53 (2H, m, C(3’)H), 1.71 – 1.77 (2H, m, C(2’)H), 3.28 (2H, t, J

= 7.7 Hz, C(1’)H), 7.44 (1H, app t, J = 7.9 Hz, C(8)H), 7.57 (1H,

d, J = 8.2 Hz, C(6)H), 7.66 – 7.72 (1H, m, C(7)H), 8.30 (1H, d, J

= 7.9 Hz, C(9)H); δ_C(100 MHz, DMSO–d₆) 13.5, 21.5, 29.6,

30.9, 112.7, 117.7, 121.4, 122.4, 130.7, 140.3, 140.9, 146.7,

167.3; m/z (ESI⁺) 281 ([M+Na]⁺, 100%); HRMS (ESI⁺)

C₁₃H₁₄N₄NaS ([M+Na]⁺) requires 281.0831; found 281.0821.

112.6, 117.3, 117.4, 121.4, 122.4, 128.1, 130.2, 130.9, 133.0,

133.2, 140.2, 141.1, 141.2, 146.4, 165.6; m/z (ESI^–) 316 ([M–H]^–,

100%); HRMS (ESI⁺) C₁₇H₁₀N₅S^–([M–H]^–) requires 316.0662;

found 316.0673.

4.3.7. 3-((3’-Cyanobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (13)

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol) and Et₃N (155 µL, 1.11

mmol) in methanol (5 mL) was added 3-cyano benzyl bromide

(146 mg, 0.74 mmol) and the resulting mixture was stirred

overnight. The precipitate was filtered and washed to give the

title compound (13) as a pale yellow solid (137 mg, 58 %); mp

(Gallenkamp) 279 – 281 °C; C₁₇H₁₁N₅S requires C, 64.34; H,

3.49; N, 22.07%; found C, 64.07; H, 3.33; N, 22.02%; ν_max(solid)

3120, 2228, 1596, 1579, 1338, 1173; δ_H(400 MHz, DMSO–d₆,

363 K) 4.62 (2H, s, CH₂), 7.44 (1H, app t, J = 7.6 Hz, C(8)H),

7.55 (1H, app t, J = 7.6 Hz, C(5’)H), 7.59 (1H, app t, J = 7.6 Hz,

C(6)H), 7.70 (1H, app t, J = 7.6 Hz, C(7)H), 7.72 – 7.75 (1H, m,

C(6’)H), 7.90 (1H, d, J = 8.2 Hz, C(4’)H), 7.99 (1H, s, C(2’)H),

8.32 (1H, d, J = 7.6 Hz, C(9)H), 12.67 (1H, br s, NH); δ_C(100

MHz, DMSO–d₆) 33.1, 111.3, 112.7, 117.6, 118.7, 121.5, 122.5,

129.7, 131.0, 131.0, 132.5, 134.0, 139.9, 140.3, 141.3, 146.6,

166.1; m/z (ESI^–) 316 ([M–H]^–, 100%); HRMS (ESI⁺)

C₁₇H₁₁N₅NaS ([M+Na]⁺) requires 340.0627; found 340.0619.

4.3.4. 3-((Cyclohexylmethyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (11)

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol) and Et₃N (155 µL, 1.11

mmol) in methanol (5 mL) was added bromomethyl cyclohexane

(104 µL, 0.74 mmol) and the resulting suspension was stirred at

rt for 16 h. The precipitate was filtered and washed to give the

title compound (11) as a pale yellow solid (149 mg, 67 %); mp

(Gallenkamp) 298 – 300 °C; ν_max(solid) 3059, 2918, 1603, 1582,

1460, 1420, 1336; HPLC (Method 1) >95 %, tR = 11.62 min; δ_H

(400 MHz, DMSO–d₆, 363 K) 1.04 – 1.25 (5H, m, 5 x CH), 1.63

– 1.73 (4H, m, 4 x CH), 1.87 – 1.91 (2H, m, 2 x CH), 3.20 (2H,

d, J = 6.7 Hz, S-CH₂), 7.43 (1H, app t, J = 7.6 Hz, C(7)H), 7.57

(1H, d, J = 7.6 Hz, C(6)H), 7.69 (1H, app t, J = 7.6 Hz, C(8)H),

8.30 (1H, d, J = 7.6 Hz, C(9)H), 12.60 (1H, br s, NH); δ_C(100

MHz, DMSO-d₆) 25.5, 25.8, 32.1, 36.7, 37.1, 112.6, 117.7,

121.4, 122.4, 130.8, 140.2, 140.9, 146.6, 167.5; m/z (ESI^–) 297

([M–H]^–, 100%); HRMS (ESI⁺) C₁₆H₁₈N₄NaS ([M+Na]⁺)

requires 321.1144; found 321.1143.

4.3.8. 3-((2’-Fluorobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (14)

To an aqueous solution of NaOH (4 %, 5 mL) was added 5H-

[1,2,4]triazino[5,6-b]indole-3-thiol 9 (150 mg, 0.74 mmol) and 2-

fluoro benzyl bromide (89.6 µL, 0.74 mmol) and the reaction was

stirred overnight at rt. The resulting precipitate was filtered and

recrystallised from methanol to yield the title compound (14) as a

light yellow solid (131 mg, 57 %); mp (Gallenkamp) 285 – 286

°C; ν_max(solid) 3054, 2800, 1492, 1461, 1323, 1176; HPLC

(Method 1) 97.0%, tR = 11.13 min; δ_H(400 MHz, DMSO–d₆)

4.59 (2H, s, CH₂), 7.16 (1H, app t, J = 7.6 Hz, C(5’)H), 7.21 –

7.24 (1H, m, C(6’)H), 7.32 – 7.35 (1H, m, C(3’)H), 7.43 (1H,

app t, J = 7.6 Hz, C(8)H), 7.58 (1H, d, J = 7.6 Hz, C(6)H), 7.65

(1H, app t, J = 7.6 Hz, C(4’)H), 7.69 (1H, app t, J = 7.6 Hz,

C(7)H), 8.30 (1H, d, J = 7.6 Hz, C(9)H), 12.67 (1H, br s, NH); δ_C

(126 Hz, DMSO–d₆) 27.6, 112.7, 115.4 (d, J = 21 Hz), 117.6,

121.5, 122.5, 124.3 (d, J = 14 Hz), 124.5, 129.5 (d, J = 8 Hz)

130.9, 131.4 (d, J = 4 Hz), 140.3, 141.2, 146.6, 160.6 (d, J = 246

Hz), 166.3; δ_F(470 MHz, DMSO–d₆) –116.6; m/z (ESI^–) 309

([M–H]^–, 100%); HRMS (ESI⁺) C₁₆H₁₁FNaN₄S ([M+Na]⁺)

requires 333.0581; found 333.0572.

4.3.5. 3-(Benzylthio)-5H-[1,2,4]triazino[5,6-b]indole (12)

To an aqueous solution of NaOH (4 %, 5.00 mL) was added

5H-[1,2,4]triazino[5,6-b]indole-3-thiol 9 (100 mg, 0.49 mmol)

followed by benzyl bromide (58.5 µL, 0.49 mmol). The reaction

was stirred at overnight at rt and resulting precipitate was filtered.

Recrystallisation from methanol yielded the product (12) as a

light yellow solid (68 mg, 75 %)¹; mp (Gallenkamp) 290 – 292

°C; ν_max(solid) 3059, 2799, 1600, 1579, 1461, 1321; HPLC

(Method 1) >99 %, tR = 11.06 min; δ_H(400 MHz, DMSO–d₆)

4.57 (2H, s, CH₂), 7.27 (1H, t, J = 7.9 Hz, C(4’)H), 7.34 (2H, app

t, J = 7.6 Hz, C(3’)H and C(5’)H), 7.44 (1H, app t, J = 7.7 Hz,

C(8)H), 7.52 (2H, d, J = 7.6 Hz, C(2’)H and C(6’)H), 7.59 (1H,

d, J = 7.7 Hz, C(6)H), 7.70 (1H, app t, J = 7.7 Hz, C(7)H), 8.31

(1H, d, J = 7.7 Hz, C(9)H), 12.66 (1H, br s, NH); δ_C(126 Hz,

DMSO–d₆) 34.0, 112.7, 117.6, 121.5, 122.5, 127.2, 128.5, 129.1,

130.9, 137.6, 140.3, 141.1, 146.6, 166.7; m/z (ESI^–) 291 ([M–H]^–,

100%); HRMS (ESI⁺) C₁₆H₁₂NaN₄S ([M+Na]⁺) requires

315.0675; found 315.0663.

4.3.9. 3-((4’-Fluorobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (15)

To an aqueous solution of NaOH (4 %, 5 mL) was added 5H-

[1,2,4]triazino[5,6-b]indole-3-thiol 9 (150 mg, 0.74 mmol) and 2-

fluoro benzyl bromide (92.5 µL, 0.74 mmol). The reaction was

stirred overnight at rt and resulting precipitate was filtered.

Recrystallisation from methanol yielded the title compound (15)

as a light yellow solid (187 mg, 82 %); mp (Gallenkamp) 261 –

264 °C; ν_max(solid) 3054, 2798, 1600, 1539, 1461, 1417; HPLC

(Method 1) >97 %, tR = 11.11 min; δ_H(400 MHz, DMSO–d₆,

363 K) 4.56 (2H, s, CH₂), 7.15 (2 H, app t, J = 8.8 Hz, C(3’)H

and C(5’)H), 7.42 (1H, app t, J = 7.9 Hz, C(8)H), 7.56 (1H, d, J =

7.9 Hz, C(6)H), 7.57 (2 H, d, J = 7.9 Hz, C(2’)H and C(6’)H),

7.68 (1H, app t, J = 7.9 Hz, C(7)H), 8.29 (1H, d, J = 7.9 Hz,

C(9)H), 12.64 (1H, br s, NH); δ_C(100 Hz, DMSO–d₆) 33.2,

112.7, 115.2 (d, J = 21 Hz), 117.6, 121.5, 122.4, 130.9, 131.0 (d,

J = 8 Hz), 133.9 (d, J = 3 Hz), 140.3, 141.1, 146.6, 161.3 (d, J =

243 Hz), 166.5; δ_F(470 MHz, DMSO–d₆) –115.3; m/z (ESI^–) 309

4.3.6.

b]indole (7, DIAS2)

3-((2’-Cyanobenzyl)thio)-5H-[1,2,4]triazino[5,6-

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (2.26 g, 11.2 mmol) and Et₃N (2.33 mL, 16.8

mmol) in methanol (40 mL) was added 2-cyanobenzyl bromide

(2.19 g, 11.2 mmol) and the resulting mixture stirred at rt for 16h.

The precipitate was filtered and washed to give the title

compound (7, DIAS2) as a pale yellow solid (3.60 g, quant); mp

(Gallenkamp) 276 – 278 °C; ν_max(solid) 3061, 2971, 2804, 2226,

1598, 1580, 1342; HPLC (Method 1) >99 %, tR = 10.85 min; δ_H

(400 MHz, DMSO–d₆, 363 K) 4.73 (2 H, s, CH₂), 7.41 – 7.49 (2

H, m, C(8)H and C(4’)H), 7.58 (1 H, d, J = 7.8 Hz, C(6’)H), 7.64

– 7.71 (2 H, m, C(3’)H and C(7)H), 7.82 (1 H, m, C(5’)H), 7.85

(1H, d, J = 7.6 Hz, C(6)H), 8.30 (1 H, d, J = 7.8 Hz, C(9)H),

12.66 (1 H, br s, NH); δ_C(100 MHz, DMSO–d₆) 32.4, 111.9,

([M–H]^–, 100%); HRMS (ESI⁺) C₁₆H₁₁FN₄NaS ([M+Na]⁺)

requires 333.0581; found 333.0576.

4.3.13. 3-((4’-Chlorobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (19)

4.3.10. 3-((2’,6’-Difluorobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (16)

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol) and Et₃N (155 µL, 1.11

mmol) in methanol (5 mL) was added 3-chloro benzyl bromide

(152 mg, 0.74 mmol) and the resulting mixture stirred at rt for 16

h. The precipitate was filtered and washed to give the title

compound (19) as a pale yellow solid (142 mg, 59 %); mp

(Gallenkamp) 248 – 250 °C; ν_max(solid) 3054, 2797, 1600, 1578,

1489, 1461, 1338; HPLC (Method 1) >98 %, tR = 11.51 min; δ_H

(400 MHz, DMSO–d₆, 363 K) 4.56 (2H, s, CH₂), 7.38, (2H, d, J

= 8.8 Hz, C(2’)H and C(6’)H), 7.44 (1H, app t, J = 7.6 Hz,

C(8)H), 7.55 (2H, d, J = 8.8 Hz, C(3’)H and C(5’)H), 7.58 (1H,

d, J = 7.6 Hz, C(6)H), 7.70 (1H, app t, J = 7.6 Hz, C(7)H), 8.31

(1H, d, J = 7.6 Hz, C(9)H), 12.66 (1H, br s, NH); δ_C(100 MHz,

DMSO–d₆) 33.2, 112.7, 117.6, 121.5, 122.5, 128.4, 128.6, 130.9,

131.8, 137.0, 140.3, 141.2, 146.6, 166.4; m/z (ESI^–) 325 ([³⁵ClM–

H]^–, 100%), 327 ([³⁷ClM–H]^–, 45%); HRMS (ESI⁺)

C₁₆H₁₀³⁵ClNaN₄S ([M+H]⁺) requires 349.0285; found 349.0284,

C₁₆H₁₀³⁷ClNaN₄S ([M+H]⁺) requires 351.0256; found 351.0258.

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol) and Et₃N (155 µL, 1.11

mmol) in methanol (5 mL), was added 2,6-difluoro benzyl

bromide (154 mg, 0.74 mmol) and the resulting mixture was

stirred at rt for 16 h. The precipitate was filtered and washed to

give the title compound (16) as a pale yellow solid (132 mg, 54

%); mp (Gallenkamp) 292 – 294 °C; ν_max(solid) 3065, 2968,

2802, 1626, 1594, 1578, 1470, 1343; HPLC (Method 1) >97 %,

tR = 11.28 min; δ_H(400 MHz, DMSO–d₆, 363 K) 4.66 (2H, s,

CH₂), 7.14 – 7.18 (2H, m, C(3’)H and C(5’)H), 7.42 – 7.46 (2H,

m, C(8)H and C(4’)H), 7.59 (1H, d, J = 7.9Hz, C(6)H), 7.71 (1H,

app t, J = 7.9 Hz, C(7)H), 8.33 (1H, d, J = 7.9 Hz, C(9)H), 12.69

(1H, br s, NH); δ_C(100 Hz, DMSO–d₆) 21.7, 111.8 (dd, J = 20, 5

Hz), 112.7, 112.8, 117.6, 121.6, 122.5, 130.2 (t, J = 11 Hz),

131.0, 140.4, 141.3, 146.6, 160.9 (dd, J = 248, 7.6 Hz), 166.0; δ_F

(470 MHz, DMSO–d₆) –113.1; m/z (ESI^–) 327 ([M–H]^–, 100%);

HRMS (ESI^–) C₁₆H₉F₂N₄S^–([M–H]^–) requires 327.0521; found

327.0536.

4.3.14. 3-((2’-Nitrobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (20)

4.3.11. 3-((2’-Chlorobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (17)

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol), and Et₃N (155 µL, 1.11

mmol) in methanol (5 mL) was added 2-nitrobenzyl bromide

(160 mg, 0.74 mmol) and the resulting mixture stirred at rt for 16

h. The resulting precipitate was filtered and washed to give the

title compound (20) as a pale yellow solid (246 mg, 98 %); mp

(Gallenkamp) 264 – 265 °C; ν_max(solid) 3066, 2965, 2793, 1602,

1524, 1336; C₁₆H₁₁N₅O₂S requires C, 56.96; H, 3.29; N, 20.76%;

found C, 56.75; H, 3.16; N, 20.55%; δ_H(400 MHz, DMSO–d₆,

363 K) 4.87 (2 H, s, CH₂), 7.40 – 7.43 (1 H, m, C(8)H), 7.55 (1H,

app t, J = 7.3 Hz, C(4’)H), 7.58 (1 H, d, J = 7.3 Hz, C(6)H), 7.66

– 7.72 (2H, m, C(7)H and C(5’)H), 7.91 (1H, dd, J = 7.3, 1.3 Hz,

C(6’)H), 8.05 (1H, d, J = 7.3 Hz, C(3’)H), 8.98 (1H, d, J = 7.3

Hz, C(9)H), 12.62 (1 H, bs, NH); δ_C(100 Hz, DMSO–d₆) 31.1,

112.4, 117.5, 121.5, 122.5, 124.9, 128.9, 131.0, 132.5, 133.1,

133.7, 140.4, 141.3, 146.5, 148.4, 166.0; m/z (ESI^–) 336 ([M–H]^–,

100%); HRMS (ESI⁺) C₁₆H₁₁NaN₅O₂S ([M+Na]⁺) requires

360.0526; found 360.0516.

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol) and Et₃N (155 µL, 1.11

mmol) in methanol (5 mL) was added 2-chloro benzyl bromide

(97 µL, 0.74 mmol) and the resulting mixture stirred at rt for 16

h. The precipitate was filtered and washed to give the title

compound (17) as a pale yellow solid (190 mg, 79 %); mp

(Gallenkamp) 266 – 267 °C; ν_max(solid) 3026, 2973, 2804, 1599,

1580, 1342, 1177; HPLC (Method 1) >99 %, tR = 11.44 min; δ_H

(400 MHz, DMSO–d₆, 363 K) 4.67 (2 H, s, CH₂), 7.32 – 7.36 (2

H, m, C(8)H and C(6’)H), 7.43 – 7.46 (1 H, m, C(7)H), 7.49 –

7.53 (1 H, m, C(4’)H), 7.59 (1 H, d, J = 8.2 Hz, C(6)H), 7.70 (1

H, d, J = 7.6 Hz, C(3’)H), 7.72 – 7.74 (1H, m, C(5’)H), 8.32 (1

H, d, J = 7.6 Hz, C(9)H), 12.69 (1 H, br s, NH); δ_C(100 MHz,

DMSO–d₆) 32.1, 112.7, 117.6, 121.5, 122.5, 127.4, 129.3, 129.5,

131.0, 131.5, 133.4, 134.9, 140.4, 141.3, 146.6, 166.3; m/z (ESI^–)

325 ([M–H]^–, 100%); HRMS (ESI⁺) C₁₆H₁₀ClNaN₄S

([³⁵ClM+Na]⁺)

requires

349.0285;

found

349.0286,

4.3.15. 3-((4’-Nitrobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (21)

C₁₆H₁₀ClNaN₄S ([³⁷ClM+Na]⁺) requires 351.0256; found

351.0262.

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol), and Et₃N (155 µL, 1.11

mmol) in methanol (15 mL) was added 4-nitrobenzyl bromide

(160 mg, 0.74 mmol) and the resulting mixture stirred at rt for 16

h. The resulting precipitate was filtered and washed to give the

title compound (21) as a light yellow solid (97 mg, 62 %); mp

(EZ Melt) 235 – 237 °C; ν_max(solid) 3455, 3336, 1680, 1584,

1466, 1438; HPLC (Method 2) >99 %, tR = 11.09 min; δ_H(500

MHz, DMSO–d₆, 363 K) 4.69 (2H, s, CH₂), 7.43 (1H, app t, J =

7.6 Hz, C(8)H), 7.58 (1H, d, J = 7.6 Hz, C(6)H), 7.70 (1H, app

td, J = 7.6, 1.3 Hz, C(7)H), 7.81 (2H, d, J = 8.8 Hz, C(2’)H and

C(6’)H), 8.18 (2H, d, J = 8.8 Hz, C(3’)H and C(5’)H), 8.30 (1H,

d, J = 7.6 Hz, C(9)H), 12.66 (1H, s, NH); δ_C(125 MHz, DMSO–

d₆) 33.6, 113.1, 117.9, 121.9, 122.9, 123.8, 130.6, 131.4, 140.7,

141.7, 146.7, 146.9, 146.9, 166.3; m/z (ESI^–) 336 ([M–H]^–,

100%); HRMS (ESI⁺) C₁₆H₁₁NaN₅O₂S ([M+Na]⁺) requires

360.0526; found 360.0516.

4.3.12. 3-((3’-Chlorobenzyl)thio)-5H-[1,2,4]triazino[5,6-

b]indole (18)

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol) and Et₃N (155 µL, 1.11

mmol) in methanol (5 mL) was added 3-chloro benzyl bromide

(97 µL, 0.74 mmol) and the resulting mixture stirred at rt for 16

h. The precipitate was filtered and washed to give the title

compound (18) as a very pale yellow solid (175 mg, 73 %); mp

(Gallenkamp) 266–267 °C; ν_max(solid) 3059, 2799, 1597, 1574,

1461, 1336; HPLC (Method 2) >99 %, tR = 11.54 min; δ_H(400

MHz, DMSO–d₆, 363 K) 4.58 (2H, s, CH₂), 7.31 – 7.34 (1H, m,

C(6’)H), 7.36 (1H, t, J = 7.6 Hz, C(5’)H), 7.44 (1H, app t, J = 7.6

Hz, C(8)H), 7.51 (1H, app d, J = 7.6 Hz, C(4’)H), 7.57 – 7.62

(2H, m, C(6)H and C(1’)H), 7.70 (1H, app t, J = 7.6 Hz, C(7)H),

8.31 (1H, d, J = 7.6, Hz C(9)H), 12.67 (1H, br s, NH); δ_C(100

MHz, DMSO–d₆) 33.3, 112.7, 117.6, 121.5, 122.5, 127.1, 127.8,

128.8, 130.3, 131.0, 132.9, 140.3, 140.5, 141.2, 146.6, 166.3; m/z

(ESI^–) 325 ([M–H]^–, 100%); HRMS (ESI⁺) C₁₆H₁₀³⁵ClN₄S ([M–

H]^–) requires 325.0320; found 325.0330, C₁₆H₁₀³⁷ClN₄S ([M–H]^–

) requires 327.0291; found 327.0306.

4.3.16. 4.3.17. 3-((2’-(Trifluoromethyl)benzyl)thio)-5H-

[1,2,4]triazino[5,6-b]indole (22)

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol) and Et₃N (155 µL, 1.11

mmol) in methanol (5 mL) was added 2-trifluoromethyl benzyl

bromide (176 mg, 0.74 mmol) and the resulting mixture was

stirred at rt for 16 h. The precipitate was filtered and washed to

give the title compound (22) as a pale yellow solid (196 mg, 74

%); mp (Gallenkamp) 269 – 270 °C; ν_max(solid) 3061, 2970,

2804, 1739, 1600, 1581, 1299; HPLC (Method 1) >99 %, tR =

11.71 min; δ_H(400 MHz, DMSO–d₆, 363 K) 4.76 (2 H, s, CH₂),

7.41 – 7.47 (1 H, m, C(8)H)), 7.49 – 7.55 (1 H, m, C(7)H), 7.59

(1 H, d, J = 8.2 Hz, C(6’)H), 7.63 – 7.72 (2 H, m, C(5’) and

C(4’)H), 7.78 (1 H, d, J = 7.9 Hz, C(3’)H), 7.87 (1 H, d, J = 7.9

Hz, C(6)H), 8.32 (1 H, d, J = 7.9 Hz, C(9)H), 12.68 (1 H, br s,

NH); δ_C(100 Hz, DMSO–d₆) 32.5, 112.7, 117.5, 121.6, 122.5,

123.0, 128.2, 130.4, 131.0, 132.1 (q, J = 284 Hz), 133.1, 133.3,

140.4, 141.4, 146.5, 149.1, 165.7; δ_F(470 MHz, DMSO–d₆) –

58.1; m/z (ESI^–) 359 ([M–H]^–, 100%); HRMS (ESI^–)

C₁₇H₁₀F₃N₄S^–([M–H]^–) requires 359.0584; found 359.0595.

NaOH (1M, 270 µL, 0.27 mmol) in H₂O (5 mL) and stirred at rt

for 16 h. The solution was acidified with aq. HCl (1M) until a

precipitate was formed, which was filtered and dried in vacuo to

yield the final product (25) (92 mg, 99%); mp (Gallenkamp)

>300°C; ν_max(solid) 3241 (v br), 1712, 1553, 1378, 1166, 1090;

HPLC (Method 2) >99 %, tR = 10.11 min; δ_H(500 MHz,

DMSO–d₆, 363 K) 4.99 (2 H, s, S-CH₂), 7.12 – 7.22 (3 H, m,

C(7)H, C(8)H and C(4’)H), 7.47 – 7.52 (3 H, m, C(6)H, C(5’)H

and C(6’)H), 7.68 – 7.72 (1 H, m, C(3’)H), 8.15 (1 H, d, J = 7.6

Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 32.1, 114.9, 119.1, 119.7,

120.7, 126.0, 127.4, 129.2, 129.5, 129.6, 130.0, 137.2, 141.1,

142.5, 150.4, 166.4, 171.6; m/z (ESI^–) 335 ([M–H]^–, 100%);

HRMS (ESI^–) C₁₇H₁₁N₄O₂S^–([M–H]^–) requires 335.0608; found

335.0612.

4.3.20. 2-(((5H-[1,2,4]Triazino[5,6-b]indol-3-

yl)sulfinyl)methyl)benzonitrile (26)

2’-(((5H-[1,2,4]Triazino[5,6-b]indol-3-yl)thio)methyl)benzo

nitrile 7 (50 mg, 0.16 mmol) was added to a solution of

trifluoroacetic acid (5 mL) and peroxytrifluoroacetic acid (4 M,

40 uL, 0.16 mmol) and stirred for 16 h at rt. The resulting

solution was concentrated in vacuo to yield the sulfoxide (26) as

a yellow solid (50 mg, 95 %); mp (EZ Melt) 237 – 239 °C; ν_max

(solid) 3244, 2228, 1621, 1591, 1570, 1416, 1183; HPLC

(Method 2) >96 %, rt = 9.22 min; δ_H(500 MHz, DMSO–d₆, 363

K) 4.64 (1H, d, J = 13.2, S-CH₂), 4.81 (1H, d, J = 13.2 Hz, S-

CH₂), 7.43 (1H, d, J = 7.6 Hz, C(6)H), 7.52 (1H, app td, J = 7.6,

1.3 Hz, C(8)H), 7.55 (1H, app t, J = 7.6 Hz, C(5’)H), 7.65 (1H,

app td, J = 7.6, 1.3 Hz, C(7)H), 7.72 (1H, d, J = 7.6 Hz, C(3’)H),

7.80 (1H, dd, J = 7.6, 1.0 Hz, C(6’)H), 7.84 (1H, app t, J = 7.6,

1.3 Hz, C(4’)H), 8.48 (1H, d, J = 7.6 Hz, C(9)H), 13.19 (1H, br s,

NH); δ_C(125 MHz, DMSO–d₆) 57.7, 112.9, 113.2, 117.0, 117.2,

122.6, 123.1, 129.0, 132.1, 132.5, 133.1, 133.1, 133.2, 141.7,

144.5, 147.1, 166.5; m/z (ESI^–) 332 ([M–H]^–, 100%); HRMS

(ESI⁺) C₁₇H₁₁N₅NaOS ([M+Na]⁺) requires 356.0577; found

356.0571.

4.3.17. 3-((2’-(Trifluoromethoxy)benzyl)thio)-5H-

[1,2,4]triazino[5,6-b]indole (23)

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (150 mg, 0.74 mmol) and Et₃N (155 µL, 1.11

mmol)

in

methanol

(5

mL)

was

added

2-

(trifluoromethoxy)benzyl bromide (119 µL, 0.74 mmol) and the

resulting mixture stirred was stirred at rt for 16 h. The precipitate

was washed to give the title compound (23) as a pale yellow solid

(139 mg, 50 %); mp (Gallenkamp) 285 – 288 °C; ν_max(solid)

3062, 2969, 2805, 1607, 1461, 1423; HPLC (Method 1) >99 %,

tR = 11.70 min; δ_H(400 MHz, DMSO–d₆, 363 K) 4.64 (2 H, s,

CH₂), 7.36 – 7.39 (1H, m, C(4’)H), 7.39 – 7.47 (3 H, m, C(8)H,

C(5’)H and C(6’)H), 7.60 (1 H, d, J = 7.9 Hz, C(6)H), 7.71 (1 H,

app t, J = 7.9 Hz, C(7)H), 7.76 (1 H, dd, J = 7.6, 1.6 Hz, C(3’)H),

8.32 (1 H, d, J = 7.9 Hz, C(9)H), 12.67 (1 H, br s, NH); δ_C(100

MHz, DMSO–d₆) 28.4, 112.7, 117.6, 120.2 (q, J = 257 Hz),

120.4, 121.5, 122.5, 127.5, 129.5, 129.9, 131.0, 131.7, 140.4,

141.3, 146.6, 146.9, 166.1; δ_F(470 MHz, DMSO–d₆) –55.9; m/z

(ESI^–) 375 ([M–H]^–, 100%); HRMS (ESI⁺) C₁₇H₁₁F₃N₄NaOS

([M+Na]⁺) requires 399.0498; found 399.0499.

4.3.21. 6-Bromo-5H-[1,2,4]triazino[5,6-b]indole-3-thiol (31)

7-Bromoisatin 27 (1.00 g, 4.45 mmol), thiosemicarbizide (405

mg, 4.45 mmol) and K₂CO₃(920 mg, 6.67 mmol) were

suspended in water (50 mL). The reaction mixture was stirred at

reflux for 16 h over which time the dark brown suspension

became a clear light brown solution. The solution was carefully

acidified by dropwise addition of acetic acid and the resulting

precipitate was filtered. The precipitate was recrystallised from

DMF to yield the title compound (31) as a red solid (416 mg, 34

%); mp (Gallenkamp) >300 °C; ν_max(solid) 2849 (br), 1629,

1603, 1581, 1386, 1151; δ_H(500 MHz, DMSO–d₆, 363 K) 7.28

(1H, app t, J = 7.6 Hz, C(8)H), 7.84 (1H, d J = 7.6 Hz, C(7)H),

8.01 (1H, d, J = 7.6 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 116.1,

117.5, 123.8, 124.9, 126.3, 135.5, 144.6, 149.9, 176.6; m/z (ESI^–)

279 ([⁷⁹BrM–H]^–, 100%), 281 ([⁸¹BrM–H]^–, 100%); HRMS

(ESI^–) C₉H₅BrN₄NaS ([⁷⁹BrM–H]^–) requires: 278.9335; found

278.9344, ([⁸¹BrM–H]^–) requires 280.9314; found 280.9321.

4.3.18. Ethyl 2’-(((5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzoate (24)

Following General Procedure 1, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 9 (200 mg, 0.99 mmol) and Et₃N (303 µL, 2.18

mmol) in methanol (10 mL) was added ethyl 2-

(bromomethyl)benzoate (361 mg, 1.53 mmol) and the resulting

mixture stirred at rt for 16 h. The resulting precipitate was

washed to give the title compound (24) as a pale yellow solid

(436 mg, 83 %); mp (Gallenkamp) 205 – 206 °C; ν_max(solid)

3151, 2989, 1707, 1597, 1250, 1079; HPLC (Method 2) >95 %,

tR = 11.36 min; δ_H(400 MHz, DMSO–d₆, 363 K) 1.31 (3 H, t, J

= 7.0 Hz, CH₃), 4.34 (2 H, q, J = 7.0 Hz, O-CH₂-CH₃), 4.88 (2 H,

s, S-CH₂), 7.37 – 7.45 (2 H, m, C(8)H and C(5’)H), 7.53 (1 H,

app t, J = 7.7 Hz, C(7)H), 7.57 (1 H, d, J = 7.7 Hz, C(6)H), 7.63

– 7.70 (1 H, m, C(4’)H), 7.75 (1 H, d, J = 7.7 Hz, C(6’)H), 7.88

(1 H, dd, J = 7.8, 1.3 Hz, C(3’)H), 8.29 (1 H, d, J = 7.8 Hz,

C(9)H), 12.62 (1 H, s, NH); δ_C(100 MHz, DMSO–d₆) 14.9, 33.4,

61.8, 113.6, 118.5, 122.3, 123.3, 128.5, 130.3, 131.4, 131.7,

132.4, 133.1, 140.0, 141.2, 142.0, 147.4, 167.5, 167.7; m/z (ESI^–)

363 ([M–H]^–, 100%); HRMS (ESI^–) C₁₉H₁₅N₄O₂S^–([M–H]^–)

requires 363.0921; found 363.0927.

4.3.22. 7-Bromo-5H-[1,2,4]triazino[5,6-b]indole-3-thiol (32)

6-Bromoisatin 28 (1.00 g, 4.45 mmol), thiosemicarbizide (405

mg, 4.45 mmol) and K₂CO₃(920 mg, 6.67 mmol) were

suspended in water (50 mL). The reaction mixture was stirred at

reflux for 16 h over which time the dark brown suspension

became a clear light brown solution. The solution was carefully

acidified by dropwise addition of acetic acid and resulting

precipitate was filtered. The precipitate was recrystallised from

DMF to yield the title compound (32) as a red solid (1.12 g, 90

%); mp (Gallenkamp) >300 °C; ν_max(solid) 2919 (br), 1589,

1422, 1359, 1161; δ_H(500 MHz, DMSO–d₆, 363 K) 7.50 (1H, d,

4.3.19. 2’(((5H-[1,2,4]Triazino[5,6-b]indol-3-

yl)thio)methyl)benzoic acid (25)

Ethyl 2’-(((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)

benzoate 24 (100 mg, 0.27 mmol), was added to a solution of aq.

J = 7.5 Hz, C(8)H), 7.60 (1H, s, C(6)H), 7.94 (1H, 1H, d, J = 7.5

Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 105.5, 120.3, 121.3,

124.8, 134.6, 136.1, 142.4, 150.2, 179.8; m/z (ESI^–) 279 ([⁷⁹BrM–

H]^–, 100%), 281 ([⁸¹BrM–H]^–, 100%); HRMS (ESI^–)

C₉H₅BrN₄NaS ([⁷⁹BrM–H]^–) requires: 278.9335; found 278.9343,

([⁸¹BrM–H]^–) requires 280.9314; found 280.9321.

4.3.26. 2–(((7-Bromo-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (36)

Following General Procedure 1, to 8-bromo-5H-

[1,2,4]triazino[5,6-b]indole-3-thiol 32 (100 mg, 0.36 mmol) and

Et₃N (75 µL, 0.54 mmol) in methanol (5 mL), 2-cyanobenzyl

bromide (70 mg, 0.36 mmol) were stirred overnight at rt for 16 h.

The resulting precipitate was filtered and washed to give the title

compound (36) as a light yellow solid (130 mg, 92 %); mp

(Gallenkamp) 289 – 291 °C; ν_max(solid) 3076, 3012, 2701, 2228,

1608, 1420; δ_H(400 MHz, DMSO–d₆) 4.75 (2 H, s, CH₂), 7.48 (1

H, app t, J = 7.9 Hz, C(5’)H), 7.60 (1 H, dd, J = 8.2, 1.6 Hz,

C(8)H), 7.68 (1 H, app t, J = 7.9 Hz, C(4’)H), 7.77 (1 H, d, J =

1.6 Hz, C(6)H), 7.84 (1 H, d, J = 7.9 Hz, C(3’)H), 7.88 (1H, dd,

J = 7.9, 1.3 Hz, C(6’)H), 8.26 (1 H, d, J = 8.2 Hz, C(9)H), 12.81

(1 H, br s, NH); δ_C(100 MHz, DMSO–d₆) 32.5, 112.0, 115.5,

116.9, 117.5, 123.2, 123.7, 125.6, 128.3, 130.4, 133.1, 133.3,

140.9, 141.1, 141.3, 146.9, 166.2; m/z (ESI^–) 394 ([⁷⁹BrM–H]^–,

100%), 396 ([⁸¹BrM–H]^–, 100%); HRMS (ESI⁺) C₁₇H₁₀BrN₅NaS

4.3.23. 8-Bromo-5H-[1,2,4]triazino[5,6-b]indole-3-thiol (33)

5-Bromoisatin 29 (1.00 g, 4.45 mmol), thiosemicarbizide (405

mg, 4.45 mmol) and K₂CO₃(920 mg, 6.67 mmol) were

suspended in water (50 mL). The reaction mixture was stirred at

reflux for 16 h over which time the dark brown suspension

became a clear light brown solution. The solution was carefully

acidified by dropwise addition of acetic acid and resulting

precipitate was filtered. The precipitate was recrystallised from

DMF to yield the title compound (33) as a red solid (1.23 g, 97

%); mp (Gallenkamp) >300 °C; ν_max(solid) 3097, 2858, 1603,

1589, 1446, 1311; δ_H(500 MHz, DMSO–d₆, 363 K) 1.90 (1H, br

s, NH), 7.38 (1H, d, J = 8.6 Hz, C(6)H), 7.74 (1H, dd, J = 8.6, 1.8

Hz, C(7)H), 8.15, (1H, d, J = 1.8 Hz, C(9)H); δ_C(125 MHz,

DMSO–d₆) 115.6, 115.9, 120.8, 125.0, 134.9, 135.8, 143.5,

150.4, 180.1; m/z (ESI^–) 279 ([⁷⁹BrM–H]^–, 100%), 281 ([⁸¹BrM–

H]^–, 100%); HRMS (ESI^–) C₉H₅BrN₄NaS ([⁷⁹BrM–H]^–)

278.9335; found 278.9343, ([⁸¹BrM–H]^–) requires 280.9314;

found 280.9320.

([⁷⁹BrM+Na]⁺)

([⁸¹BrM+Na]⁺) requires 419.9712; found 419.9718.

requires

417.9732;

found

417.9738,

4.3.27. 2-(((8-Bromo-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (37)

Following General Procedure 1, to 8-bromo-5H-

[1,2,4]triazino[5,6-b]indole-3-thiol 33 (100 mg, 0.36 mmol),

Et₃N (75 µL, 0.54 mmol) in methanol (5 mL) was added 2-

cyanobenzyl bromide (70 mg, 0.36 mmol) and were stirred at rt

for 16 h. The resulting precipitate was washed to give the title

compound (37) as a light yellow solid (110 mg, 78 %); mp

(Gallenkamp) 286 – 288 °C; ν_max(solid) 3091, 2967, 2796, 2225,

1591; HPLC (Method 2) >96 %, tR = 11.72 min; δ_H(400 MHz,

DMSO–d₆) 4.73 (2H, s, CH₂), 7.47 (1H, t, J = 7.6 Hz, C(4’)H),

7.53 (1H, d, J = 8.6 Hz, C(6)H), 7.66 (1H, t, J = 7.6 Hz, C(5’)H),

7.78 – 7.88 (3H, m, C(7)H, C(3’)H and C(6’)H), 8.43 (1H, s,

C(9)H), 12.80 (1H, br s, NH); δ_C(100 MHz, DMSO–d₆) 33.4,

112.9, 115.4, 115.7, 118.3, 120.4, 124.7, 129.11, 131.3, 134.0,

134.2, 140.0, 142.0, 147.5, 153.7, 155.0, 167.4; m/z (ESI^–) 394

([⁷⁹BrM–H]^–, 100%), 396 ([⁸¹BrM–H]^–, 100%); HRMS (ESI⁺)

C₁₇H₁₀BrN₅NaS ([⁷⁹BrM+Na]⁺) requires 417.9732; found

417.9740, ([⁸¹BrM+Na]⁺) requires 419.9713; found 419.9796.

4.3.24. 9-Bromo-5H-[1,2,4]triazino[5,6-b]indole-3-thiol (34)

4-Bromoisatin 30 (1.00 g, 4.45 mmol), thiosemicarbizide (405

mg, 4.45 mmol) and K₂CO₃(920 mg, 6.67 mmol) were

suspended in water (50 mL). The reaction mixture was stirred at

reflux for 16 h over which time the dark brown suspension

became a clear light brown solution. The solution was carefully

acidified by dropwise addition of acetic acid and resulting

precipitate was filtered. The precipitate was recrystallised from

DMF to yield the title compound (34) as a red solid (1.18 g, 95

%); mp (Gallenkamp) >300 °C; ν_max(solid) 3389 (br), 3297,

1604, 1573, 1140; δ_H(500 MHz, DMSO–d₆, 363 K) 7.43– 7.52

(3H, m, C(6)H, C(7)H), and C(8)H), 14.68 (1H, br s, NH); δ_C

(125 MHz, DMSO–d₆) 113.0, 117.0, 118.3, 127.4, 133.5, 136.1,

145.3, 149.9, 180.0; m/z (ESI^–) 279 ([⁷⁹BrM–H]^–, 100%), 281

([⁸¹BrM–H]^–, 100%); HRMS (ESI^–) C₉H₅BrN₄S ([⁷⁹BrM–H]^–)

requires: 278.9335; found 278.9343, ([⁸¹BrM–H]^–) requires

280.9314; found 280.9320.

4.3.28. 2’-(((9-Bromo-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (38)

4.3.25. 2’-(((6-Bromo-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (35)

Following

General

Procedure

1,

9-bromo-5H-

[1,2,4]triazino[5,6-b]indol-3-thiol 34 (100 mg, 0.36 mmol) and

Et₃N (75 µL, 0.54 mmol) in methanol (5 mL), 2-cyanobenzyl

bromide (70 mg, 0.36 mmol) was added at rt and stirred for 16 h.

The resulting precipitate was filtered and washed to give the title

compound (38) as a light yellow solid (91 mg, 64 %); mp

(Gallenkamp) 283 – 284 °C; ν_max(solid) 3269 (br), 3076, 3039,

2232, 1583, 1304, 1168; HPLC (Method 2) >99 %, tR = 11.42

min; δ_H(400 MHz, DMSO–d₆, 363 K) 4.76 (2 H, s, S–CH₂), 7.48

(1 H, app t, J = 7.8 Hz, C(5’)H), 7.55 – 7.64 (3 H, m, C(6)H,

C(7)H, C(9)H), 7.67 (1 H, app t, J = 7.8 Hz, C(4’)H), 7.83 (1 H,

d, J = 7.8 Hz, C(3’)H), 7.88 (1 H, dd, J = 7.8, 1.0 Hz, C(6’)H),

12.89 (1 H, br s, NH); δ_C(100 MHz, DMSO–d₆) 33.4, 112.8,

112.9, 116.9, 117.9, 118.3, 127.0, 129.1, 131.2, 132.5, 134.0,

134.2, 141.9, 142.5, 147.3, 149.1, 167.0; m/z (ESI^–) 394 ([⁷⁹BrM–

H]^–, 100%), 396 ([⁸¹BrM–H]^–, 100%); HRMS (ESI⁺)

C₁₇H₁₀BrN₅NaS ([⁷⁹BrM+Na]⁺) requires 417.9732; found

417.9740, ([⁸¹BrM+Na]⁺) requires 419.9713; found 419.9783.

Following General Procedure 1, to 6-bromo-5H-

[1,2,4]triazino[5,6-b]indol-3-thiol 31 (100 mg, 0.36 mmol) and

Et₃N (75 µL, 0.54 mmol) in methanol (5 mL) was added 2-

cyanobenzyl bromide (70 mg, 0.36 mmol) and the resulting

mixture stirred at rt for 16 h. The resulting precipitate was

filtered and washed to give the title compound (35) as a light

yellow solid (128 mg, 87 %); mp (Gallenkamp) 226 – 228 °C;

ν_max(solid) 3065, 2780, 2228, 1600, 1401, 1436; HPLC (Method

2) >98 %, tR = 11.47 min; δ_H(500 MHz, DMSO–d₆, 363 K) 4.74

(2H, s, S–CH₂), 7.34 (1H, app t, J = 7.8 Hz, C(8)H), 7.47 (1H,

app t, J = 8.1 Hz, C(4’)H), 7.66 (1H, app t, J = 7.8 Hz, C(5’)H),

7.84 – 7.89 (3H, m, C(7)H, C(3’)H and C(6’)H), 8.29 (1H, d, J =

7.6 Hz, C(9)H), 12.94 (1H, br s, NH); δ_C(125 MHz, DMSO–d₆)

33.45, 106.0, 112.8, 118.31, 120.6, 121.5, 124.7, 129.1, 131.4,

134.0, 134.2, 134.2, 139.9, 141.9, 142.2, 148.0, 167.4; m/z (ESI^–)

394 ([⁷⁹BrM–H]^–, 100%), 396 ([⁸¹BrM–H]^–, 100%); HRMS

(ESI⁺) C₁₇H₁₀BrN₅NaS ([⁷⁹BrM+Na]⁺) requires 417.9732; found

417.9733, ([⁸¹BrM+Na]⁺) requires 419.9713; found 419.9717.

4.3.29. 9-Bromo-3-((2’-nitrobenzyl)thio)-5H-

[1,2,4]triazino[5,6-b]indole (39)

Following

General

Procedure

1,

9-bromo-5H-

methyphenyltrifluoroborate (123 mg, 0.62 mmol) and K₃PO₄

(338 mg, 1.59 mmol) followed by Pd(PPh₃)₂Cl₂(31 mg, 0.04

mmol). The reaction vessel was sealed and heated by microwave

irradiation for 4 h at 130 °C. The reaction mixture was cooled to

rt, diluted with EtOAc (5 mL) and filtered through Celite^®. The

organic solution was washed with brine (5 mL) and the resulting

aqueous layer was further extracted with EtOAc (2 x). The

combined organic layers were dried (MgSO₄), filtered and

concentrated in vacuo to give the crude product. The product was

purified via flash column chromatography (eluent 30–40 °C

petrol/acetone, 4:1) to afford the title compound (42) as an

orange solid (72 mg, 68 %); mp 178 – 179 °C; ν_max(solid) 3274,

3051, 2906, 1741, 1609, 1486; δ_H(400 MHz, CDCl₃) 2.43 (3H, s,

CH₃), 6.88 (1H, d, J = 8.0 Hz, C(7)H), 7.08 (1H, d, J = 8.0 Hz,

C(5)H), 7.28 (2H, d, J = 8.5 Hz, C(3’)H and C(5’)H), 7.45 (2H,

d, J = 8.5 Hz, C(6’)H and C(2’)H), 7.55 (1H, app t, J = 8.0 Hz,

C(6)H), 8.70 (1H, br s, NH); δ_C(125 MHz, CDCl₃) 21.4, 110.8,

114.4, 125.9, 128.8, 129.0, 133.1, 138.0, 139.4, 143.8, 154.2,

159.2, 178.3; m/z (ESI¯) 236 ([M–H]¯, 100%); HRMS (ESI^–)

C₁₅H₁₀NO₂S¯ ([M–H]¯) requires 236.0717; found 236.0716.

[1,2,4]triazino[5,6-b]indol-3-thiol 34 (100 mg, 0.36 mmol) and

Et₃N (75 µL, 0.54 mmol) in methanol (5 mL), 2-nitrobenzyl

bromide (70 mg, 0.36 mmol) was added at rt and stirred for 16 h.

The resulting precipitate was filtered and washed to give the title

compound (39) as a light yellow solid (98 mg, 66 %); mp

(Gallenkamp) 285 – 286 °C; ν_max(solid) 3111, 2877, 2832, 1606,

1521, 1338; C₁₆H₁₀BrN₅O₂S requires C, 46.17; H, 2.42; N,

16.82%; found C, 46.12; H, 2.29; N, 16.63%; δ_H(400 MHz,

DMSO–d₆, 363 K) 4.90 (2 H, s, S–CH₂), 7.42 – 7.62 (4 H, m,

C(6)H, C(7)H, C(8)H and C(4’)H), 7.69 (1 H, app t, J = 7.8 Hz,

C(5’)H), 7.93 (1 H, dd, J = 7.8, 1.3 Hz, C(6’)H), 8.06 (1 H, dd, J

= 7.8, 1.3 Hz, C(3’)H); δ_C(100 MHz, DMSO–d₆) 32.0, 112.6,

116.9, 118.1, 125.8, 126.7, 128.5, 129.7, 132.3, 133.3, 134.6,

141.2, 142.4, 147.2, 149.2, 167.1; m/z (ESI^–) 414 ([⁷⁹BrM–H]^–,

100%),

416

([⁸¹BrM–H]^–,

100%);

HRMS

(ESI^–)

C₁₆H₁₀BrN₅NaO₂S ([⁷⁹BrM–H]^–) requires 413.9666; found

413.9680, ([⁸¹BrM–H]^–) requires 415.9646; found 415.9658.

4.3.30. 4-Bromo-1-(4-methoxybenzyl)indoline-2,3-dione

(40)⁷²

4.3.33. 4–(3’-Thienyl)indoline-2,3-dione (43)

4-Bromoisatin 30 (2.00 g, 8.85 mmol) was added to a suspension

of NaH (60 % dispersion in mineral oil, 550 mg, 8.85 mmol) in

DMF (20 mL) at 0 °C. The resulting purple solution was stirred

at 0 °C for 10 min before dropwise addition of p-methoxybenzyl

chloride (1.12 mL, 8.23 mmol). The reaction mixture was stirred

for 30 min at 0 °C, heated to 40 °C for 3 h, cooled down and

quenched with water. The solution was extracted with EtOAc (3

x 30 mL), the combined organic phases were washed with brine,

dried (Na₂SO₄), filtered and concentrated in vacuo to give the

crude compound as an orange solid. Recrystallisation in

EtOAc/petrol afforded the isatin 40 as an orange powder (2.10 g,

6.09); δ_H(CDCl₃, 400 MHz) 3.80 (3H, s, O-CH₃), 4.88 (2H,s, N-

CH₂), 6.76 (1H, d, J = 7.8 Hz, C(7)H), 6.88 (d, J = 8.3 Hz, 2H,

phenyl ArH), 7.22 (d, J = 8.1 Hz, 1H, C(5)H), 7.26 (d, J = 8.3

Hz, 2H, phenyl ArH), 7.30 (t, J = 8.1 Hz, 1H, C(6)H); LRMS m/z

(ESI⁺) 347 [M+H]⁺, 715 [2M+Na]⁺.

To a solution of 4-bromoisatin 30 (200 mg, 0.88 mmol) in

THF/water (3:1,

3

mL) was added potassium 3-

thienyltrifluoroborate (235 mg, 1.24 mmol) and K₃PO₄(675 mg,

3.19 mmol) followed by Pd(PPh₃)₂Cl₂(62 mg, 0.09 mmol). The

reaction vessel was sealed and heated by microwave irradiation

for 4 h at 130 °C. The reaction mixture was cooled to rt, diluted

with EtOAc (10 mL) and filtered through Celite^®. The organic

solution was washed with brine (10 mL) and the resulting

aqueous layer was further extracted with EtOAc (2 x). The

combined organic layers were dried (MgSO₄), filtered and

concentrated in vacuo to give the crude product. The product was

purified via flash column chromatography (eluent 30–40 °C

petrol/acetone, 4:1) to afford the title compound (43) as an

orange solid (120 mg, 59%); mp 213 – 216 °C; ν_max(solid) 3098,

2927, 1727, 1584; δ_H(500 MHz, DMSO–d₆) 6.85 (1H, dd, J =

7.9, 0.6 Hz, C(7)H), 7.20 (1H, dd, J = 7.9, 0.6 Hz, C(5)H), 7.50

(1H, dd, J = 4.9, 1.3 Hz, C(4’)H), 7.58 (1H, app t, J = 7.9 Hz,

C(6)H), 7.62 (1H, dd, J = 4.9, 2.9 Hz, C(5’)H), 8.07 (1H, dd, J =

2.9, 1.3 Hz, C(2’)H), 11.13 (1H, br s, NH); δ_C(125 MHz,

DMSO–d₆) 110.8, 113.9, 123.8, 125.6, 126.3, 128.4, 135.8,

137.2, 138.0, 151.6, 158.9, 183.0; m/z (ESI¯) 228 ([M–H]¯,

100%); HRMS (ESI¯) C₁₂H₆N₄O₂S¯ ([M–H]¯) requires 228.0125;

found 228.0127.

4.3.31. 4-(2’-Methylphenyl) indoline-2,3-dione (41)

To a solution of 4-bromoisatin 30 (100 mg, 0.44 mmol) in

degassed THF/water (3:1, 2.5 mL) was added potassium 2-

methylphenyltrifluoroborate (114 mg, 0.62 mmol) and K₃PO₄

(338 mg, 1.59 mmol) followed by Pd(PPh₃)₂Cl₂(31 mg, 0.04

mmol). The reaction vessel was sealed and heated by microwave

irradiation for 4 h at 130 °C. The reaction mixture was cooled to

rt, diluted with EtOAc (5 mL) and filtered through Celite^®. The

organic solution was washed with brine (5 mL) and the resulting

aqueous layer was further extracted with EtOAc (2 x). The

combined organic layers were dried (MgSO₄), filtered and

concentrated in vacuo to give the crude product. The product was

purified via flash column chromatography (eluent 40–60 °C,

petrol/EtOAc, 4:1) to afford the title compound (41) as an orange

solid (40 mg, 42 %); mp 161 – 162 °C; ν_max(solid) 3249, 3017,

2926, 1727, 1603, 1480; δ_H(500 MHz, DMSO–d₆) 2.15 (3H, s,

CH₃), 6.93 (2H, m, C(5)H and C(7)H), 7.14 (1H, d, J = 7.6 Hz,

C(6’)H), 7.20 – 7.26 (1H, m, C(4’)H), 7.30 (1H, d, J = 7.3 Hz,

C(3’)H), 7.32 – 7.37 (1H, m, C(5’)H), 7.56 (1H, app t, J = 7.9 Hz

C(6)H), 8.84 (1H, br s, NH); δ_C(125 MHz, DMSO–d₆) 19.7,

111.2, 115.7, 125.6, 126.3, 128.6, 128.8, 130.1, 135.7, 136.4,

137.9, 142.8, 149.3, 159.2, 182.0; m/z (ESI¯) 236 ([M–H]¯, 60%);

HRMS (ESI¯) C₁₅H₁₀NO₂¯ ([M–H]¯) requires 236.0717; found

236.0719.

4.3.34. 4–Cyclopropyl-1-(4-methoxybenzyl)indoline-2,3-

dione (44)

Bromoisatin 30 (683 mg, 1.98 mmol), potassium

cycloproplytrifluoroborate (410 mg, 2.77 mmol), K₃PO₄(1.51 g,

7.13 mmol) were added sequentially to a microwave vial before

addition of degassed THF/H₂O (3:1, 9 mL). The reaction mixture

was further degassed before addition of Pd(dppf)Cl₂(141 mg,

0.193 mmol). The reaction vessel was sealed and heated by

microwave irradiation for 4 h at 130 °C. The reaction mixture

was cooled to room temperature, filtered through Celite^®, using

EtOAc as an eluent. The solution was washed with brine (20

mL), dried (Na₂SO₄), filtered and concentrated in vacuo to give

the crude compound as a black green oil. Purification on silica

gel (CH₂Cl₂) afforded the title compound (44) as an orange solid

(586 mg, 96%). mp 175 – 178 °C; ν_max(solid) 1727, 1608, 1590,

1514, 1454, 1247, 1182, 1030; δ_H(500 MHz, acetone–d₆) 0.85

(2H, ddd, J = 9.0, 6.6, 4.4 Hz, C(2’)H or C(3’)H), 1.14 (2H, ddd,

J = 8.5, 6.8, 4.4 Hz, C(2’)H or C(3’)H), 2.96 (1H, app tt, J = 8.5,

5.0 Hz, C(1’)H), 3.76 (3H, s, O-CH₃), 4.88 (2H, s, N-CH₂), 6.55

(1H, d, J = 8.0 Hz, C(7)H), 6.74 (1H, dd, J = 7.9, 0.8 Hz, C(5)H),

4.3.32. 4-(4’-Methylphenyl)indoline-2,3-dione (42)

To a solution of 4-bromoisatin 30 (100 mg, 0.44 mmol) in

degassed THF/H₂O (3:1, 3 mL) was added potassium 4-

6.90 (2H, d, J = 8.7 Hz, ArH), 7.38 (3H, m, ArH and C(6)H); δ_C

(125 MHz, , acetone–d₆) 11.4, 43.6, 55.6, 108.4, 115.0, 116.7,

118.4, 128.6, 129.8, 138.7, 149.0, 151.8, 159.0, 160.4, 185.0;

resulting solution heated to 90 °C in a sealed tube for overnight.

After being cooled, the reaction was treated carefully with

NaHCO₃(sat. aq.) and extracted with EtOAc (3 x 30 mL). The

combined organic phase was dried (Na₂SO₄), filtered and

concentrated in vacuo to give the crude compound as a dark

solid. Purification on silica gel (acetone/CH₂Cl₂, 1/50) afforded

the title compound 48 as a orange solid (136 mg, 67%) and

recovered starting material 44 (75 mg, 21%); mp 143 – 145 °C;

ν_max(solid) 3268, 2360, 1755, 1724, 1619, 1591, 1497; δ_H(500

MHz, acetone–d₆) 0.86 (2H, ddd, J = 6.4, 5.0, 4.4 Hz, C(2’)H or

C(3’)H), 1.13 (2H, ddd, J = 8.5, 6.6, 4.4 Hz, C(2’)H or C(3’)H),

2.97 (1H, app tt, J = 8.5, 5.0 Hz, C(1’)H), 6.54 (1H, d, J = 8.1

Hz, C(7)H), 6.73 (1H, dd, J = 7.7, 0.8 Hz, C(5)H), 7.43 (1H, t, J

= 8.1 Hz, C(6)H); δ_C(125 MHz, acetone–d₆) 11.4, 109.4, 117.2,

118.0, 139.1, 149.1, 151.4, 159.8, 185.5; LRMS m/z (ESI⁺) 188

+

LRMS m/z (ESI⁺) 308 ([M+H]⁺); HRMS (ESI⁺) C₁₉H₁₇NNaO₃

([M+Na]⁺) requires 330.1110; found 330.1110.

4.3.35. 9-(2’-Methylphenyl-5H-[1,2,4]triazino[5,6-b]indole-

3-thiol (45)

4-(2’-Methylphenyl)indoline-2,3-dione 41 (70 mg, 0.31

mmol), thiosemicarbizide (29 mg, 0.31 mmol) and K₂CO₃(65

mg, 0.47 mmol) were suspended in water (7 mL) and heated at

reflux overnight. The solution was acidified and the resulting

precipitate was filtered to yield the crude product which was

recrystallised from DMF to afford the title compound (45) as a

yellow solid (59 mg, 67 %); mp 297 – 299 °C; ν_max(solid) 3204,

3045, 2922, 2361, 1473; δ_H(500 MHz, DMSO–d₆) 2.26 (3H, s,

CH₃), 7.12 (1H, dd, J = 7.8, 0.6 Hz, C(6)H), 7.25 – 7.28 (2H, m,

C(2’)H and C(3’)H or C(4’)H), 7.31 – 7.34 (2H, m, C(3’) or

C(4’)H and C(5’)H), 7.45 (1H, dd, J = 7.8, 0.6 Hz, C(8)H), 7.68

(1H, app t, J = 7.8 Hz, C(7)H), 12.52 (1H, br s, NH), 14.27 (1H,

s, SH); δ_C(125 MHz, DMSO–d₆) 19.5, 111.7, 115.9, 124.5,

125.7, 128.1, 129.1, 130.0, 131.6, 135.4, 135.7, 137.9, 138.4,

143.1, 149.0, 178.7; m/z (ESI¯) 291 ([M–H]¯, 100%); HRMS

(ESI¯) C₁₆H₁₁N₄S¯ ([M–H]¯) requires 291.0710; found 291.0715.

+

([M+H]⁺); HRMS (ESI⁺) C₁₁H₉NNaO₂([M+Na]⁺) requires

210.0525; found 210.0524.

4.3.39. 9-Cyclopropyl-5H-[1,2,4]triazino[5,6-b]indole-3-

thiol (49)

To a stirred suspension of 4-cyclopropylindoline-2,3-dione 48

(135 mg, 0.722 mmol) and K₂CO₃(150 mg, 1.08 mmol) in water

(10 mL), was added thiosemicarbizide (72 mg, 0.794 mmol). The

mixture was heated under rapid reflux for 16 h. Upon cooling,

the solution was acidified (pH~6) with glacial acetic acid to

afford a precipitate, which was filtered and washed with water,

followed by cold acetone and Et₂O. The resulting solid was

triturated with DMF, filtered and dried to yield the title

compound (49) as a bright yellow solid (124 mg, 71 %); mp

>300 °C; ν_max(solid) 3012, 2882, 1611, 1584, 1306, 1157; δ_H

(500 MHz, DMSO–d₆) 0.90 (2H, ddd, J = 6.4, 4.9, 4.4 Hz,

C(2’)H or C(3’)H), 1.16 (2H, ddd, J = 8.3, 6.6, 4.4 Hz, C(2’)H or

C(3’)H), 2.77 (1H, app tt, J = 8.3, 5.0 Hz, C(1’)H), 6.77 (1H, d, J

= 8.0 Hz, C(7)H), 7.18 (1H, dd, J = 7.9, 0.8 Hz, C(5)H), 7.48

(1H, t, J = 8.0 Hz, C(6)H), 12.45 (1H, br s, NH), 14.57 (1H, br s,

SH); δ_C(125 MHz, , DMSO–d₆) 10.4, 12.4, 109.5, 116.2, 131.9,

136.8, 142.6, 142.9, 149.1, 178.5; LRMS m/z (ESI⁺) 243

([M+H]⁺); HRMS (ESI⁺) C₁₂H₁₀N₄SNa⁺([M+Na]⁺) requires

265.0518; found 265.0523.

4.3.36. 9-(4’-Methylphenyl)-5H-[1,2,4,]triazino[5,6-b]indole-

3-thiol (46)

4-(4-Methylphenylindoline-2,3-dione 42 (38 mg, 0.16 mmol),

thiosemicarbizide (15 mg, 0.16 mmol) and K₂CO₃(33 mg, 0.24

mmol) were suspended in water (4 mL) and stirred at reflux

overnight. The solution was acidified and resulting precipitate

was filtered to yield the crude product which was recrystallised

from DMF to afford the title compound (46) as a yellow solid (14

mg, 30 %); mp >300 °C; ν_max(solid) 3224, 3049, 2890, 2571,

1488; δ_H(500 MHz, DMSO–d₆) 2.39 (3H, s, CH₃) 7.25 (1H, d, J

= 7.9 Hz, C(6)H), 7.29 (2H, d, J = 7.9 Hz, C(3’)H and C(5’)H),

7.41 (1H, d, J = 8.2 Hz, C(8)H), 7.58 (2H, d, J = 8.2 Hz, C(2’)H

and C(6’)H), 7.67 (1H, app t, J = 7.9 Hz, C(7)H), 12.50 (1H, s,

NH), 14.31 (1H, s, SH); δ_C(125 MHz, DMSO–d₆) 20.9, 111.5,

114.8, 124.2, 128.7, 128.8, 131.8, 135.2, 135.9, 137.6, 138.8,

143.7, 149.0, 178.6; m/z (LRMS, ESI¯) 291 ([M–H]¯, 60 %);

HRMS (ESI¯) C₁₆H₁₁N₄S¯ ([M–H]¯) requires 291.0710; found

291.0716.

4.3.40. 2’’-(((9-(2’-Methylphenyl-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (50)

Following General Procedure 1, 9-(4’-methylphenyl)-5H-

[1,2,4]triazino[5,6-b]indole-3-thiol 45 (40 mg, 0.14 mmol), Et₃N

(30 µL, 0.22 mmol), methanol (2.5 mL) and 2-

(bromomethyl)benzonitrile (28 mg, 0.14 mmol) gave the title

compound (50) as a pale yellow solid (36 mg, 63%); mp 217 –

219 °C; ν_max(solid) 3203, 3121, 2936, 2225, 1581; δ_H(500 MHz,

DMSO–d₆) 2.06 (3H, s, C(2’)CH₃), 4.71 (2H, s, S-CH₂), 7.22

(1H, dd, J = 7.9, 0.6 Hz, C(6)H), 7.25 – 7.33 (2H, m, C(2’)H and

C(3’)H or C(4’)H), 7.33 – 7.39 (2H, m, C(3’)H and C(3’)H or

C(4’)H), 7.48 (1H, app td, J = 7.6, 0.9 Hz, C(5”)H), 7.61 (1H, dd,

J = 7.9, 0.6 Hz, C(8)H), 7.66 (1H, app td, J = 7.6, 1.3 Hz,

C(4”)H), 7.75 (1H, app t, J = 7.9 Hz, C(7)H), 7.78 (1H, d, J = 7.6

Hz, C(3”)H), 7.86 (1H, dd, J = 7.6, 1.3 Hz, C(6”)H), 12.81 (1H,

s, NH); δ_C(125 MHz, DMSO–d₆) 19.6, 32.4, 111.5, 111.9, 115.8,

117.4, 123.9, 125.5, 127.8, 128.3, 129.3, 129.8, 130.4, 130.7,

133.2, 133.4, 135.7, 137.8, 139.0, 140.4, 141.0, 141.6, 146.3,

165.4; m/z (ESI⁺) 408 ([M+H]⁺, 20%); HRMS (ESI^–) C₂₄H₁₆N₅S¯

([M–H]¯) requires 406.1132; found 406.1125.

4.3.37. 9-(3’-Thienyl)-5H-[1,2,4]triazino[5,6-b]indol-3-thiol

(47)

4-(3’-Thienyl)indoline-2,3-dione 43 (137 mg, 0.60 mmol),

thiosemicarbizide (54 mg, 0.60 mmol) and K₂CO₃(124 mg, 0.90

mmol) were suspended in water (5 mL) and stirred at reflux

overnight. The solution was acidified by dropwise addition of

acetic acid and the resulting precipitate was filtered to yield the

crude product which was recrystallised from DMF to afford the

title compound (47) as an orange solid (148 mg, 87%); mp >300

°C; ν_max(solid) 3214, 3016, 2926, 1593; δ_H(500 MHz, DMSO–

d₆) 7.40 (1H, dd, J = 7.9, 0.6 Hz, C(6)H), 7.46 (1H, dd, J = 7.9,

0.6 Hz, C(8)H), 7.63 (1H, app td, J = 7.9, 1.2 Hz, C(7)H), 7.65 –

7.69 (2 H, m, C(4’)H and C(5’)H), 8.12 (1H, dd, J = 3.2, 1.3 Hz,

C(2’)H), 12.54 (1H, br s, NH), 14.45 (1H, br s, SH); δ_C(125

MHz, DMSO–d₆) 111.6 (C6), 114.4 (C9a), 123.8 (C8), 125.3

(C2’), 125.8 (C5’), 128.4, 131.8, 133.2, 135.8, 138.6, 143.8,

148.9, 178.5; m/z (ESI¯) 283 ([M–H]¯, 100%) HRMS (ESI¯)

C₁₃H₇N₄S₂¯ ([M–H]¯) requires 283.0118; found 283.0115.

4.3.41. 2–(((9-(4’-Methylphenyl)-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (51)

4.3.38. 4-Cyclopropylindoline-2,3-dione (48)

Following General Procedure 1, 9-(4’-methylphenyl)-5H-

[1,2,4]triazino[5,6-b]indole-3-thiol 46 (14 mg, 0.05 mmol), Et₃N

(10 µL, 0.07 mmol), 2-(bromomethyl)benzonitrile (10 mg, 0.05

4-Cyclopropyl-1-(4-methoxybenzyl)indoline-2,3-dione

(330 mg, 1.08 mmol) was dissolved in TFA (3 mL) and the

44

mmol) in methanol (1.5 mL) gave the title compound (51) as a

pale yellow solid (12 mg, 56%); mp (Gallenkamp) 239 – 241 °C;

ν_max(solid) 3335, 3035, 2916, 2224, 1611; δ_H(500 MHz,

DMSO–d₆) 2.42 (3H, s, CH₃), 4.74 (2H, s, S-CH₂), 7.33 (2H, d, J

= 8.1 Hz, C(3’)H and C(5’)H), 7.36 (1H, dd, J = 7.6, 0.8 Hz,

C(6)H), 7.49 (1H, app td, J = 7.9, 0.9 Hz, C(5’’)H), 7.57 (1H, dd,

J = 7.6, 0.8 Hz, C(8)H), 7.67 (1H, app td, J 7.9, 1.3 Hz,

C(4’’)H), 7.71 (2H, d, J = 8.1 Hz, C(2’)H and C(6’)H), 7.74 (1H,

app t, J = 7.6 Hz, C(7)H), 7.80 (1H, d, J = 7.9 Hz, C(6’’)H), 7.88

(1H, dd, J = 7.9, 0.9 Hz, C(3’’)H), 12.79 (1H, s, NH); δ_C(125

MHz, DMSO–d₆) 20.9, 32.5, 111.3, 111.9, 114.6, 117.4, 123.7,

128.3, 128.7, 129.1, 130.4, 130.9, 133.2, 133.4, 135.6, 137.5,

138.8, 141.0, 141.1, 142.0, 146.3, 165.3; m/z (ESI¯) 406 ([M–

H]¯, 100%); HRMS (ESI¯) C₂₄H₁₆N₅S¯ ([M–H]¯) requires

406.1123; found 406.1125.

dried (MgSO₄), filtered and concentrated in vacuo. The resulting

solid was washed with methanol to yield the title compound (54)

as a yellow solid (72 mg, 69 %); mp (Gallenkamp) 219 – 221 °C;

ν_max(solid) 3392, 2221, 1573, 1467, 1358, 1325; HPLC (Method

1) >99 %, rt = 10.93 min; δ_H(400 MHz, pyr–d₅, 363 K) 3.68 (2H,

s, S-CH₂), 4.99 (3H, s, N-CH₃), 7.30 (1H, t, J = 7.8 Hz, C(5’)H),

7.42 (1H, t, J = 7.8 Hz, C(8)H), 7.46 (1H, d, J = 7.8 Hz, C(6)H),

7.52 (1H, t, J = 7.8 Hz, C(4’)H), 7.66 (1H, t, J = 7.8 Hz, C(7)H),

7.74 (1H, d, J = 7.8 Hz, C(3’)H), 7.95 (1H, d, J = 7.8 Hz,

C(6’)H), 8.43 (1H, d, J = 7.8 Hz, C(9)H); δ_C(100 MHz, pyr–d₅)

28.7, 35.1, 112.4, 118.2, 118.6, 120.2, 123.6, 124.8, 129.0, 129.9,

132.4, 132.7, 134.6, 134.9, 143.6, 144.1, 144.9, 167.2; m/z (ESI⁺)

354 ([M+Na]⁺, 100%); HRMS (ESI⁺) C₁₈H₁₃N₅NaS ([M+Na]⁺)

requires 354.0784; found 354.0786.

4.3.45. 2’-(((5-Ethyl-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (55)

4.3.42. 2”-(((9-(3’-Thienyl)-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (52)

Following General Procedure 2, 2’(((5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) was

added to DMF (5 mL) and cooled to 0 °C. NaH (60 % dispersion

in mineral oil, 13.8 mg, 0.34 mmol) was added to the cooled

solution and stirred at 0 °C for 10 min. Ethyl iodide (38 µL, 0.47

mmol) was added and reaction mixture was allowed to warm to rt

and stirred for 16 h. The reaction mixture was quenched by

dropwise addition of water until a precipitate was formed, this

was collected by filtration and dried to yield the title compound

(55) as a white powder (85 mg, 78 %); mp (EZ Melt) 187 – 189

°C; ν_max(solid) 2923, 2853, 2225, 1570, 1335, 1169; HPLC

(Method 2) >97 %, tR = 11.31 min; δ_H(500 MHz, DMSO–d₆,

363 K) 1.34 (3H, t, J = 7.3 Hz, CH₃), 4.45 (2H, q, J = 7.3 Hz, N-

CH₂), 4.77 (2H, s, S-CH₂), 7.45 – 7.48 (1H, m, C(8)H), 7.49 (1H,

app t, J = 7.6 Hz, C(5’)H), 7.67 (1H, app td, J = 7.6, 1.3 Hz,

C(4’)H), 7.78 (1H, app t, J = 7.6 Hz, C(7)H), 7.82 – 7.88 (3H, m,

C(6)H, C(3’)H and C(6’)H), 8.34 (1H, d, J = 7.6 Hz, C(9)H); δ_C

(125 MHz, DMSO–d₆) 13.4, 32.4, 36.0, 111.2, 112.0, 117.4,

117.6, 121.7, 122.9, 128.1, 130.2, 131.1, 133.0, 133.3, 140.5,

141.1, 141.6, 145.5, 165.9; m/z (ESI⁺) 368 ([M+Na]⁺, 100%);

HRMS (ESI⁺) C₁₉H₁₅N₅NaS ([M+Na]⁺) requires 368.0940; found

368.0936.

Following General Procedure 1, 9-(3’-thienyl)-5H-

[1,2,4]triazino[5,6-b]indole-3-thiol 47 (20 mg, 0.07 mmol), Et₃N

(15 µL, 0.11 mmol), methanol (5 mL) and 2-

(bromomethyl)benzonitrile (14 mg, 0.07 mmol) gave the title

compound (52) as a yellow solid (16 mg, 57 %); mp

(Gallenkamp) 274 – 276 °C; ν_max(solid) 3217, 3095, 2863, 2236,

1579; δ_H(500 MHz, DMSO–d₆) 4.76 (2H, s, S-CH₂), 7.39 (1H, d,

J = 7.9 Hz, C(6)H), 7.49 (1H, app t, J = 7.6 Hz, C(5’’)H), 7.55

(1H, d, J = 7.9 Hz, C(8)H), 7.62 (1H, t, J = 7.6 Hz, C(4’’)H),

7.64 – 7.77 (2H, m, C(7)H and C(4’)H), 7.77 – 7.86 (2H, m,

C(3”)H and C(6”)H), 8.12 (1H, d, J = 2.9 Hz, C(5’)H), 8.46 (1H,

d, J = 2.9 Hz, C(2’)H), 12.85 (1H, br s, NH); δ_C(125 MHz,

DMSO–d₆) 31.6, 111.0, 111.6, 117.0, 117.4, 124.8, 125.7, 125.9,

128.6, 129.8, 130.8, 132.2, 133.7, 133.2, 133.5, 135.0, 139.7,

140.9, 141.2, 143.9, 163.6; m/z (ESI¯) 398 ([M–H]¯, 50 %);

HRMS (ESI¯) C₂₁H₁₂N₅S₂¯ ([M–H]¯) requires 398.0540; found

398.0533.

4.3.43. 2-(((9-Cyclopropyl-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (53)

Following General Procedure 1, 9-cyclopropyl-5H-

[1,2,4]triazino[5,6-b]indole-3-thiol 49 (40 mg, 0.165 mmol) and

Et₃N (35 µL, 0.248 mmol) in methanol (5 mL), 2-cyanobenzyl

bromide (32 mg, 0.165 mmol) were added at rt and stirred for 16

h. The resulting precipitate was filtered and washed with EtOH,

Et₂O and petrol to give the title compound (53) as a light green

solid (57 mg, 97%); mp (Gallenkamp) 227 – 229 °C, ν_max(solid)

3067, 1584, 1517, 1447, 1328, 1175; δ_H(500 MHz, DMSO–d₆)

0.94 (2H, m, C(2’)H or C(3’)H), 1.22 (2H, m, C(2’)H or C(3’)H),

3.35 (1H, m, C(1’)H), 4.75 (2H, s, S-CH₂), 6.87 (1H, d, J = 7.7

Hz, C(7)H), 7.32 (1H, d, J = 7.9 Hz, C(5)H), 7.48 (1H, t, J = 7.6

Hz, C(5”)H), 7.55 (1H, t, J = 7.9 Hz, C(6)H), 7.67 (1H, t, J = 7.6

Hz, C(4”)H), 7.83 (1H, d, J = 7.9 Hz, C(3”)H), 7.87 (1H, J = 7.6

Hz, C(6”)H), 12.84 (1H, br s, NH); δ_C(125 MHz, , DMSO–d₆)

10.5, 12.6, 32.5, 109.1, 111.9, 115.9, 116.2, 117.5, 128.2, 130.3,

131.0, 133.2, 133.4, 140.3, 141.3, 142.7, 143.0, 146.5, 164.9;

LRMS m/z (ESI⁺) 358 ([M+H]⁺); HRMS (ESI⁺) C₂₀H₁₅N₅SNa⁺

([M+Na]⁺) requires 380.940; found 380.939.

4.3.46. 2-(((5-Butyl-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (56)

Following General Procedure 2, 2’(((5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol),

was added to DMF (5 mL) and cooled to 0 °C. To the cooled

solution was added NaH (60 % dispersion in mineral oil, 13.8

mg, 0.35 mmol) and stirred at 0 °C for 10 min. Iodobutane (54

µL, 0.47 mmol) was added and reaction mixture was stirred for

16 h at rt, quenched and the resulting precipitate was filtered and

dried to yield the title compound (56) as a white powder (79 mg,

68 %); mp (EZ Melt) 159 – 161 °C; ν_max(solid) 2954, 2925,

2221, 1561, 1466, 1171; HPLC (Method 2) >95 %, tR = 11.97

min; δ_H(500 MHz, DMSO–d₆, 363 K) 0.86 (3H, t, J = 7.6 Hz,

CH₃), 1.22 – 1.30 (2H, m, C(3’’)H₂), 1.70 – 1.76 (2H, m,

C(2’’)H₂), 4.38 (2H, t, J = 7.6 Hz, N-CH₂), 4.76 (2H, s, S-CH₂),

7.47 (1H, app t, J = 7.9 Hz, C(8)H), 7.48 (1H, app t, J = 7.6 Hz,

C(5’)H), 7.66 (1H, app t, J = 7.6 Hz, C(4’)H), 7.75 (1H, app t, J

= 7.9 Hz, C(7)H), 7.81 (1H, d, J = 7.9 Hz, C(6)H), 7.84 (1H, d, J

= 7.6 Hz, C(3’)H), 7.87 (1H, dd, J = 7.6, 1.3 Hz, C(6’), 8.33 (1H,

d, J = 7.9 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 13.6, 19.5, 29.9,

32.4, 40.8, 111.4, 111.9, 117.3, 117.6, 121.6, 122.9, 128.1, 130.0,

131.0, 133.0, 133.3, 140.8, 140.9, 141.5, 145.9, 165.9; m/z (ESI⁺)

396 ([M+Na]⁺, 100%); HRMS (ESI⁺) C₂₁H₁₉N₅NaS ([M+Na]⁺)

requires 396.1253; found 396.1247.

4.3.44. 4.3.45. 2-(((5-Methyl-5H-[1,2,4]triazino[5,6-b]indol-

3-yl)thio)methyl)benzonitrile (54)

Following General Procedure 2, to 5H-[1,2,4]triazino[5,6-

b]indole-3-thiol 7 (100 mg, 0.32 mmol) in THF (5 mL) was

added NaH (60 % dispersion in mineral oil, 13.8 mg, 0.35 mmol)

followed by methyl iodide (29 µL, 0.47 mmol) at 0 °C and the

resulting mixture stirred at rt for 16 h. The reaction mixture was

quenched with water (30 mL) and CH₂Cl₂(30 mL) added. The

organic layer was separated and washed with brine (30 mL),

4.3.47. 2’-((5-(Cyclopropylmethyl)-5H–[1,2,4,]triazino[5,6-

b]indol-3- ylthio)methyl)benzonitrile (57)

C(6’’)H), 7.42 – 7.45 (1 H, m, C(8)H), 7.47 – 7.51 (1 H, m,

C(4’)H), 7.53–7.56 (1 H, m, C(7)H), 7.69 – 7.74 (2 H, m, C(3’)H

and C(6’)H), 7.75 – 7.81 (2 H, m, C(6)H and C(5’)H), 8.37 (1 H,

d, J = 7.9 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 32.4, 44.2,

111.7, 111.9, 117.5, 117.6, 119.3, 121.7, 123.2, 127.3, 127.7,

128.1, 128.8, 130.2, 133.1, 133.2, 135.8, 140.7, 141.2, 141.5,

146.2, 166.2; m/z (ESI⁺) 430 ([M+Na]⁺, 100%); HRMS (ESI⁺)

C₂₄H₁₇N₅NaS ([M+H]⁺) requires 430.1097; found 430.1081.

Following General Procedure 2, 2’-(((5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) was

added to DMF (5 mL), cooled to 0 °C followed by the addition of

NaH (60 % dispersion in mineral oil, 13.8 mg, 0.35 mmol) and

stirred at 0 °C for 10 min. Bromomethylcyclopropane (31 µL,

0.32 mmol) was added and the resulting mixture stirred at rt for

16 h. The reaction mixture was quenched with water and

resulting precipitate was filtered and washed with methanol to

yield the title compound (57) as a pale yellow solid (62 mg, 52

%); mp (Gallenkamp) 149 – 150 °C; ν_max(solid) 3030, 2923,

2251, 1740; HPLC (Method 2) >99 %, tR = 1.66 min; δ_H(500

MHz, DMSO–d₆, 363 K) 0.37 – 0.50 (4H, m, C(2”)H₂and

C(3”)H₂), 1.19 – 1.33 (1H, m, C(1”)H), 4.32 (1H, d, J = 6.9 Hz,

N-CH₂), 4.73 (2H, s, S-CH₂), 7.48 (2H, m, C(8)H and C(4’)H),

7.67 (1H, app t, J = 7.8 Hz, C(7)H), 7.77 (1H, app t, J = 7.6 Hz,

C(5’)H), 7.8 (1H, d, J = 7.8 Hz, C(6)H), 7.81 – 7.99 (2H, m,

C(3’)H and C(6’)H), 8.34 (1H, d, J = 7.8 Hz, C(9)H); δ_C(125

MHz, DMSO–d₆) 3.8, 10.2, 32.4, 45.2, 111.6, 111.9, 117.3,

117.6, 121.6, 122.9, 128.1, 130.0, 131.0, 133.0, 133.4, 140.9,

141.0, 141.5, 145.8, 166.0; m/z (ESI⁺) 394 ([M+Na]⁺, 100%);

HRMS (ESI⁺) C₂₀H₂₀N₅NaS ([M+Na]⁺) requires 394.1094; found

394.1095.

4.3.50. 5-Benzyl-3-(2’-fluorobenzylthio)-5H-

[1,2,4]triazino[5,6-b]indole (60)

Following General Procedure 2, to 3-((2-fluorobenzyl)thio)-

5H-[1,2,4]triazino[5,6-b]indole 14 (50 mg, 0.16 mmol) in THF at

0 °C, was added NaH (60 % dispersion in mineral oil, 10 mg,

0.24 mmol) followed by benzyl bromide (25 µL, 0.24 mmol) and

the resulting mixture stirred at rt for 16 h. The reaction mixture

was quenched with water (30 mL) and CH₂Cl₂(30 mL) added.

The organic layer was separated and washed with brine (30 mL),

dried (MgSO₄), filtered and concentrated in vacuo to yield the

title compound (60) as a light yellow solid (26 mg, 40 %); mp

(Gallenkamp) 139 – 141 °C; ν_max(solid) 3064, 1572, 1491, 1467,

1351, 1333, 1175; HPLC (Method 2) >98 %, tR = 12.17 min; δ_H

(500 MHz, DMSO–d₆, 363 K) 4.62 (2 H, s, S-CH₂), 5.67 (2 H, s,

N-CH₂), 7.02 – 7.05 (1 H, m, C(5’)H), 7.18 (1 H, app t, J = 7.6

Hz, C(4’)H), 7.25 – 7.35 (6 H, m, C(8)H, C(2’’)H, C(3’’)H,

C(5’’)H, C(6’’)H and C(6’)H), 7.49 (1 H, app t, J = 6.9 Hz,

C(7)H), 7.56 – 7.59 (1 H, m, C(4’)H), 7.68 – 7.76 (2 H, m, C(6)H

and C(3’)H), 8.37 (1 H, d, J = 7.9 Hz, C(9)H); δ_C(125 MHz,

DMSO–d₆) 27.5, 44.2, 111.6, 115.3 (d, J = 21 Hz), 117.6, 121.7,

123.1, 124.4 (d, J = 2.9 Hz), 124.7, 127.3, 127.8, 128.8, 129.4 (d,

J = 8.6 Hz), 131.0, 131.3 (d, J = 3.8 Hz), 135.9, 140.7 (d, J = 31

Hz), 141.0, 146.2, 160.5 (d, J = 246 Hz), 166.7; δ_F(470 MHz,

DMSO-d₆) –116.9; m/z (ESI⁺) 423 ([M+Na]⁺, 100%); HRMS

(ESI⁺) C₂₃H₁₇FN₄NaS ([M+Na]⁺) requires 423.1050; found

423.1042.

4.3.48. 2’-(((5-(Cyclohexylmethyl)-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (58)

Following General Procedure 2, 2’(((5H-[1,2,4]triazino[5,6-

b]indol-3- yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol)

was added to DMF (5 mL), cooled to 0 °C followed by the

addition of NaH (60 % dispersion in mineral oil, 13.8 mg, 0.34

mmol) and stirred at 0 °C for 10 min. Bromomethyl cyclohexane

(44 µL, 0.32 mmol) was added, stirred for 16 h and quenched

with water. The resulting precipitate was filtered and dried to

yield the title compound (58) as a white solid (25 mg, 19 %); mp

(EZ Melt) 201 – 203 °C; ν_max(solid) 2926, 2850, 2219, 1571,

1170; HPLC (Method 2) >99 %, tR = 12.40 min; δ_H(500 MHz,

DMSO–d₆, 363 K) 0.97 – 1.12 (6H, m, 6xCH), 1.45 – 1.50 (2H,

m, 2xCH), 1.56 – 1.63 (4H, m, 4xCH), 1.85 – 1.92 (1H, m,

C(1’’)H), 4.21 (1H, d, J = 7.3 Hz, N-CH₂), 4.77 (2H, s, S-CH₂),

7.46 – 7.52 (2H, m, C(5’)H and C(8)H), 7.68 (1H app td, J = 7.6,

1.3 Hz, C(4’)H), 7.76 (1H, app t, J = 7.6 Hz, C(7)H), 7.81 – 7.87

(2H, m, C(3’)H and C(6)H), 7.90 (1H, dd, J = 7.6, 1.0 Hz,

C(6’)H), 8.35 (1H, d, J = 7.6 Hz, C(9)H); δ_C(125 MHz, DMSO–

d₆) 25.1, 25.6, 30.0, 32.4, 36.6, 46.9, 111.8, 111.9, 117.3, 117.7,

121.5, 122.9, 128.1, 129.8, 131.0, 133.0, 133.4, 140.8, 141.2,

141.6, 146.2, 166.1; m/z (ESI⁺) 414 ([M+H]⁺, 100%); HRMS

(ESI⁺) C₂₄H₂₃N₅NaS ([M+Na]⁺) requires 436.1566; found

436.1555.

4.3.51. 2’-(((5-(2’’-Nitrobenzyl)-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (61)

Following

General

Procedure

2,

to

2’(((5H-

(60

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile

7

mg, 0.19 mmol) in THF (5 mL) was added NaH (60 % dispersion

in mineral oil, 11 mg, 0.28 mmol) followed by 2-nitro benzyl

bromide (40 mg, 0.21 mmol) at 0 °C and the resulting mixture

stirred for 16 h at rt. The reaction mixture was quenched with

water until a precipitate formed. The precipitate was filtered and

washed with methanol to give the title compound (61) as an off

white solid (79 mg, 96 %); mp (Gallenkamp) 225 – 226 °C (dec);

ν_max(solid) 3061, 2804, 2226, 1580, 1340, 1175; HPLC (Method

2) >96 %, tR = 11.72 min; δ_H(500 MHz, pyr–d₅, 363 K) 4.65 (2

H, s, S–CH₂), 6.02 (2 H, s, N-CH₂), 6.71 (1 H, d, J = 7.1 Hz,

C(6’’)H), 7.42 - 7.72 (7 H, m, 7xAr-H), 7.81 – 7.89 (2 H, m,

C(3’’ and C(6)H), 8.31 (1 H, d, J = 7.8 Hz, C(3’)H), 8.40 (1 H, d,

J = 7.6 Hz, C(9)H); δ_C(125 MHz, pyr–d₅) 35.0, 45.2, 112.9,

115.0, 119.3, 119.9, 120.7, 124.0, 126.7, 129.3, 129.8, 130.4,

132.2, 133.0, 134.8, 134.8, 135.4, 141.1, 142.7, 143.7, 143.9,

148.9, 155.1, 169.2; m/z (ESI⁺) 452 ([M+H]⁺, 10%), m/z (ESI^–)

4.3.49. 2’-(((5-Benzyl-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (59)

Following General Procedure 2, to 3-((2’-cyanobenzyl)thio)-

5H-[1,2,4]triazino[5,6-b]indole 7 (50 mg, 0.15 mmol) in THF at

0 °C, was added NaH (60 % dispersion in mineral oil, 10 mg,

0.24 mmol) followed by benzyl bromide (25 µL, 0.24 mmol) and

the resulting mixture stirred at rt for 16 h. The reaction mixture

was quenched with water (30 mL) and CH₂Cl₂(30 mL) added.

The organic layer was separated and washed with brine (30 mL),

dried (MgSO₄), filtered and concentrated in vacuo to yield the

title compound (59) as a light yellow solid (49 mg, 80 %); mp

(Gallenkamp) 197 – 200 °C; ν_max(solid) 2215, 1571, 1520, 1468,

1328; HPLC (Method 2) >98 %, tR = 11.96 min; δ_H(500 MHz,

DMSO–d₆, 363 K) 4.77 (2 H, s, S–CH₂), 5.68 (2 H, s, N-CH₂),

7.23 – 7.33 (5 H, m, C(2’’)H, C(3’’)H, C(4’’), C(5’’)H and

316

([M–(2’’–NO₂Bn)H]^–,

100%);

HRMS

(ESI⁺)

C₂₄H₁₆N₆NaO₂S ([M–H]^–) requires 475.0948; found 475.954.

4.3.52. 2’-(((5-(Phenylsulfonyl)-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (62)

Following General Procedure 2, 2’(((5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) was

added to DMF (5 mL), cooled to 0 °C followed by the addition of

NaH (60 % dispersion in mineral oil, 13.8 mg, 0.35 mmol) and

stirred at 0 °C for 10 min. Phenyl sulfonyl chloride (40 µL, 0.32

mmol) was added, stirred for 16 h and quenched with water. The

resulting precipitate was filtered and dried to yield the title

compound (62) as a white solid (121 mg, 84 %); mp (EZ Melt)

176 – 177 °C; C₂₃H₁₅N₅NaO₂S₂requires C, 60.38; H, 3.30; N,

15.31%; found C, 60.26; H, 3.21; N, 15.16%; ν_max(solid) 2220,

1568, 1448, 1354, 1174; δ_H(500 MHz, DMSO–d₆, 363 K) 4.81

(2H, s, S-CH₂), 7.52 (1H, app t, J = 7.9 Hz, C(5’)H), 7.63 (1H,

app t, J = 7.6 Hz, C(8)H), 7.63 (2H, app td, J = 8.8, 1.0 Hz,

C(3’’)H and C(5’’)H), 7.69 (1H, app td, J = 7.9, 1.6 Hz, C(4’)H),

7.78 (1H, tt, J = 8.8, 1.0 Hz, C(4’’)H), 7.85 (1H, td, J = 7.6, 1.3

Hz, C(7)H), 7.89 (1H, d, J = 7.9 Hz, C(3’)H), 7.91 (1H, dd, J =

7.9, 1.0 Hz, C(6’)H), 8.10 (2H, dd, J = 8.8, 1.0 Hz, C(2’’)H and

C(6’’)H), 8.33 (1H, d, J = 7.6 Hz, C(6)H), 8.37 (1H, d, J = 7.6

Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 32.8, 112.0, 114.5, 117.5,

119.3, 121.8, 125.8, 127.4, 128.4, 130.0, 130.2, 132.2, 133.3,

133.4, 135.6, 136.8, 137.9, 140.7, 141.9, 147.1, 167.2; m/z (ESI⁺)

480 ([M+Na]⁺, 100%); HRMS (ESI⁺) C₂₃H₁₅N₅NaO₂S₂

([M+Na]⁺) requires 480.0559; found 480.0549.

1.3 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 32.8, 112.0, 115.0,

117.4, 119.0, 121.8, 125.9, 126.0, 128.5, 129.4, 130.4, 132.3,

132.6, 133.2, 133.4, 133.5, 137.4, 138.6, 140.4, 141.9, 147.0,

147.1, 167.0; m/z (ESI^–) 501 ([M–H]^–, 20%), 316 ([M–(2’’–

NO₂PhSO₂)H]^–, 100%); HRMS (ESI⁺) C₂₃H₁₄N₆NaO₄S₂

([M+Na]⁺) requires 525.0410; found 525.0425.

4.3.55. Benzyl-3-((2’-cyanobenzyl)thio)- 5H-

[1,2,4]triazino[5,6-b]indole-5-carboxylate (65)

Following

General

Procedure

2,

to

2’(((5H-

(60

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile

7

mg, 0.19 mmol) in THF (5 mL) was added NaH (60 %

dispersion in mineral oil, 11.0 mg, 0.28 mmol) followed by

benzyl chloroformate (23.4 µL mg, 0.21 mmol) at 0 °C and the

resulting mixture stirred for 16 h at rt. The reaction mixture was

quenched with water and the resulting precipitate was filtered and

washed with methanol to yield the title compound (65) an off

white solid (82 mg, 96%); mp (Gallenkamp) 97 – 99 °C; ν_max

(solid) 3426, 3063, 2232, 1739, 1582, 1392, 1195; δ_H(500 MHz,

DMSO–d₆, 363 K) 4.54 (2H, s, S-CH₂), 5.61 (2H, s, O-CH₂),

7.26 (1H, t, J = 7.6 Hz, C(4’’)H), 7.35 (2 H, app t, J = 7.6 Hz,

C(3’’)H and C(5’’)H), 7.49 (1 H, app t, J = 7.6 Hz, C(4’)H), 7.60

– 7.64 (4H, m, C(2’’)H and C(6’’)H, C(8)H and C(5’)H), 7.73

(1H, d, J = 7.6 Hz, C(6’)H), 7.82 (1H, app t, J = 7.6 Hz, C(7)H),

7.89 (1H, d, J = 7.6 Hz, C(3’)H), 8.37 (1H, d, J = 7.6 Hz, C(6)H),

8.39 (1H, d, J = 8.2 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 32.4,

69.3, 111.9, 116.1, 117.4, 119.3, 121.3, 125.4, 128.4, 128.4,

128.4, 128.5, 130.6, 131.9, 133.1, 133.4, 134.7, 138.7, 140.8,

142.2, 147.0, 149.9, 167.3; m/z (ESI⁺) 474 ([M+Na]⁺, 20%), m/z

(ESI^–) 316 ([M–(PhCH₂CO₂)H]^–, 100%); HRMS (ESI⁺)

C₂₅H₁₇N₅NaO₂S ([M+Na]⁺) requires 474.0995; found 474.0993.

4.3.53. 2’-(((5-Tosyl-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (63)

Following General Procedure 2, 2’(((5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile 7 (60 mg, 0.19 mmol) was

added to THF (5 mL), cooled to 0 °C followed by the addition of

NaH (60 % dispersion in mineral oil, 11.0 mg, 0.28 mmol) and

stirred at 0 °C for 10 min. p-toluene sulfonyl chloride (40 mg,

0.21 mmol) was added, stirred for 16 h and quenched with water

(50 mL) and extracted with EtOAc (50 mL). The organic layer

was separated, washed with brine (30 mL), dried (MgSO₄),

filtered and concentrated in vacuo. The resulting solid was

washed with methanol to yield the title compound (63) as a white

crystalline solid (82 mg, 92%); mp (Gallenkamp) 220 – 222 °C;

ν_max(solid) 2230, 1595, 1449, 1370, 1177; HPLC (Method 2) >99

%, tR = 12.37 min; δ_H(500 MHz, DMSO–d₆, 363 K) 2.28 (3 H,

s, C(4’)CH₃), 4.83 (2 H, s, S-CH₂), 7.37 (2 H, d, J = 8.3 Hz,

C(3’’)H and C(5’’)H), 7.49 (1 H, app t, J = 7.8 Hz, C(4’)H), 7.59

(1 H, t, J = 7.8 Hz, C(8)H), 7.66 (1 H, app t, J = 7.8 Hz, C(5’)H),

7.82 (1 H, app t, J = 7.8 Hz, C(7)H), 7.87 – 7.92 (2 H, m, C(3’)H

and C(6’)H), 8.01 (2 H, d, J = 8.3 Hz, C(2’’)H and C(6’’)H),

8.31 – 8.37 (2 H, m, C(6)H and C(9)H); δ_C(125 MHz, DMSO–

d₆) 21.2, 32.7, 112.0, 114.5, 117.5, 119.2, 121.8, 125.8, 127.4,

128.4, 130.2, 130.4, 132.2, 133.3, 133.4, 133.8, 137.9, 140.7,

141.8, 146.7, 147.0, 167.2; m/z (ESI⁺) 494 ([M+Na]⁺, 45%), m/z

(ESI^–) 316 ([M–(2CN–Bn)]^–, 100%); HRMS (ESI⁺)

C₂₄H₁₇N₅NaO₂S₂([M+Na]⁺) requires 494.0716; found 494.0738.

4.3.56. 2’(((5-Acetyl-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (66)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3- yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by

acetyl chloride (33.8 µL, 0.47 mmol) at 0 °C and the resulting

mixture stirred for 16 h. The reaction mixture was quenched with

water until a precipitate formed. The precipitate was filtered and

dried to give the title compound (66) as a pale yellow solid (89

mg, 77 %); mp (Gallenkamp) 207 – 209 °C; ν_max(solid) 2227,

1721, 1377, 1177; HPLC (Method 2) >99 %, tR = 11.53 min; δ_H

(500 MHz, DMSO–d₆, 363 K) 2.94 (3 H, s, CH₃), 4.82 (2 H, s, S–

CH₂), 7.51 (1 H, app t, J = 7.6 Hz, C(5’)H), 7.63 (1 H, app t, J =

7.8 Hz, C(8)H), 7.70 (1 H, app t, J = 7.6, Hz, C(4’)H), 7.81 (1H,

app t, J = 7.8 Hz, C(7)H), 7.83 (1H, d, J = 7.6 Hz, C(3’)H), 7.90

(1 H, d, J = 7.6 Hz, C(6’)H), 8.38 (1 H, d, J = 7.8 Hz, C(6)H),

8.58 (1 H, d, J = 7.8 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 27.4,

32.8, 112.0, 117.2, 117.4, 119.3, 121.2, 125.7, 128.4, 130.2,

132.1, 133.2, 133.5, 139.3, 140.5, 142.5, 147.3, 166.6, 170.3; m/z

(ESI⁺) 382 ([M+Na]⁺, 100%); HRMS (ESI⁺) C₁₉H₁₃N₅NaOS

([M+Na]⁺) requires 382.0733; found 382.0724.

4.3.54. 2’-(((2’’-Nitrophenylsulfonyl-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (64)

Following

General

Procedure

2,

to

2’(((5H-

(60

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile

7

mg, 0.19 mmol) in THF (5 mL) was added NaH (60 % dispersion

in mineral oil, 11.0 mg, 0.28 mmol) at 0 °C and stirred for 10

min. 2-Nitrophenyl sulfonyl chloride (47 mg, 0.21 mmol) was

added and the resulting mixture stirred at rt for 16 h. The reaction

mixture was quenched dropwise with water (50 mL). The

resulting precipitate was filtered and washed with methanol to

yield the title compound (64) as an off white solid (79 mg, 83%);

mp (Gallenkamp) 217 – 218 °C; ν_max(solid) 2921, 2853, 2228,

1546, 1373, 1184; HPLC (Method 2) >97 %, tR = 12.07 min; δ_H

(500 MHz, DMSO–d₆, 363 K) 4.70 (2H, s, S–CH₂), 7.50 (1H,

app t, J = 7.6 Hz, C(4’)H), 7.63 (1H, app t, J = 7.6 Hz, C(5’)H),

7.67 (1H, app t, J = 7.6 Hz, C(4’’)H), 7.76 (1H, d, J = 7.6 Hz,

C(6’)H), 7.84 – 7.91 (2H, m, C(3’)H and C(5’’)H), 7.97 (1H, app

t, J = 7.6 Hz, C(8)H), 8.06 (1H, app t, J = 7.6 Hz, C(7)H), 8.09

(1H, d, J = 8.5 Hz, C(6’’)H), 8.18 (1H, dd, J = 7.6, 1.3 Hz,

C(6)H), 8.42 (1H, d, J = 7.6 Hz, C(3’’)H), 8.49 (1H, dd, J = 7.6,

4.3.57. 2’-(((5-(Cyclopropanecarbonyl)-5H-

[1,2,4]triazino[5,6-b]indole-3-yl)thio)methyl)benzonitrile (67)

Following General Procedure 2, 2’(((5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) was

added to DMF (5 mL), cooled to 0 °C followed by the addition of

NaH (60 % dispersion in mineral oil, 13.8 mg, 0.35 mmol) and

stirred at 0 °C for 10 min. Cyclopropane carbonyl chloride (29

µL, 0.32 mmol) was added, stirred for 16 h at rt and quenched

with water. The resulting precipitate was filtered and washed

with methanol to yield the title compound (67) as a white solid

(83 mg, 76 %); mp (Gallenkamp) 183 – 184 °C; ν_max(solid) 3045,

2924, 2224, 1702; HPLC (Method 2) >99 %, tR = 11.89 min; δ_H

(500 MHz, DMSO–d₆, 363 K) 1.16 – 1.28 (4H, m, C(2”)H₂and

C(3”)H₂), 3.50 – 3.56 (1H, m, C(1”)H), 4.78 (2H, s, S-CH₂), 7.5

(1H, t, J = 7.9 Hz, C(8)H), 7.58 (1H, app t, J = 7.7 Hz, C(5’)H),

7.69 (1H, app td, J = 7.7, 1.2 Hz, C(4’)H), 7.74 (1H, app t, J =

7.9 Hz, C(7)H), 7.80 (1H, d, J = 7.9 Hz, C(6)H), 7.89 (1H, dd, J

= 7.7, 1.2 Hz, C(3’)H), 8.33 (1H, d, J = 7.7 Hz, C(6’)H), 8.46

(1H, d, J = 7.9 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 11.6, 16.1,

32.8, 111.8, 117.1, 117.4, 119.1, 121.1, 125.6, 128.4, 130.0,

130.5, 131.9, 133.2, 133.5, 139.3, 142.5, 147.4, 166.4, 174.0; m/z

(ESI⁺) 386 ([M+H]⁺, 100%); HRMS (ESI⁺) C₂₁H₁₆N₅OS

([M+H]⁺) requires 386.1073; found 386.1073.

4.3.60. Methyl 4’’-(3-((2’-cyanobenzyl)thio)-5H-

[1,2,4]triazino[5,6-b]indol-5-yl)-4’’-oxobutanoate (70)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (150

mg, 0.47 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil,20.8 mg, 0.52 mmol) followed by

methyl-5-chloro-5-oxopentoate (87 µL, 0.71 mmol) at 0 °C and

the resulting mixture stirred at rt for 16 h. The reaction mixture

was quenched with water until a precipitate formed. The

precipitate was filtered and dried to give the title compound (70)

as a pale yellow solid (165 mg, 81 %); mp (Gallenkamp) 144 –

145 °C; ν_max(solid) 2953, 2228, 1739, 1713, 1379, 1188; HPLC

(Method 2) >99 %, tR = 11.76 min; δ_H(500 MHz, DMSO–d₆,

363 K) 2.80 (2 H, t, J = 6.3 Hz, N-C(O)-CH₂), 3.62 (3 H, s, CH₃),

3.69 (2 H, t, J = 6.3 Hz, CH₂-C(O)-O), 4.82 (2 H, s, S-CH₂), 7.51

(1 H, app t, J = 7.6 Hz, C(8)H), 7.62 (1 H, app t, J = 7.6 Hz,

C(5’)H), 7.70 (1 H, t, J = 7.6 Hz, C(4’)H), 7.80 ( H, app t, J = 7.6

Hz, C(7)H), 7.83 (1H, d, J = 7.6 Hz, C(3’)H), 7.90 (1 H, d, J =

7.6 Hz, C(6’)H), 8.38 (1 H, d, J = 7.6 Hz, C(6)H), 8.56 (1 H, d, J

= 7.6 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 27.9, 32.9, 34.2,

51.5, 111.9, 117.2, 117.4, 119.4, 121.2, 125.8, 128.4, 130.2,

132.1, 133.2, 133.5, 139.2, 140.5, 142.5, 147.3, 166.5, 172.3,

172.4; m/z (ESI⁺) 454 ([M+Na]⁺, 100%); HRMS (ESI⁺)

C₂₂H₁₇N₅NaO₃S ([M+Na]⁺) requires 454.0944; found 454.0924.

4.3.58. 2’-(((5-(Cyclopentanecarbonyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (68)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by

cyclopentane carbonyl chloride (58 µL, 0.47 mmol) at 0 °C and

the resulting mixture stirred for 16 h at rt. The reaction mixture

was quenched with water until a precipitate formed. The

precipitate was filtered and dried to give the title compound (68)

as a pale yellow solid (56 mg, 42 %); mp (Gallenkamp) 173 –

174 °C; ν_max(solid) 2871, 2229, 1720, 1577, 1380; HPLC

(Method 2) >99 %, tR = 12.32 min; δ_H(500 MHz, DMSO–d₆,

363 K) 1.56 – 1.64 (2 H, m, C(3’’)H and C(4’’)H), 1.66 – 1.74 (2

H, m, C(3’’)H and C4’’)H), 1.87 – 2.03 (4 H, m, C(2’’)H and

C(5’’)H), 4.35 – 4.42 (1 H, m, C(1’’)H), 4.81 (2 H, s, S-CH₂),

7.52 (1 H, app t, J = 7.6 Hz, C(8)H), 7.62 (1 H, app t, J = 7.6 Hz,

C(5’)H), 7.71 (1 H, app td, J = 7.6, 1.1 Hz, C(4’)H), 7.79 (1H,

app t, J = 7.6 Hz, C(7)H), 7.80 (1H, d, J = 7.6 Hz, C(3’)H), 7.92

(1 H, d, J = 7.6 Hz, C(6’)H), 8.38 (1 H, d, J = 7.6 Hz, C(6)H),

8.58 (1 H, d, J = 7.6 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 25.6,

29.5, 32.7, 45.9, 111.9, 117.4, 117.5, 119.5, 121.1, 125.7, 128.4,

130.4, 132.0, 133.2, 133.6, 139.6, 140.2, 142.5, 147.0, 166.6,

176.3; m/z (ESI⁺) 436 ([M+Na]⁺, 100%); HRMS (ESI⁺)

C₂₃H₁₉N₅NaOS ([M+Na]⁺) requires 436.1203; found 436.1198.

4.3.61. Methyl 5’’-(3-((2’-cyanobenzyl)thio)-5H-

[1,2,4]triazino[5,6-b]indol-5-yl)-5’’-oxopentanoate (71)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (150

mg, 0.47 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil,20.8 mg, 0.52 mmol) followed by

methyl-5-chloro-5-oxopentoate (98 µL, 0.71 mmol) at 0 °C and

the resulting mixture stirred at rt for 16 h. The reaction mixture

was quenched with water until a precipitate formed. The

precipitate was filtered and dried to give the title compound (71)

as a pale yellow solid (170 mg, 81 %); mp (Gallenkamp) 166 –

167 °C; ν_max(solid) 2954, 2231, 1730, 1716, 1381; HPLC

(Method 2) >99 %, tR = 11.53 min; δ_H(500 MHz, DMSO–d₆,

363 K) 1.96 – 2.04 (2H, m, –CH₂-) 2.44 (2H, t, J = 7.4 Hz, CH₂-

C(O)-O), 3.47 (2H, t, J = 7.4 Hz, CH₂-C(O)-N), 3.54 (3H, s, O-

CH₃), 4.78 (2H, s, S-CH₂), 7.51 (1H, app t, J = 7.7 Hz, C(8)H),

7.60 (1H, t, J =7.6 Hz, C(5’)H), 7.69 (1H, app td, J = 7.7, 1.3 Hz,

C(4’)H), 7.79 (1H, app td, J = 7.6, 1.3 Hz, C(7)H), 7.81 (1H, d, J

= 7.6 Hz, C(3’)H), 7.91 (1H, d, J = 7.6 Hz, C(6’)H), 8.33 (1H, d,

J = 7.6 Hz, C(6)H), 8.54 (1H, d, J = 7.8 Hz, C(9)H); δ_C(125

MHz, DMSO–d₆) 19.0, 32.3, 32.8, 38.9, 51.3, 112.0, 117.3,

117.4, 119.3, 121.1, 125.7, 128.5, 130.3, 132.1, 133.3, 133.6,

139.2, 140.4, 142.4, 147.1, 166.6, 172.9, 173.0; m/z (ESI⁺) 468

([M+Na]⁺, 100%); HRMS (ESI⁺) C₂₂H₁₇N₅NaO₃S ([M+Na]⁺)

requires 468.1101; found 468.1107.

4.3.59. 2’-(((5-(Cyclohexanecarbonyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (69)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by

cyclohexane carbonyl chloride (63 µL, 0.47 mmol) at 0 °C and

the resulting mixture stirred for 16 h at rt. The reaction mixture

was quenched with water until a precipitate formed. The

precipitate was filtered and dried to give the title compound (69)

as a pale yellow solid (98 mg, 71 %); mp (Gallenkamp) 194 –

197 °C; ν_max(solid) 2932, 2854, 2226, 1720, 1453, 1379; HPLC

(Method 2) >95 %, tR = 12.67 min; δ_H(500 MHz, DMSO–d₆,

363 K) 1.18 – 1.38 (4 H, m, C(3’’)H₂and C(5’’)H₂), 1.45 – 1.55

(2 H, m, C(4’’)H₂), 1.75 – 2.07 (4 H, m, C(2’’)H₂and C(6’’)H₂),

4.04 – 4.12 (1H, m, C(1’’)H), 4.84 (2 H, s, S-CH₂), 7.53 (1 H,

app td, J = 7.6, 1.1 Hz, C(8)H), 7.60 – 7.65 (1 H, m, C(4’)H or

C(6’)H), 7.71 (1 H, app td, J = 7.6, 1.1 Hz, C(7)H), 7.77 – 7.85

(2 H, m, C(3’)H and C(4’)H or C(6’)H), 7.89 – 7.93 (1 H, m,

C(5’)H), 8.38 (1 H, d, J = 6.9 Hz, C(6)H), 8.59 (1 H, d, J = 8.5

Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 25.1, 25.3, 28.5, 32.8,

44.7, 112.0, 112.8, 117.4, 117.5, 119.6, 121.1, 125.7, 128.5,

130.4, 132.1, 133.6, 139.6, 140.3, 142.6, 147.0, 166.5, 176.2; m/z

(ESI⁺) 450 ([M+Na]⁺, 100%); HRMS (ESI⁺) C₂₄H₂₁N₅NaOS

([M+Na]⁺) requires 450.1364; found 450.1362.

4.3.62. 2’-(((5-(Benzoyl-5H-[1,2,4]triazino[5,6-b]indol-3-

yl)thio)methyl)benzonitrile (72)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3- yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.52 mmol) followed by

benzoyl chloride (55 µL, 0.47 mmol) at 0 °C and the resulting

mixture stirred for 16 h. The reaction mixture was quenched with

water until a precipitate formed. The precipitate was filtered and

dried to give the title compound (72) as a pale yellow solid (97

mg, 72 %); mp (Gallenkamp) 201 – 202 °C; ν_max(solid) 3056,

2228, 1699, 1600, 1290; C₂₄H₁₅N₅OS requires C, 68.39; H, 3.59;

N, 16.62%; found C, 68.19; H, 3.45; N, 16.62%; δ_H(500 MHz,

DMSO–d₆, 363 K) 4.06 (2 H, s, S-CH₂), 7.34 (1 H, d, J = 7.9 Hz,

C(6’)H), 7.47 (1 H, app t, J = 7.6 Hz, C(8)H), 7.52 (2 H, app t, J

= 7.3 Hz, C(3’’)H and C(5’’)H), 7.60 (1H, app t, J = 7.9 Hz,

C(5’)H), 7.63 (1H, app t, J = 7.9 Hz, C(4’)H), 7.66 (1H, app t, J

= 7.6 Hz, C(7)H), 7.82 – 7.86 (2H, m, C(3’)H and C(4’’)H), 7.94

(2 H, d, J = 7.3 Hz, C(2’’)H and C(6’’)H), 8.30 (1 H, d, J = 7.6

Hz, C(6)H), 8.44 (1 H, d, J = 7.6 Hz, C(9)H); δ_C(125 MHz,

DMSO–d₆) 31.8, 111.7, 116.2, 117.2, 119.7, 121.3, 125.6, 128.3,

128.4, 130.0, 130.3, 131.6, 133.2, 133.2, 133.3, 133.9, 139.4,

140.2, 142.1, 147.2, 166.4, 167.8; m/z (ESI⁺) 444 ([M+Na]⁺,

100%); HRMS (ESI⁺) C₂₄H₁₅N₅NaOS ([M+Na]⁺) requires

444.0890; found 444.0885.

4.3.65. 2’-(((5-(2’’-Trifluoromethyl-4’’-fluorobenzoyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (75)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by 2-

trifluoromethyl-4-fluoro benzoyl bromide (72 µL, 0.47 mmol) at

0 °C and the resulting mixture stirred at rt for 16 h. The reaction

mixture was quenched with water until a precipitate formed. The

precipitate was filtered, washed with methanol and dried to give

the title compound (75) as a pale yellow solid (67 mg, 42 %); mp

(EZ Melt) 193 – 194 °C; ν_max(solid) 2230, 1716, 1384, 1292;

HPLC (Method 2) >99 %, tR = 12.18 min; δ_H(500 MHz,

DMSO–d₆, 363 K) 4.01 (2H, s, S-CH₂), 7.50 – 7.54 (2H, m,

C(8)H and C(3’)H), 7.65 (1H, app td, J = 7.6, 1.3 Hz, C(5’)H),

7.67 – 7.71 (1H, m, C(5’’)H), 7.73 (1H, app t, J = 7.6 Hz,

C(4’)H), 7.83 (1H, dd, J = 7.6, 1.3 Hz, C(3’’)H), 7.88 (1H, dd, J

= 7.6, 1.6 Hz, C(6’)H), 7.92 (1H, app td, J = 7.6, 1.3 Hz, C(7)H),

8.01 – 8.03 (1H, m, C(6’’)H), 8.46 (1H, d, J = 7.6 Hz, C(6)H),

8.62 (1H, d, J = 7.6 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 31.8,

111.8, 114.5 (dq, J = 26, 4.8 Hz), 117.0, 117.0, 120.0 (d, J = 21

Hz), 120.2 (q, J = 276 Hz), 121.5, 123.7, 126.6, 128.4 (qd, J =

32, 7.6 Hz), 128.6, 130.2 (m), 130.2, 132.0 (d, J = 8.6 Hz), 132.4,

133.2, 133.5, 138.9, 139.3, 142.5, 147.1, 161.7, 163.7, 165.7 (d, J

= 238 Hz); δ_F(470 MHz, DMSO–d₆) –102.4 (q, J = 8.7 Hz), –

53.8; m/z (ESI^–) 542 ([M+Cl]^–, 40%); HRMS (ESI⁺)

C₂₅H₁₃F₄N₅NaOS ([M+Na]⁺) requires 530.0669; found 530.0678.

4.3.63. 2’-(((5-(4’’-Fluorobenzoyl)-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (73)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3- yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by 4-

fluoro benzoyl bromide (56 µL, 0.47 mmol) at 0 °C and the

resulting mixture stirred for 16 h. The reaction mixture was

quenched with water until a precipitate formed. The precipitate

was filtered and dried to give the title compound (73) as a pale

yellow solid (95 mg, 67 %); mp (Gallenkamp) 202 – 204 °C; ν_max

(solid) 2940, 2227, 1702, 1599, 1573, 1318; C₂₄H₁₄FN₅OS

requires C, 65.59; H, 3.21; N, 15.94%; found C, 65.31; H, 3.18;

N, 15.98%; δ_H(500 MHz, DMSO–d₆, 363 K) 4.16 (2 H, s, CH₂),

7.34 (2H, app t, J = 8.8 Hz, C(3’’)H and C(5’’)H), 7.41 (1H, d, J

= 7.6 Hz, C(3’)H), 7.48 (1H, app td, J = 7.6, 1.3 Hz, C(5’)H),

7.62 (1H, td, J = 7.6, 1.3 Hz, C(8)H), 7.66 (1H, app t, J = 7.6 Hz,

C(4’)H), 7.82 – 7.86 (2H, m, C(6’)H and C(7)H), 7.98 – 8.06 (2

H, m, C(2’’)H and C(6’’)H), 8.28 (1 H, d, J = 7.8 Hz, C(6)H),

8.43 (1 H, d, J = 7.6 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 31.9,

111.7, 115.5 (d, J = 23 Hz), 116.3, 117.2, 119.7, 121.4, 125.6,

128.4, 130.2, 130.3 (d, J = 2.9 Hz), 131.7, 133.2, 133.2 (d, J =

9.5 Hz), 133.4, 139.5, 140.1, 142.2, 147.3, 165.1 (d, J = 253 Hz),

166.4, 166.7; δ_F(470 MHz, DMSO–d₆) –110.4; m/z (ESI⁺) 462

([M+Na]⁺, 70%); HRMS (ESI⁺) C₂₄H₁₄FN₅NaOS ([M+Na]⁺)

requires 462.0795; found 462.0794.

4.3.66. 2’-(((5-(2’’-Nitrobenzoyl)-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (76)

Following General Procedure 2, 2’(((5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) was

added to DMF (5 mL), cooled to 0 °C followed by the addition of

NaH (60 % dispersion in mineral oil, 13.8 mg, 0.35 mmol) and

stirred at 0 °C for 10 min. 2-nitrobenzoyl bromide (41.6 µL,

0.32mmol) was added, stirred for 16 h and quenched with water.

The resulting precipitate was filtered to yield the title compound

(76) as a white solid (119 mg, 81 %); mp (EZ Melt) 210 – 213

°C; ν_max(solid) 2261, 1711, 1524, 1453, 1383, 1346, 1292; δ_H

(500 MHz, DMSO–d₆, 363 K) 3.97 (2H, s, S-CH₂), 7.48 – 7.53

(2H, m, C(3’)H and C(5’)H), 7.63 (1H, app td, J = 7.6, 1.3 Hz,

C(4’)H), 7.75 (1H, app t, J = 7.6 Hz, C(7)H), 7.80 (1H, app td, J

= 7.6, 1.3 Hz, C(4’’)H), 7.87 (1H, d, J = 7.6 Hz, C(6’)H), 7.93

(1H, d, J = 7.6 Hz, C(6’’)H, 7.94 (1H, app td, J = 7.6, 1.3 Hz,

C(8)H), 7.97 (1H, app td, J = 7.6, 1.3 Hz, C(5’’)H), 8.27 (1H, d,

J = 7.6 Hz, C(3’’)H), 8.47 (1H, d, J = 7.6 Hz, C(6)H), 8.73 (1H,

d, J = 7.6 Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 31.9, 111.8,

117.2, 119.8, 121.6, 124.4, 126.6, 128.6, 129.0, 130.3, 131.6,

131.7, 132.5, 133.2, 133.5, 135.6, 138.8, 139.4, 142.3, 144.7,

146.7, 146.8, 164.7, 166.8; m/z (ESI⁺) 489 ([M+Na]⁺, 100%);

HRMS (ESI⁺) C₂₄H₁₄N₆NaO₃S ([M+Na]⁺) requires 489.0740;

found 489.0733.

4.3.64. 2’-(((5-(3’’-Trifluoromethyl)benzoyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (74)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in THF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by 3-

(trifluoromethyl)benzoyl chloride (71 µL, 0.47 mmol) at 0 °C and

the resulting mixture stirred for 16 h. The reaction mixture was

quenched with water until a precipitate formed. The precipitate

was filtered, washed with methanol and dried to give the title

compound (74) as a pale yellow solid (89 mg, 58 %); mp

(Gallenkamp) 155 – 156 °C; ν_max(solid) 2228, 1701, 1592, 1580,

1377, 1339; HPLC (Method 2) >96 %, tR = 12.19 min; δ_H(500

MHz, DMSO–d₆, 363 K) 4.74 (2 H, s, S-CH₂), 7.36 (1 H, d, J =

8.0 Hz, C(6’)H), 7.46 –7.68 (4 H, m, C(7)H, C(8)H, C(4’) and

C(5’’)H), 7.73 – 7.78 (2 H, m, C(3’)H and C(5’)H), 7.95 (1 H, s,

C(2’’)H), 8.18 – 8.23 (2 H, m, C(4’’)H and C(6’’)H), 8.40 (1 H,

d, J = 8.4 Hz, C(6)H), 8.43 (1H, d, J = 7.3 Hz, C(9)H); δ_C(125

MHz, DMSO–d₆) 31.8, 111.7, 116.6, 117.1, 119.8, 121.4, 123.7

(q, J = 273 Hz), 125.9, 126.6 (q, J = 3.8 Hz), 128.4, 129.1 (q, J =

3.8 Hz), 129.1 (q, J = 32 Hz), 129.5, 130.2, 131.8, 133.2, 133.4,

133.6, 135.3, 139.4, 140.0, 142.5, 147.4, 166.2, 166.6; δ_F(470

MHz, DMSO–d₆) –61.2; m/z (ESI⁺) 512 ([M+Na]⁺, 100%);

HRMS (ESI⁺) C₂₅H₁₄F₃N₅NaOS ([M+Na]⁺) requires 512.0769;

found 512.0759.

4.3.67. 2’-(((5-(4’’-(Trifluoromethoxy)benzoyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (77)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by 4-

(trifluoromethoxy)benzoyl chloride (74 µL, 0.47 mmol) at 0 °C

and the resulting mixture stirred for 16 h at rt. The reaction

mixture was quenched with water until a precipitate formed. The

precipitate was filtered and dried to give the title compound (77)

as a white solid (109 mg, 67 %); mp (Gallenkamp) 169 – 170 °C;

ν_max(solid) 2926, 2224, 1701, 1458; HPLC (Method 2) >97 %, tR

= 12.07 min; δ_H(500 MHz, DMSO–d₆, 363 K) 4.08 (2 H, s, S-

CH₂), 7.39 (1 H, d, J = 7.6 Hz, C(6’)H), 7.45 – 7.53 (3 H, m,

C(3’’)H, C(5’’)H and C(8)H), 7.61 (1 H, app td, J = 7.7, 1.2 Hz,

C(7)H), 7.68 (1 H, app t, J = 7.4 Hz, C(4’)H), 7.81 – 7.88 (2 H,

m, C(3’)H and C(5’)H), 8.08 – 8.14 (2 H, m, C(2’’)H and

C(6’’)H), 8.37 (1 H, d, J = 7.7 Hz, C(6)H), 8.43 (1 H, d, J = 7.3

Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 32.5, 111.7, 116.5, 117.1,

119.7, 120.4, 120.7, 121.3, 125.8, 128.5, 129.5 (q, J = 258 Hz),

130.3, 131.7, 132.4, 133.1, 133.4, 139.4, 140.0, 142.2, 147.3,

151.4, 166.6, 170.3; δ_F(470 MHz, DMSO–d₆) –56.8; m/z (ESI⁺)

528 ([M+Na]⁺, 50%); HRMS (ESI⁺) C₂₅H₁₄F₃N₅NaO₂S

([M+Na]⁺) requires 528.0718; found 528.0713.

4.3.70. 2’-(((5-(Furan-2’’-carbonyl)-5H-[1,2,4]triazino[5,6-

b]indol-3-yl)thio)methyl)benzonitrile (80)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by

furan-2-carbonyl chloride (46 µL, 0.47 mmol) at 0 °C and the

resulting mixture stirred for 16 h. The reaction mixture was

quenched with water until a precipitate formed. The precipitate

was filtered and dried to give the title compound (80) as a pale

yellow solid (103 mg, 78 %); mp (Gallenkamp) 177 – 178 °C;

ν_max(solid) 2924, 2227, 1692, 1382, 1309; HPLC (Method 2) >97

%, tR = 11.63 min; δ_H(500 MHz, DMSO–d₆, 363 K) 4.49 (2 H, s

S-CH₂), 6.83 (1 H, dd, J = 3.8, 1.8 Hz, C(4’’)H), 7.46 (1 H, app

td, J = 7.6, 1.3 Hz, C(5’)H), 7.53 (1H, d, J = 7.6 Hz, C(6’)H),

7.59 (1H, app t, J = 7.8 Hz, C(8)H), 7.60 – 7.64 (1H, m, C(4’)H),

7.73 – 7.80 (2 H, m, C(7)H and C(5’’)H), 7.84 (1 H, dd, J = 7.6,

1.0 Hz, C(3’)H), 8.02 (1 H, d, J = 8.3 Hz, C(6)H), 8.20 (1H, dd, J

= 3.8, 0.8 Hz, C(3’’)H), 8.38 (1 H, d, J = 7.8 Hz, C(9)H); δ_C(125

MHz, DMSO–d₆) 33.3, 112.7, 114.0, 116.1, 118.2, 120.0, 122.3,

125.1, 126.2, 129.2, 131.1, 132.4, 134.1, 134.2, 139.9, 141.3,

142.7, 146.5, 147.8, 150.8, 156.4, 167.3; m/z (ESI⁺) 434

([M+Na]⁺, 100%); HRMS (ESI⁺) C₂₂H₁₃N₅NaO₂S ([M–H]^–)

requires 434.0682; found 434.0680.

4.3.68. 2’-(((5-(4’’-Dimethylamino)benzoyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (78)

To a stirred suspension of 2’(((5H- [1,2,4] triazino [5,6-b]

indol-3-yl) thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol),

DMAP (0.8 mg, 0.06 mmol) and Et₃N (86 µL, 0.63 mmol) in

CH₂Cl₂(5 mL) at rt was added 4-dimethylaminobenzoyl chloride

(83 mg, 0.48 mmol). The resulting mixture was stirred at rt for 16

h. CH₂Cl₂(30 mL) was added to the suspension and washed with

H₂O (30 mL). The organic layer was washed with brine (30 mL),

dried (MgSO₄), filtered and concentrated in vacuo. The resulting

solid was washed with methanol and dried to yield the title

compound (78) as a white solid (81 mg, 55 %); mp (Gallenkamp)

196 – 197 °C; ν_max(solid) 2917, 2229, 1679, 1603, 1184; HPLC

(Method 2) >95 %, tR = 12.15 min; δ_H(400 MHz, pyr–d₅, 363 K)

2.84 (6H, s, N–CH₃), 5.04 (2H, s, S-CH₂), 6.74 (2H, d, J = 9.1

Hz, C(3’’)H and C(5’’)H), 7.28 (1 H, app t, J = 7.6 Hz, C(8)H),

7.45 (1H, app t, J = 7.6 Hz, C(7)H), 7.56 (1H, app t, J = 7.6 Hz,

C(4’)H), 7.59 (1H, d, J = 7.6 Hz, C(3’)H), 7.62 – 7.71 (2 H, m,

C(5’)H and C(6’)H), 8.08 (2 H, d, J = 9.1 Hz, C(2’’)H and

C(6’’)H), 8.19 (1 H, d, J = 7.6 Hz, C(6)H), 8.48 (1 H, d, J = 7.6

Hz, C(9)H); δ_C(125 MHz, pyr–d₅) 33.5, 39.7, 111.2, 113.2,

116.2, 118.1, 119.4, 120.2, 121.9, 125.1, 128.4, 131.1, 131.5,

133.2, 133.5, 134.0, 140.9, 141.7, 142.4, 147.6, 154.9, 166.8,

168.0; m/z (ESI^–) 463 ([M–H]^–, 50%); HRMS (ESI⁺)

C₂₆H₂₀N₆NaOS ([M+Na]⁺) requires 487.1312; found 487.1310.

4.3.71. 2’-(((5-(Isoxazole-5’’-carbonyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (81)

Following

General

Procedure

2,

to

2’(((5H-

[1,2,4]triazino[5,6-b]indol-3-yl) thio) methyl)benzonitrile 7 (100

mg, 0.32 mmol) in DMF (5 mL) was added NaH (60 %

dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by

isoxazole-5-carbonyl chloride (62 µL, 0.47 mmol) at 0 °C and the

resulting mixture stirred for 16 h. The reaction mixture was

quenched with water until a precipitate formed. The precipitate

was filtered and dried to give the title compound (81) as a pale

yellow solid (112 mg, 85 %); mp (Gallenkamp) 195 – 196 °C;

ν_max(solid) 2923, 2227, 1709, 1299; HPLC (Method 2) >95 %, tR

= 11.47 min; δ_H(500 MHz, DMSO–d₆, 363 K) 4.44 (2 H, s,

CH₂), 7.48 (1H, d, J = 1.9 Hz, C(5’’)H), 7.49 (1H, app td, J =

7.6, 1.3 Hz, C(5’)H), 7.60 – 7.67 (2H, m, C(8)H and C(7)H),

7.69 (1H, app t, J = 7.6 Hz, C(4’)H), 7.83 – 7.88 (2 H, m, C(3’)H

and C(6’)H), 8.33 (1 H, d, J = 7.6 Hz, C(6)H), 8.44 (1 H, d, J =

7.6 Hz, C(9)H), 8.91 (1 H, d, J = 1.9 Hz, C(4’’)H); δ_C(125 MHz,

DMSO–d₆) 32.4, 110.8, 111.9, 116.4, 117.3, 119.8, 121.6, 126.4,

128.5, 130.4, 132.1, 133.3, 133.5, 138.6, 140.2, 142.3, 147.3,

151.6, 155.6, 160.5, 166.6; m/z (ESI⁺) 413 ([M+Na]⁺, 100%);

HRMS (ESI⁺) C₂₁H₁₂N₆NaO₂S ([M+Na]⁺) requires 413.0815;

found 340.0610 (corresponds to parent compound).

4.3.69. 2’-(((5-(3’’, 4’’-Dimethoxybenzoyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (79)

To a stirred suspension of 2’(((5H-[1,2,4]triazino[5,6-b]indol-

3-yl) thio) methyl)benzonitrile 7 (100 mg, 0.32 mmol), DMAP

(0.8 mg, 0.06 mmol) and Et₃N (86 µL, 0.63 mmol) in CH₂Cl₂(5

mL) at rt was added 3,4-dimethoxybenzoyl chloride (95 mg, 0.48

mmol). The resulting mixture was stirred at rt for 16 h. CH₂Cl₂

(30 mL) was added to the suspension and washed with water (30

mL). The organic layer was washed with brine (30 mL), dried

(MgSO₄), filtered and concentrated in vacuo. The resulting solid

was washed with methanol and dried to yield the title compound

(79) as a white solid (87 mg, 56 %); mp (Gallenkamp) 234 – 235

°C; ν_max(solid) 3115, 2932, 2229, 1697, 1514, 1378; HPLC

(Method 2) >99 %, tR = 11.81 min; δ_H(500 MHz, DMSO–d₆,

363 K) 3.73 (3H, s, O-CH₃), 3.80 (3H, s, O-CH₃), 4.25 (2H, s, S-

CH₂), 7.09 (1H, d, J = 8.5 Hz, C(5’’)H), 7.35 (1H, d, J = 7.9 Hz,

C(3’)H), 7.46 (1H, app td, J = 7.6, 1.3 Hz, C(5’)H), 7.55 (1H, d,

J = 1.9 Hz, C(2’’)H), 7.59 (1H, td, J = 7.6, 1.3 Hz, C(4’)H), 7.63

– 7.67 (2H, m, C(8)H and C(6’’)H), 7.80 – 7.85 (2H, m, C(7)H

and C(6)’), 8.14 (1H, d, J = 8.5 Hz, C(6)H), 8.43 (1H, d, J = 7.9

Hz, C(9)H); δ_C(125 MHz, DMSO–d₆) 32.1, 55.8, 55.9, 110.8,

111.7, 113.4, 115.9, 117.2, 119.5, 121.4, 125.1, 125.3, 125.5,

128.3, 130.2, 131.5, 133.2, 133.2, 139.7, 140.5, 142.2, 147.3,

148.4, 153.8, 166.3, 166.8; m/z (ESI⁺) ([M+Na]⁺, 100%); HRMS

(ESI⁺) C₂₆H₁₉N₅NaO₃S ([M+Na]⁺) requires 504.1110; found

504.1107.

4.3.72. 2’-(((5-(Morpholine-4’’-carbonyl)-5H-

[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile (82)

Following General Procedure 2, 2’(((5H-[1,2,4]triazino[5,6-

b]indol-3- yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol)

was added to DMF (5 mL), cooled to 0 °C followed by the

addition of NaH (60 % dispersion in mineral oil, 13.8 mg, 0.34

mmol) and stirred at 0 °C for 10 min. 1-Morpholine carbonyl

chloride (47 µL, 0.32 mmol) was added, stirred for 16 h, cooled

in an ice bath and quenched slowly with dropwise addition of

water. The resulting precipitate was filtered and dried to yield the

title compound (82) as a white solid (98 mg, 72 %); mp (EZ

Melt) 191 – 193 °C; ν_max(solid) 2928, 2867, 2223, 1688, 1570,

1389; HPLC (Method 2) >99 %, tR = 10.86 min; δ_H(500 MHz,

DMSO–d₆, 363K) 3.40 – 3.79 (8H, m, 8xCH), 4.78 (2H, s, S-

CH₂), 7.50 (1H, app td, J = 7.8, 1.3 Hz, C(5’)H), 7.57 (1H, app t,

J = 7.6 Hz, C(8)H), 7.68 (1H, app td, J = 7.8, 1.3 Hz, C(4’)H),

7.76 (1H, d, J = 7.8 Hz, C(3’)H), 7.79 (1H, app t, J = 7.6 Hz,

C(7)H), 7.83 (1H, d, J = 7.6 Hz, C(6)H), 7.89 (1H, dd, J = 7.8,

1.3 Hz, C(6’)H), 8.37 (1H, d, J = 7.6 Hz, C(9)H); δ_C(125 MHz,

DMSO–d₆) 32.7, 44.7, 66.1, 112.0, 113.9, 117.5, 118.5, 121.6,

124.3, 128.3, 130.4, 131.5, 133.2, 133.3, 139.4, 140.9, 141.8,

145.7, 148.4, 166.3; m/z (ESI⁺) 453 ([M+Na]⁺, 100%); HRMS

(ESI⁺) C₂₂H₁₈N₆NaO₂S ([M+Na]⁺) requires 453.1104; found

453.1087.

solution was diluted in DMSO to produce a range of

concentrations. These were then added to phosphate buffered

saline at pH 7.4 (final test compound concentrations = 1 µM, 3

µM, 10 µM, 30 µM, 100 µM, final DMSO concentration = 1%, 7

replicates per concentration) and incubated for 2 h at 37 °C. At

the end of the incubation period, the absorbance at 620 nm was

read for each concentration and each replicate. The assay

provided a precipitation range, where the mid-point was used as

the estimated precipitation concentration and recorded in µM.

4.4. Pharmacology

4.5.1

Microsomal Stability Studies

4.4.1 hCB₂R and hCB₁R cAMP assay

The microsomal stability assay was performed by Cyprotex plc.

The microsomes used were derived from mouse liver and were

incubated with the test compound at 37 °C in the presence of

added co-factor, NADPH. The reaction was terminated by the

addition of methanol containing an internal standard. Following

centrifugation, the supernatant was analysed by LC-MS/MS. The

disappearance of test compound was monitored at 0, 5, 15 30, 45

min time points. Diazepam and diphenhydramine were used as

the positive controls. Compound stability in the absence of added

co-factor was measured at the 45 min time point.

The cAMP Hunter eXpress GPCR assay kit and cells used are

from DiscoveRx Corporation (DiscoveRx Corporation,

Birmingham, UK) and used according to the manufacturer's

protocol.

The CB₂R agonist 4-[4-(1,1-dimethylheptyl)-2,6-

dimethoxyphenyl]-6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-

methanol (HU-308 from Tocris Bioscience, UK) and the CB₁R

agonist

N-(2-Chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide

(ACEA from Tocris Bioscience, UK) were used as positive

controls in the hCB₂R and hCB₁R assays respectively. Briefly,

hCB2R or hCB1R CHO-K1 cells were plated in the provided

medium (15000 cells per well) in half-area 96-well plates

(Corning, UK) and placed overnight in an incubator 37 °C 5 %

CO₂. From this stage onwards, the assays for CB₁R and CB₂R

CHO-K1 cells were identical. Next day, the medium was

aspirated and replaced with a 1:3 solution of anti-cAMP antibody

in provided buffer. In the presence of 20 µΜ forskolin, freshly

reconstituted compounds or solvent alone (final concentration 0.2

% DMSO) were added to the wells and incubated for 30 minutes

at 37 °C, 5 % CO₂. After the incubation, a mixture of provided

lysis solution and substrates was added to the wells and incubated

in the dark for one hour at room temperature. The provided

detection reagent EA was then added to the wells and incubated

in the dark for 3 hours at room temperature. The luminescence

was then read using a standard luminometer (Perkin-Elmer, Seer

Green, UK). The luminescence of the wells incubated only with

forskolin and solvent (control wells) were normalised as 100%

and the dose-response curves were expressed as a percentage of

the control wells. The EC₅₀values shown are from pooling 3-4

independent experiments and were identified using GraphPad

prism non-linear regression curve fit (4 PL parameters).

4.5.1

Cellular Permeability Studies

Cell permeability studies were carried out by Cyprotex using

MDR1-transfected (MDR1-MDCK) cells derived from Madin

Darby canine kidney (MDCK) cells. The cells were plated on a

Multiscreen plate (Millipore, MA, USA) and formed a confluent

monolayer over a period of 4 days before the experiment. On day

4, the test compounds were applied to the apical side of the

membrane and the transport of the compound across the

monolayer was monitored over a 60 minute time period. The

efflux ratio of the desired compound was measured by

determining transport of the compound from the basolateral area

to the apical compartment.

4.6. Molecular Modelling

Energy minimisations were performed for 51, 52 and 53 using

Maestro molecular modelling software (version 9.0 Schrodinger).

The calculations were performed ignoring solvent interactions,

and the force field used was MM2. In each case an evaluation of

the model was performed by a Ramachandran plot. The selection

of most the plausible structural pose(s) was done by observation

of the energy minimization score results.

4.4.2 Radioligand binding assays: hCB₁R and hCB₂R

Radioligand binding assays were contracted to CEREP (Celle

l'Evescault, France) where membranes from CHO-cells

expressing either hCB₁R or hCB₂R were used to assess binding to

the two cannabinoid receptors. Competition binding studies were

performed by incubating membranes expressing hCB₁R or

hCB₂R with unlabeled compounds and tritiated CP 55,940 (0.5

nM) or WIN-55212 (0.8 nM) respectively, for two hours at 37

°C. For CB₁R and CB₂R, non-specific binding was assessed

using WIN-55212 at 10 and 5 µΜ respectively. Two hours later,

the binding of tritiated ligands was assessed by scintillation

counting. The competition binding data for each compound are

expressed as the percentage of the remaining bound tritiated

ligand after the compound’s incubation with the membrane. K_i

values (equilibrium dissociation constants for each compound)

were calculated from CEREP using the Cheng-Prusoff equation

and the K_dvalue (equilibrium dissociation constant for each

radioligand).

Acknowledgments

We thank Dr Raman Parkesh for technical advice in setting up

the virtual screen. We also thank the British Heart Foundation for

the provision of a studentship (I.C., FS/08/067) and a programme

grant (D.R.G., RG/10/15/28578), the British Heart Foundation

Centre of Research Excellence at the University of Oxford for an

award (A.J.R. and D.R.G.), Research Councils’ UK for a

Fellowship (A.J.R.).

Supplementary Material

Virtual and selected actual screening data, EC₅₀curves and

off-target receptor and affinity data and enzyme activity data for

DIAS2 is provided in the supplementary material.

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Ligand-based virtual screening identifies a family of selective cannabinoid receptor 2 agonists

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