Rational modification, synthesis and biological evaluation of N-substituted phthalazinone derivatives designed to target interleukine-15 protein

Bioorg. Med. Chem. 39 (2021) 116161

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

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

Rational modiﬁcation, synthesis and biological evaluation of N-substituted

phthalazinone derivatives designed to target interleukine-15 protein

a

,

b

,

c

,

1

, Agn `e s Qu ´e m ´e ner^a

,

c

,

*

,

1

a

,

c

,

d

b

Jimmy Smadja

, Mike Maillasson

, Benoit Sicard ,

b

a,

c,

d

b

Aur ´e lien Leray , Laurence Arzel , Jacques Lebreton , Erwan Mortier

, Didier Dubreuil ,

Monique Math ´e -Allainmat^b

,*

a

Universit ´e de Nantes, CNRS, Inserm, CRCINA, F-44000 Nantes, France

Universit ´e de Nantes, CNRS, Chimie et Interdisciplinarit ´e : Synth `e se, Analyse, Mod ´e lisation (CEISAM), UMR CNRS 6230, 2, rue de la Houssini `e re – BP 92208-44322,

b

Nantes, France

c

LabEx IGO, Immunotherapy, Graft, Oncology, Nantes, France

Universit ´e de Nantes, Inserm, CNRS, CHU Nantes, SFR Sant ´e , FED 4203, Inserm UMS 016, CNRS, UMS 3556, IMPACT Platform, Nantes, France

d

A R T I C L E I N F O

A B S T R A C T

Keywords:

Interleukin (IL)-15 is a pleiotropic cytokine structurally close to IL-2 and sharing with the IL-2Rβ and γc receptor

Protein-protein interaction

Interleukin-15

(

R) subunits. IL-15 plays important roles in innate and adaptative immunity, supporting the activation and

+

proliferation of NK, NK-T, and CD8 T cells. Over-expression of IL-15 has been shown to participate to the

development of inﬂammatory and autoimmune diseases and diverse T cell malignancies. This study is in con-

tinuity of our previous work through which a family of small-molecule inhibitors impeding IL-15/IL-2Rβ

interaction with sub-micromolar activity has been identiﬁed using pharmacophore-based virtual screening and

hit optimization methods. With the aim to improve the efﬁcacy and selectivity of our lead inhibitor, speciﬁc

modiﬁcations have been introduced on the basis of optimized SAR and modelisation. The new series of com-

pounds generated have been evaluated for their capacity to inhibit the proliferation as well as the down-stream

signaling of IL-15-dependent cells and to bind to IL-15.

IL-15Rβ

IL-15Rβ inhibitor

Inﬂammatory and autoimmune diseases

1

. Introduction

phosphorylates STAT3 (p-Stat3) via the IL-2Rβ chain and JAK3 that

6

,7

subsequently phosphorylates STAT5 (p-Stat5) via the γc chain.

Interleukin(IL)-15 is a proinﬂammatory cytokine, belonging to the

Several approaches have been developed to inhibit IL-15 activity,

including soluble IL-15R ,

IL-15 mutants,⁹IL-15- or IL-15R-directed

α

8

IL-2 cytokine family, involved in the stimulation of T and natural killer

1

,2

10

(

NK) cell activity.

Dysregulation of IL-15 expression has been

monoclonal antibodies and fusion proteins, IL-2 mutant or peptide

modiﬁed to block IL-15 interaction with its receptors (H9-RETR, BNZ-1

described in a range of autoimmune inﬂammatory diseases, including

rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus,

alopecia areata, vitiligo, psoriasis, type 1 diabetes and celiac diseases

and in patients with leukemia such as large granular lymphocyte (LGLL),

cutaneous T cell lymphoma (CTCL) and human T cell lymphotropic virus

and 2)¹

soluble form of the murine IL-15R

contribution of IL-15 in the rejection of fully vascularized cardiac allo-

grafts in a murine model. Administration of sIL-15R to CBA/Ca (H-2 k)

1,12

and JAK inhibitors. Smith et al. have reported the use of a

chain (sIL-15R ) to investigate the

13

α

3

I (HTLV-1) driven adult T cell leukemia (ATL). IL-15 binds a hetero-

recipients completely prevented rejection of minor histocompatibility

complex-mismatched B10.BR (H-2 k) heart grafts and led to a state of

trimeric receptor composed of a non-transducing, private

α

chain (IL-

) and a IL-2Rβ/γc-signal transducing complex. The binding of IL-

5 to IL-2Rβ/γc heterodimer induces JAK1 activation that subsequently

4

,5

14

1

5R

α

donor speciﬁc immunologic tolerance. In addition, administration of

sIL-15R profoundly suppressed the development of collagen-induced

α

Abbreviations: IL, Interleukin; R, Receptor; NK, Natural Killer; LGLL, Large Granular lymphocyte; CTCL, Cutaneous T Cell Lymphoma; HTLV-1, Human T Cell

Lymphotropic Virus 1; ATL, Adult T cell Leukemia; Stat, Signal Transducers and Activators of Transcription; HAM/TSP, HTLV-1-Associated Tropical Spastic para-

paresis; PPIs, Protein-Protein Interactions; SPR, Surface Plasmon Resonance.

*

Corresponding authors.

E-mail addresses: agnes.quemener@univ-nantes.fr (A. Qu ´e m ´e ner), monique.mathe@univ-nantes.fr (M. Math ´e -Allainmat).

These authors contributed equally to this work.

1

https://doi.org/10.1016/j.bmc.2021.116161

Received 20 January 2021; Received in revised form 8 April 2021; Accepted 9 April 2021

Available online 20 April 2021

0

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

8

also prevented psoriasis¹⁵as well as

arthritis in mice. Soluble IL-15R

α

benzamide 2 and its alkoxy or benzoate analogs 2A, 2B and 2C, which

gave a good activity in a cell proliferation assay and IC50 at a nanomolar

range in a p-Stat5 assay (Fig. 2, Series 3), we proposed to complete our

initial series²⁹with a compound bearing a benzoyl substituent giving

also access to a benzophenone derivative which could be regarded as a

allergic inﬂammation.¹⁶In this context, Amgen has developed an anti-

IL-15 antibody (AMG-714 or HuMax-IL15) targeting IL-15 in the re-

gion involved in γc chain binding. AMG-714 has ﬁrst shown efﬁcacy in a

xenograft model of human psoriasis and rheumatoid arthritis as well

as coeliac disease.¹⁸In psoriasis, it led to a better clinical outcome than

the standard cyclosporine A therapy. Likewise, a humanized antibody

1

7

30

potential photocrosslinker tool (Fig. 2, R3 modulation).

(

Hu-Mik-Beta-1) speciﬁc for IL-2Rβ was developed and was shown to

2. Results and discussion

1

9,20,21

prolong cardiac allograft survival in cynomolgus monkeys.

This

antibody is currently being tested in patients with refractory coeliac

disease and HTLV-1-associated tropical spastic paraparesis (HAM/TSP)

2.1. Chemistry

(

clinicaltrials.gov). As an alternative and challenging approach, design

of original small molecule inhibitors of protein–protein interactions

PPIs) has recently been applied to target IL-15/IL-15R interfaces. Some

potent small-molecule inhibitors of the IL-2/IL-2R interface have

To access to the ﬁrst series of N-substituted triazole analogues of

compounds 1 or 2, we chose to condense the dihydro-phthalazinone

hydrazide 3²⁹with an isothiocyanate diethylacetal reagent providing

a masked carbonyl group. So, isothiocyanate 4 was condensed with

hydrazide 3 to give the N-substituted mercapto-triazole derivative 5 in

80% yield. This strategy allowed the access to the desired targeted

compounds 10 to 14 from this common precursor (Scheme 1). Both

(

α

already been identiﬁed by chemical or DNA-encoded chemical library

screening, fragment-based approaches or virtual library screening fol-

lowed by chemical optimizations.²

2–26

Concerning IL-15/IL-15R in-

terfaces, a study using in silico pharmacophore-based virtual screening

methods directed to IL-15/IL-15R interface has revealed putative IL-15

inhibitors in an in vitro primary cellular assay among which cefazolin, a

2

9

alkylating agents 6 (n = 1) and 7 (n = 7) efﬁciently reacted by

nucleophilic substitution with the thiol 5 in basic conditions, without

identiﬁcation of the N-substituted regioisomer. The resulting acetal

derivatives 8 and 9 were then subjected to hydrolysis in acidic condi-

tions to yield the corresponding aldehydes 10 and 11, respectively. The

targeted aldehyde 10 with the short linker chain (n = 1) was obtained

α

2

7

ﬁrst-generation cephalosporin antibiotic. However, it turned that

cefazolin was not selective to IL-15 but was rather a broad common

gamma chain cytokine inhibitor.²⁸

◦

Meanwhile, using a similar approach coupled to hit optimization, we

discovered original low MW non-peptidic inhibitors targeting the IL-15/

with a good 95% yield after 2 h stirring at a temperature of 40 C. The

poor solubility of the acetal 9, bearing a longer linker chain (n = 7), in

THF or acetone gave aldehyde 11 in a moderate 59% yield after

completion of the reaction (24 h). We also observed that in these acidic

conditions retro-Michael process occurred leading to the NH free

analogue 12. Intermediate 10 was either reduced with sodium boro-

hydride in an ethanolic medium to furnish the alcohol 13 (67%), or

oxidized in Dalcanale conditions³¹into the corresponding carboxylic

acid 14 (60%). However, reduction or oxidation of the aldehyde 11 into

the corresponding alcohol or carboxylic acid derivatives was not suc-

cessful due to poor solubility and slow progress of the reaction in both

conditions. Consequently, efﬁcient puriﬁcation of the reaction products

by ﬂash column chromatography failed.

2

9

IL-2Rβ interface. In this previous work, we designed a novel family of

IL-15 inhibitors with a thio-triazolyl-methyl-phthalazinone backbone

bearing variable alkyl amide fragments. Starting from selected hit de-

rivative 1, identiﬁed through the virtual screening strategy targeting the

IL-2Rβ binding site of IL-15, SAR investigation has given access to a

potent lead 2 and other derived analogues showing inhibitory activities

against IL-15 with IC50 values ranging between 50 and 150 nM in cell-

based experiments (p-Stat5). Unfortunately, none of these IL-15 in-

hibitors expressed selectivity versus the competitive IL-2.

Based on our docking model (Fig. 1), we proposed to further inves-

tigate some options to improve the inhibitory potency of the compounds

introducing speciﬁc modiﬁcations directed by a modelisation design

progress. This process led us to target the triazole or phthalazinone

heterocycle cores by substitution of the N-Me with N-alkylated baits,

offering a terminal hydrophilic function, in order to probe comple-

mentary hydrogen bond interactions with the polar asparagine side

chain residues of the IL-15 protein (Fig. 1B). For this purpose, two novel

series of analogues of compounds 1 and 2, such as N-substituted triazole

To access to the targeted N-substituted triazolyl analogues of the lead

2 (n = 7), we turned back to a linear synthesis starting from hydrazide 3

33

3

2

which was condensed with the isothiocyanate reagents 15 and 16

bearing a terminal alcohol or amino function, respectively (Scheme 2).

Following this strategy, mercapto-triazole derivatives 17 and 18 were

efﬁciently obtained in 80% yields and subsequently alkylated with

bromo amide fragment 7 leading to the silylated intermediate 19 and

the targeted N-dimethylamino propyl analogue 20 in 95% yields.

Finally, the alcohol 21 was quantitatively isolated after desilylation of

(

Fig. 2, Series 1) and phthalazinone (Fig. 2, Series 2) derivatives have

been targeted. Furthermore, based on the inhibitor potency of

Fig. 1. Binding mode of compound 1. (A) Connolly surface of IL-15 coloured according to the hydrophobicity index of the exposed residues (hydrophobic in brown;

2

9

hydrophilic in blue) showing the docking mode of compound 1 and hydrophilic areas surrounding the phthalazinone-triazole moiety. (B) Ribbon drawing of the

predicted binding mode of compound 1 on IL-15 showing IL-15 residues in the vicinity of the triazole and the phthalazinone heterocycles of 1. Binding interactions

are depicted (dashed lines: green = hydrogen bond, light green = carbon hydrogen bond, pink = hydrophobic interaction). (For interpretation of the references to

colour in this ﬁgure legend, the reader is referred to the web version of this article.)

2

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

Fig. 2. From IL-15 inhibitors 1 and 2 to the targeted molecules: chemical modiﬁcations introduced on the N-triazole moiety (series 1, R

1

), N-phthalazinone moiety

(

series 2, R ) and on the terminal amidic moiety in lead 2 (series 3, R ).

2

3

1

9 by Olah’s reagent prior to be oxidized into the corresponding acid 22

compounds bearing heptyl-amido linker (n = 7). Starting from the

precursor 24, N-alkylation was performed with protected bromopropa-

nol 33³⁵or mesylated N-dimethylaminopropanol 34 . Alkylation of 24

using TEMPO/DAIB system.

36

The second series of N-substituted phthalazinone analogues of

compounds 1 or 2 was then investigated. For the short alkyl chain series

2 3

monitored by K CO as base did not work well and sodium hydride was

(

n = 1), the strategy involved the dihydro-phthalazinone carboxylic

required to improve the yield of alkylated phthalazinones 35 and 36 to

60 and 75%, respectively (Scheme 4). The formation of the N-methyl-

mercaptotriazole heterocycle was efﬁcient in two steps giving the cor-

responding thiols 37 (70%) and 38 (89%), from 35 and 36, respectively.

Here again the thio-alkylation of thiol 37 and 38 using the bromo re-

agent 7, in Williamson-type conditions proceeded smoothly giving the

protected intermediate 39 and the dimethylaminopropyl phthalazinone

analogue 40 in moderate 52 and 47% yields after 24 h of stirring at room

temperature. From the protected alcohol derivative 39, desilylation

using Olah’s reagent quantitatively led to the targeted N-triazolyl

propanol compound 41 from which a ﬁrst oxidation with Dess-Martin

periodinane (DMP) reagent afforded the aldehyde analogue 42 and a

second oxidation step in Dalcanale conditions gave the carboxylic acid

43.

3

4

ester 24 quantitatively obtained by the condensation of one equivalent

of hydrazine hydrate with the conjugated ester 23 (Scheme 3). Here

again a reagent bearing an acetal function regarded as a suitable pro-

moter of a key aldehyde was preliminary introduced on the phthalazi-

none ring. Thereby, commercial bromo acetal 25 was used to alkylate

the phthalazinone prior inducing the formation of the triazol ring in a

two steps procedure involving hydrazine hydrate addition followed by a

cyclisation with methylisothiocyanate (89% overall yield). Thio-

alkylation under Williamson-type conditions in the presence of the

bromo amide reagent 6 afforded the key intermediate 28. Once again,

the hydrolysis of the acetal 27 in aqueous HCl medium failed to give

aldehyde 29, however, success was encountered in 81% yield using

◦

APTS (0.5 equiv.) in an acetone/water solvent mixture, at 60 C over-

night. Starting from the aldehyde 29, both targeted alcohol 30 and

carboxylic acid 31 analogues (n = 1) were obtained in the same

reduction and oxidation conditions, as previously described for the N-

substituted triazolyl analogues. Alternatively, reductive amination of 29

with dimethylamine in the presence of sodium borohydride also affor-

ded N-dimethylaminopropyl analogue 32 conveniently.

To access to the free NH–phthalazinone analogue 46 of the lead

compound 2, a similar strategy was applied starting from compound 23

via the key intermediate 44 obtained in 82% yield in a reﬂuxing alco-

holic solution of hydrazine. The expected analogue 46 was then ob-

tained in 26% overall yield following a same three steps reaction

sequence previously described (Scheme 5). Finally, the targeted

benzophenone analogue 49 was quantitatively obtained after alkylation

of the intermediate 47²⁹with the iodinated amidic chain 48 (Scheme 6).

To overcome problems highlighted in precedent attempts, a linear

strategy was adopted to reach the targeted N-substituted phthalazino

3

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

Scheme 1. Synthesis of the N-triazolyl propionaldehydes 10 and 11 (n = 1 and 7), N-triazolyl propanol 13 and N-triazolyl propionionic acid 14 (n = 1).

2

.2. Structure-activity analyses

triazole and phthalazinone moieties of compound 1 (Fig. 1A), we

envisioned to graft hydrophilic substituents on chemically modulable

nitrogens of both heterocycles. Besides these modiﬁcations, we also

prepared a benzophenone analogue of compound 2, in order to complete

our previous SAR study²⁹with a bulky terminal amidic fragment.

The effectiveness of the novel compounds was evaluated using the IL-

15- and IL-2-dependent cell proliferation and Stat5 phosphorylation

assays and, for some of them, their binding to IL-15 was determined by

SPR technology.

In a previous work, we identiﬁed the ﬁrst IL-15 small molecules in-

hibitors by a pharmacomodulation study focused on the terminal amidic

part of our hit 1 and the linker chain relying it to the triazole-

phthalazinone main core of the molecule. Several novel compounds

with IC50 values ranging between 50 and 150 nM in cell-based experi-

ments (p-Stat5), were identiﬁed and among them a compound 2 (n = 7)

homolog of the hit 1 (n = 1) appeared as the ﬁrst promising inhibitor

candidate. Nevertheless, any of these inhibitors disclosed IL-15 selec-

tivity, displaying the same inhibitory activity on the IL-2 protein. Based

on the physicochemical properties of the exposed residues to IL-2Rβ

binding site on IL-15 and IL-2 and on the docking of compound 1 to IL-15

2.2.1. In vitro inhibition of cell proliferation and Stat5 phosphorylation

First of all, similar IC50 values were retrieved for the reference

2

9

compounds 1 and 2, as compared with those previously published

both in the cell proliferation and p-Stat5 assays (IC50 = 22.5 and 4.5

for 1, respectively and IC50 = 2.2 and 0.19 M for 2, respectively),

(

Fig. 1), we have pursued our SAR study towards higher IL-15 inhibitor

μM

efﬁciency and selectivity. Exploring the protein surfaces close to the

μ

4

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

Scheme 2. Synthesis of the N-triazolyl dimethyl propylamine 20, N-triazolyl propanol 21 and N-triazolyl propionionic acid 22 compounds (n = 7).

Scheme 3. Synthesis of the N-phthalazino propionaldehyde 29 and derivatives 30–32 (n = 1).

ascertaining the good stability of the two compounds as well as of the

two cell lines used (Tables 1 and 2).

favor hydrophobic interactions with the isoleucine 68 and leucine 69

residues of IL-15. Moreover asparagine residues (Fig 1B, residues N1, N4

and N72) were also pointing in the vicinity of the phthalazinone moiety.

To experimentally sustain the binding mode of this family of inhibitors,

we chose to change the N-methyl group with hydrophilic substituents

sequentially on the triazole or phthalazinone heterocycles to develop

In the predicted binding mode of the hit 1 (Fig. 1B), the methyl

substituent of the triazole heterocycle was found to share hydrogen bond

with the carbamoyl group of the residue asparagine 65 (N65) of IL-15,

and the methyl substituent of the phthalazinone moiety seemed to

5

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

Scheme 4. Linear synthesis of the N-phthalazino substituted compounds with the longer linker chain (n = 7): 40 (N-dimethylaminopropyl residue), 41 (N-propanol

residue), 42 (N-propionaldehyde residue), and 43 (N-propionic acid residue).

Scheme 5. Synthesis of the NH-phthalazinone analogue 46.

Scheme 6. Synthesis of the benzophenone analogue 49.

two series of analogues of compounds 1 and 2 (Fig. 2, series 1 and series

As noticed in our previous SAR study,²⁹some analogues of lead 2

having a longer alkyl chain (n = 7), appeared among the most inter-

esting molecules. In these novel series, introduction of an acetal group

(compound 9) on the N-substituent of the triazole, a protected or not

protected hydroxyl function on either of the heterocyles (compounds 19,

39 or compounds 21, 41) were found deleterious since low or no

inhibitory effect was measured either in the cell proliferation and p-

2

, respectively).

The targeted analogues of hit 1 bearing the shorter chain (n = 1),

uniformly gave no inhibition of cell proliferation (Table 1) as well as p-

Stat5 (Table 2). One exception was the N-triazolyl substituted analogue

1

0, with an aldehyde residue, giving a similar inhibitor potency (IC50

values of 6.0 and 4.2 M, respectively) than the hit 1.

μ

6

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

Table 1

Effect of the compounds on 32Dβ cell proliferation.

R

1

R

2

Inhibition of 32β cell proliferation

a

IC50 (µM)

b

c

b

c

Cpd

RLI

IL-2

Cpd

RLI

IL-2

n ¼ 1

n ¼ 7

Me

1

8

22.5 ± 4.5

> 30

21.1 ± 3.4

> 30

2

9

2.2 ± 0.4

3.9 ± 1.2

12.1 ± 2.8

16.4 ± 4.7

Me

10

6.0 ± 1.2

6.4 ± 1.3

11

2.5 ± 0.6

5.5 ± 1.1

H

Me

12

19

1.1 ± 0.1

> 30

3.5 ± 0.3

> 30

Me

13

14

> 30

21

22

> 30

23.5 ± 4.9

Me

20

3.7 ± 1.4

5.5 ± 2.0

Me

28

29

> 30

Me

39

42

> 30

~ 30

8.9 ± 1.4

11.7 ± 1.9

Me

30

31

> 30

41

43

> 30

Me

32

18.8 ± 2.4

16.7 ± 2.9

40

2.3 ± 0.5

3.2 ± 0.3

Me

H

46

49

> 30

Me

6.7 ± 3.0

20.6 ± 5.6

a

b

c

All values are the mean of at least three independent experiments. RLI (100 pM). IL-2 (1.5 nM).

Stat5 assays (Tables 1 and 2). Some improvement was observed with the

introduction of a carboxylic function as depicted on the triazole

binding model (Fig. 1B). Interestingly, in the N-substituted phthalazi-

none series 2, the NH-free analogue 46 appeared absolutely inactive in

cell proliferation and Stat5 assays and 42 with the propionaldehyde

analogue 22 (IC50 = 9.5

analogue 43 (IC50 > 30

μ

M for p-Stat5) but not for the phthalazinone

μ

M for p-Stat5). Furthermore, the dimethyla-

chain gave values in the range of 10

2).

μM in both bioassays (Tables 1 and

minopropyl residue gave similar results in the cell proliferation assay

M) whatever grafted on the triazole (compound 20) or on

(

IC50 = 2–4

μ

The methyl group on the phthalazinone heterocycle (series 2) of

compound 2 seems to have an essential role since its substitution with

more polar groups (compounds 39, 40, 41, 43) or its withdrawal

(compound 46) led to compounds unable to inhibit IL-15-dependent cell

the phthalazinone (compound 40) heterocycles (Table 1) albeit no

inhibitory effect of these compounds was detectable in the p-Stat5 assay

(

Table 2, IC50 > 30 M). This discrepancy could reﬂect a non-speciﬁc

μ

effect of these compounds affecting the proliferation of the cells inde-

pendently to Stat5 signaling pathway and consequently of IL-15 or IL-2

stimulation.

proliferation as well as Stat5 phosphorylation (Table 2, IC50 > 30 M).

μ

These results can strengthen the hypothetic binding model which

pointed on the interaction of lipophilic residues such as isoleucine 68

and leucine 69 with the N-methyl-phthalazinone moiety.

As observed with the propionaldehyde derivative 10 in series 1, the

analogue 11 with a seven carbons chain appeared as the most efﬁcient

inhibitor in this series with its NH-free triazolyl analogue 12, which gave

p-Stat5 inhibition of 280 nM compared to 710 nM for 11. Because this

NH-free triazolyl analogue 12 derived from the propionaldehyde 11

following a retro-Michael process, some doubt remains on the biological

stability of the aldehyde 11 and so its efﬁciency level in these bioassays.

At least, compounds 11 and the NH-analogue 12 behaved similarly to

compound 2 suggesting the involvement of plausible hydrogen bonds

with the carbamoyl function of asparagine N65, as depicted in the

Satisfyingly, data obtained from cell proliferation assays correlate

those found in the p-Stat5 assay, highlighting that the proliferative

inhibitory effects measured for IL-15 and IL-2 with these analogues

resulted from the inhibition of these cytokines signaling pathway. Un-

fortunately, no selectivity of IL-15 versus IL-2 could be obtained with

these novel series of derivatives.

Finally the substitution of the terminal 2,4-dichlorophenyl group of

compound 2 with a benzophenone moiety led to the compound 49

which displayed efﬁciency in the same range of its parent 2 (Tables 1-2,

7

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

Table 2

Effect of the compounds on Stat5 phosphorylation in NK-92 cells.

R

1

R

2

Inhibition of p-Stat5 in NK-92 cells

a

IC50 (µM)

b

c

b

c

Cpd

IL-15

IL-2

Cpd

IL-15

IL-2

n ¼ 1

n ¼ 7

Me

1

8

4.5 ± 0.7

> 30

7.7 ± 1.0

> 30

2

9

0.19 ± 0.05

0.35 ± 0.09

> 30

~ 30

Me

10

4.2 ± 0.6

8.6 ± 0.7

11

0.71 ± 0.15

0.96 ± 0.16

H

Me

12

19

0.28 ± 0.08

> 30

0.45 ± 0.09

> 30

Me

13

14

> 30

21

22

> 30

9.5 ± 0.7

14.3 ± 2.7

Me

20

> 30

Me

28

29

~ 30

~ 20

> 30

Me

39

42

> 30

14.9 ± 2.6

21.9 ± 6.0

Me

30

31

~ 30

> 30

41

43

> 30

Me

32

> 30

40

> 30

Me

H

46

49

~ 20

> 30

Me

0.05 ± 0.02

0.08 ± 0.03

a

b

c

All values are the mean of at least three independent experiments. IL-15 at 50 pM. IL-2 at 250 pM.

Fig. 3). It can be classiﬁed such as the alkoxy analogues 2A and 2B or the

carboxylate analogue 2C (Fig.2), among the best representatives of this

family with an IC50 of 50 nM in the p-Stat5 bioassay and no selectivity

observed versus IL-2 (IC50 = 80 nM). However, such compounds may be

of therapeutic interest in pathologies where both cytokines are known to

be involved.³⁷

With the efﬁcient N-Me inhibitors such as 2, the benzophenone

analogue 49, and the NH-triazolyl analogue 12 the protein afﬁnity

values were good and closed to 2–3

aldehyde function such as 11 and 42, displayed higher K

5.9 and 7.8 M, respectively) in agreement with their IC50 values in the

μ

M. The compounds bearing an

d

values (K

d

=

μ

p-Stat5 assay. Moreover, these data allow us to conﬁrm that aldehyde 11

is able to bind to the IL-15 protein attesting that the observed biological

results (Tables 1 and 2) do not result from its optional metabolite 12.

2

.2.2. Binding of selected compounds to IL-15

To complete our study, we focussed on the binding of selected

compounds 2, 11–12, 41–42, 46 and 49 to IL-15 using SPR technology.

The afﬁnity of these compounds for IL-15 was determined and compared

to their pharmacological proﬁle measured in the p-Stat5 assay (Table 3).

Thus, recombinant IL-15 was covalently immobilized to sensor chips

and the binding at increasing concentrations of the compounds was

monitored. Satisfyingly, we could observe that the NH-free phthalazi-

none compound 46 lacking activity in both cell proliferation and p-Stat5

assays did not bind to IL-15 protein. However, the propanol analogue 41

3. Conclusion

Starting from IL-15 inhibitors identiﬁed in our ﬁrst SAR study and

based on docking structural data obtained with our hit 1, we have

developed two novel series of potential IL-15 inhibitors with speciﬁc

modiﬁcations on the N-triazole or N-phthalazinone heterocycles and we

also completed our initial series. Twenty-ﬁve compounds have been

prepared and tested in two functional bioassays, cell proliferation and p-

Stat5 bioassay. In the novel series, the best compounds were found in the

N-triazole modiﬁed family, with the nitrogen substituted with a pro-

pionaldehyde chain or free. We also identiﬁed a novel benzophenone

analogue of our lead compound 2, with a very good functional activity.

with similar proﬁle was found to be able to bind to IL-15 with a K

d

value

of 3.9 M. Its behaviour must probably proceed by a different and less

μ

speciﬁc mode than that of the lead compound 2, since it was not efﬁcient

enough to impair the functional effects of the cytokine.

8

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

Fig. 3. Inhibition of IL-15-dependent cell pro-

liferation and Stat5 phosphorylation. (A-B)

3

2Dβ cell proliferation induced by a ﬁxed con-

centration of the IL-15 agonist RLI (100 pM)

and increasing concentrations of indicated

compounds was assessed by the Alamar Blue

reduction assay. Data showed the effect of

indicated compounds calculated as the per-

centage of the proliferation response induced

by 100 pM of RLI. (C-D) Phosphorylation of

Stat5 was evaluated in NK-92 cells stimulated

for 1 h by a ﬁxed concentration of IL-15 (50

pM) after a 30 min incubation with increasing

concentrations of indicated compounds. Data

showed the effect of indicated compounds

calculated as the percentage of the p-Stat5

response induced by 50 pM of IL-15. All data

are the mean ± SEM of at least 3 independent

experiments.

Table 3

a

IL-15 binding (K

d

) of compounds 2, 11–12, 41, 42, 46 and 49 associated with their p-Stat5 inhibitory effect (IC50).

R

1

R

2

Cpd

Inhibition of p-Stat5 in NK-92 cells

Direct interaction SPR

IL-15 binding

IL-15

IC50 (µM)

K

d

(µM)

Me

H

Me

H

2

0.19 ± 0.05

0.05 ± 0.02

0.28 ± 0.08

~ 20

1.3

3.5

2.4

49

12

46

11

Me

no binding

5.9

Me

0.71 ± 0.15

Me

41

42

> 30

3.9

7.8

14.9 ± 2.6

a

Table S1 (supporting information) detailed molecular properties (ALogP, … ) of these compounds.

These compounds were conﬁrmed as good binders of the targeted pro-

tein IL-15. Despite our efforts, we did not succeed in improving IL-15

selectivity in respect with IL-2. However, such inhibitors targeting

both IL-15 and IL-2 may be of therapeutic interest in pathologies such as

HAM/TSP,¹²CTCL, LGLL and HTLV-1 driven ATL, for which these two

cytokines have been shown to be involved.

performed on silica-covered aluminum sheets (Kieselgel 60F254,

MERCK). Eluted TLC was revealed using UV radiation (λ = 254 nm), or

molybdate solution. Flash column chromatography was performed on

silica gel 60 ACC 40–63 µm (SDS-CarloErba). NMR spectra were recor-

ded on an Avance I 300 MHz Bruker, on an Avance III 400 MHz Bruker

and an Avance III 500 MHz Bruker 500 at room temperature, on samples

dissolved in an appropriate deuterated solvent. References of tetrame-

3

1

13

4

. Experimental section

thylsilane (TMS) for H and deuterated solvent signal for C were used.

Chemical displacement values (δ) are expressed in parts per million

(ppm), and coupling constants (J) in Hertz (Hz). High-Resolution Mass

Spectrometry (HRMS in Da unit) analyses were recorded on a Xevo-Q-

Tof Waters in the CEISAM Laboratory (AMaCC platform, Nantes) or on

4

.1. Chemistry

General. All solvents used were reagent grade and TLC was

9

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

a LC-Q-TOF (Synapt-G2 HDMS, Waters) in the IRS-UN center (Mass

Spectrometry platform, Nantes).

MHz, CDCl

3

): δ(ppm) 167.3 (CO), 159.5 (CO), 152.9 (CIV-C=N), 151.5

(CIV-C=N), 140.9 (CIV-C=N), 134.0 (CIV), 133.4 (Car-Phthal), 132.0 (Car-

Phthal), 129.5 (CIV), 129.1 (CIV and Car), 128.0 (CIV), 127.5 (Car), 127.3

(Car-Phthal), 125.2 (Car-Phthal), 124.8 (CIV), 123.2 (Car), 100.1 (CH), 62.1

4

.1.1. 1,1-diethoxy-3-isothiocyanatopropane (4)

To a solution of 3,3-diethoxypropan-1-amine (1 eq, 11 mmol, 1.78

(2 × CH

29.9 (CH

2

), 40.1 (NCH

2

), 39.5 (NMe), 36.0 (SCH

2

), 33.1 (NCH

2 2

CH ),

+

mL) and anhydrous triethylamine (3.3 eq, 33 mmol, 5 mL) in THF (1.1

2

), 15.3 (2 × CH

3

). HRMS (ESI ): calcd for C27

H31Cl N O S

2 6 4

ꢀ 1

◦

+

mol.L ) at 0 C under argon atmosphere was added carbon disulﬁde (1

[M+Na] m/z 605.1499, found: 605.1503.

eq, 11 mmol, 0.66 mL) over a period of 30 min and then stirred at r.t for

◦

2

h. The mixture was cooled to 0 C using an ice-bath before tosyl

4.1.4. N-(2,4-dichlorophenyl)-8-((4-(3,3-diethoxypropyl)-5-((3-methyl-4-

oxo-3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)

octanamide (9)

chloride (1.1 eq, 12.1 mmol, 2.3 g) was added in one portion, and the

reaction was stirred at r.t for another 30 min. 1 N HCl (10 mL) and t-

butyl methyl ether (MTBE) (10 mL) were added to the mixture. The

aqueous layer was extracted with MTBE (10 mL) and the combined

To a solution of thiol 5 (1 eq, 0.46 mmol, 187 mg) in anhydrous DMF

(0.08 mol.L ¹) at room temperature under argon atmosphere was added

ꢀ

organic layers dried over Na

2

SO

4

, ﬁltered and concentrated. The crude

K

2

CO

3

(1.5 eq, 0.69 mmol, 95 mg), followed by a solution of bromoa-

◦

oil was puriﬁed by ﬂash column chromatography on silica gel (PE/

mide 7 (1.1 eq, 0.51 mmol, 188 mg) in DMF at 0 C. The mixture was

stirred 4 h at room temperature, quenched with water and extracted

with AcOEt. The combined organic layers were washed with brine, dried

AcOEt, 98:2 to 96:4) to afford the desired product 4 as a colorless oil

1

(

9.68 mmol, 1.83 g, 88%). H NMR (300 MHz, CDCl

3

): δ(ppm) 4.61 (t,

, 2 × OCH ), 2.01–1.93

). C NMR (75 MHz,

3

J = 5.5 Hz, 1H, CH), 3.73–3.46 (m, 6H, NCH

2

over MgSO

4

, ﬁltrered and concentrated under reduced pressure. The

3

13

1

(

m, 2H, CH

2

), 1.21 (t, J = 7.0 Hz, 6H, 2 × CH

3

product 9 was obtained in quantitave yield without any puriﬁcation. H

3

4

CDCl

3

): δ(ppm) 130.2 (SCN), 100.2 (CH), 62.4 (OCH

2

), 41.3 (CH

2

), 34.2

NMR (400 MHz, CDCl

3

): δ(ppm) 8.43 (dd, J = 7.7 Hz, J = 1.2 Hz, 1H,

+

3

(

CH

2

), 15.4 (2 × CH

3

). HRMS (ESI ): calcd for C

8

H

2

15NO NaS [M+Na]

H

ar-Phthal), 8.32 (d, J = 8.7 Hz, 1H, Har), 8.22 (d, J = 7.7 Hz, 1H, Har-

3

m/z 212.0716; found 212.0709.

Phthal), 7.77 (m, 2H, Har-Phthal), 7.60 (bs, 1H, NH), 7.36 (d, J = 2.35 Hz,

H, Har), 7.23 (dd, ³J = 2.35 Hz, J = 8.88 Hz, 1H, Har), 4.53 (s, 2H,

4

1

4

.1.2. 4-((4-(3,3-diethoxypropyl)-5-mercapto-4H-1,2,4-triazol-3-yl)

CH

2

), 4.50 (t, ³J = 5.2 Hz, 1H, CH), 4.05 (t, 2H, NCH

2

), 3.80 (s, 3H,

3

methyl)-2-methylphthalazin-1(2H)-one (5)

NMe), 3.61 (m, 2H, 2 × OCH

2

), 3.45 (m, 2H, 2 × OCH

2

), 3.23 (t, J = 7.3

CO), 1.93 (m, 2H,

), 1.17 (t, 6H, 2

To a suspension of hydrazide 3 (1 eq, 0.82 mmol, 190 mg) in

Hz, 2H, SCH

NCH CH

× CH

2

), 2.40 (t, ³J = 7.30 Hz, 2H, CH

2

ꢀ

1

anhydrous ethanol (0.21 mol.L ) at room temperature under argon

atmosphere was added isothiocyanate 4 (1 eq, 0.82 mmol, 155 mg)

followed by triethylamine (5 eq, 4.1 mmol, 0.57 mL). The mixture was

reﬂuxed overnight under argon atmosphere. After completion of the

reaction, the mixture was allowed to cool to room temperature,

concentrated, and directly puriﬁed by ﬂash column chromatography on

silica gel (DCM/AcOEt, 50:50) to afford the desired product 5 as a white

2

), 1.72 (m, 4H, 2 × CH

2

), 1.37 (m, 6H, 3 × CH

2

). ¹C NMR (100 MHz, CDCl

3

): δ(ppm) 171.4 (CO); 159.6 (C0),

3

151.9 (CIV-C=N), 151.8 (CIV-C=N), 141.1 (CIV-C=N), 133.6 (Car-Phthal),

133.4 (CIV), 131.9 (Car-Phthal), 129.2 (CIV), 128.8 (Car), 128.0 (2 × CIV),

128.0 (Car), 127.2 (Car-Phthal), 125.5 (Car-Phthal), 123.3 (CIV), 122.6 (Car),

100.2 (CH), 61.9 (2 × OCH

33.3 (NCH CH ), 33.1 (SCH

28.8 (CH ), 28.5 (CH

for C33 SCl

2

), 40.1 (NCH

), 30.0 (CH

2 2

), 39.5 (NMe), 37.9 (CH CO),

2

), 29.5 (2 × CH

), 15.4 (2 × CH

2

), 29.0 (CH

2

),

powder (0.66 mmol, 265 mg, 80%). ¹H NMR (300 MHz, DMSO‑d

δ(ppm) 13.55 (s, 1H, SH), 8.34–8.26 (m, 1H, Har-Phthal), 8.05–7.98 (m,

):

), 25.4 (CH

+

6

2

3

). HRMS (ESI ): calcd

+

H

42

N

6

O

4

2

[M+H] m/z 689.2444, found 689.2448.

3

1

H, Har-Phthal), 7.96–7.84 (m, 2H, Har-Phthal), 4.57 (t, J = 5.1 Hz, 1H,

CH), 4.52 (s, 2H, CH ), 4.01 (m, 2H, NCH ), 3.67 (s, 3H, NMe),

), 3.43–3.35 (m, 2H, OCH ), 2.04–1.95 (m, 2H,

2

4.1.5. N-(2,4-dichlorophenyl)-2-((5-((3-methyl-4-oxo-3,4-

dihydrophthalazin-1-yl)methyl)-4-(3-oxopropyl)-4H-1,2,4-triazol-3-yl)

thio)acetamide (10)

3

.58–3.47 (m, 2H, OCH

2

3

13

NCH

2

CH

2

), 1.02 (t, J = 7.0 Hz, 6H, 2 × CH

2 3

CH ). C NMR (75 MHz,

DMSO‑d

6

): δ(ppm) 166.6 (CIV-C=N), 158.4 (CO), 149.3 (CIV-C=N), 140.6

To a solution of ketal 8 (1 eq, 0.17 mmol, 100 mg) in THF (0.04 mol.

ꢀ

1

(

C

IV-C=N), 133.2 (CHar-Phthal), 132.0 (CHar-Phthal), 128.8 (CIV), 127.1

IV), 126.1 (CHar-Phthal), 125.6 (CHar-Phthal), 100.0 (CH), 60.6 (2 ×

L

) at room temperature was added a 1 N solution of hydrochloric acid

◦

C

(33% v/v). The mixture was stirred 2 h at 40 C and quenched by

CH

2

CH

3

), 39.6 (NCH

CH

2

), 38.8 (NMe), 30.7 (NCH

2

CH

2

), 28.8 (CH

2

), 15.1

addition of a saturated solution of Na

extracted with DCM. The organic layers were combined and washed

with brine, dried over Na SO and concentrated. The crude product was

2 3

CO until pH 7. The product was

+

S [M+Na]⁺m/z

(

2 × CH

2

3

). HRMS (ESI ): calcd for C19

H

25

N

5

NaO

3

4

26.1570; found 426.1566.

2

4

puriﬁed by ﬂash column chromatography on silica gel (DCM/MeOH,

4

.1.3. N-(2,4-dichlorophenyl)-2-((4-(3,3-diethoxypropyl)-5-((3-methyl-4-

97:3) to afford the desired compound 10 as a white powder (0.12 mmol,

1

oxo-3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)

acetamide (8)

64 mg, 71%). H NMR (300 MHz, CDCl

3

): δ(ppm) 9.97 (s, 1H, NH), 9.77

(s, 1H, CHO), 8.47–8.42 (m, 1H, Har-Phthal), 8.23–8.16 (m, 2H, Har-Phthal

To a solution of thiol 5 (1 eq, 0.57 mmol, 230 mg) in DMF (0.06 mol.

and Har), 7.86–7.75 (m, 2H, Har-Phthal), 7.31 (d, ⁴J = 2.4 Hz, 1H, Har),

ꢀ

1

3

4

3

L

) under argon atmosphere at room temperature was added K

1.5 eq, 0.86 mmol, 118 mg) and a solution of -bromoamide 6 (1.1 eq,

.63 mmol, 177 mg) in DMF (0.7 mol.L ) was added. The mixture was

2

CO

3

7.19 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H, Har), 4.62 (s, 2H, CH

2

), 4.30 (t, J

3

(

α

= 7.0 Hz, 2H, NCH

7.0 Hz, 2H, NCH CH

166.9 (CONH), 159.5 (C

2

), 4.05 (s, 2H, SCH

2

), 3.76 (s, 3H, NMe), 2.88 (t, J =

ꢀ 1

13

0

2

2 3

). C NMR (75 MHz, CDCl ): δ(ppm) 197.9 (CHO),

–

O), 153.0 (CIV-C=N), 151.3 (CIV-C=N), 140.9

then stirred at room temperature for 1 h until completion. A saturated

solution of ammonium chloride was added and the product was

extracted twice with AcOEt. The organic layer was washed several times

(CIV-C=N), 133.9 (CIV), 133.5 (CHar-Phthal), 132.2 (CHar-Phthal), 129.6

(CIV), 129.1 (CHar), 129.0 (CIV), 128.0 (CIV), 127.6 (CHar), 127.4 (CHar-

with brine, dried over Na

2

SO

4

and concentrated. The crude product was

2

Phthal), 125.2 (CHar-Phthal), 124.8 (CIV), 123.2 (CHar), 42.6 (NCH ), 39.5

+

puriﬁed by ﬂash column chromatography on silica gel (DCM/MeOH,

(NMe), 37.6 (NCH

for C23 21Cl

2

CH

2

), 36.1 (SCH

2

), 30.0 (CH

2

). HRMS (ESI ): calcd

+

9

3

8

8:2) to afford the desired compound 8 as a white powder (0.55 mmol,

H

2

N

6

O

3

S [M+H] m/z 531.0773, found 531.0784.

1

3

18 mg, 96%). H NMR (300 MHz, CDCl ): δ(ppm) 10.13 (s, 1H, NH),

.50–8.39 (m, 1H, Har-Phthal), 8.26–8.15 (m, 2H, Har-Phthal and Har),

4.1.6. N-(2,4-dichlorophenyl)-8-((5-((3-methyl-4-oxo-3,4-

dihydrophthalazin-1-yl)methyl)-4-(3-oxopropyl)-4H-1,2,4-triazol-3-yl)

thio)octanamide (11) and N-(2,4-dichlorophenyl)-2-((5-((3-methyl-4-oxo-

3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)acetamide

(12)

4

7

.85–7.73 (m, 2H, 2 × Har-Phthal), 7.30 (d, J = 2.3 Hz, 1H, Har), 7.18 (dd,

3

4

3

J = 8.9 Hz, J = 2.3 Hz, 1H, Har), 4.54 (s, 2H, CH

2

), 4.50 (t, J = 4.8 Hz,

1

3

H, CH), 4.13–4.01 (m, 4H, NCH

.66–3.54 (m, 2H, 2 × OCH

2 2

and SCH ), 3.80 (s, 3H, NMe),

2

), 3.49–3.38 (m, 2H, 2 × OCH ), 2.02–1.88

2

3

13

(

m, 2H, NCH

2

CH

2

), 1.15 (t, J = 7.0 Hz, 6H, 2 × CH

3

). C NMR (75

To a solution of ketal 9 (1 eq, 0.3 mmol, 206 mg) in acetone (0.04

1

0

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

mol.L ¹, 7.5 mL) was added para-toluenesulfonic acid (0.1 eq, 0.03

mmol, 5.1 mg) and then a 1 N solution of hydrochloric acid (3 mL, 10 eq,

the mixture became clear), at room temperature. The reaction was

stirred for 24 h at room temperature. The mixture was quenched by

ꢀ

4.1.8. 3-(3-((2-((2,4-dichlorophenyl)amino)-2-oxoethyl)thio)-5-((3-

methyl-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-4-yl)

propanoic acid (14)

To a solution of aldehyde 10 (1 eq, 0.15 mmol, 80 mg) in a mixture of

ꢀ 1

addition of a saturated solution of Na

product was extracted with AcOEt and organic layers washed with brine,

dried over Na SO and concentrated. The crude product was puriﬁed by

ﬂash column chromatography on silica gel (CHCl /MeOH, 99:1 to 98:2)

2

CO

3

until pH 7–8 (10 mL). The

tert-butanol/water (3:1, 0.02 mol.L ) at room temperature was added

2-methyl-2-butene (50 eq, 7.5 mmol, 0.8 mL), followed by NaH PO

2

4

2

4

dihydrate (10 eq, 1.5 mmol, 235 mg). The mixture was stirred 20 min.

before sodium chlorite (4.5 eq, 0.68 mmol, 61 mg) was added. The

mixture was stirred 2 h. at room temperature and ethyl acetate was

added followed by an aqueous 1 N HCl solution. The aqueous layer was

extracted with ethyl acetate. The organic layers were combined and

3

to give a ﬁrst fraction 12 (0.054 mmol, 30 mg, 17%) and the desired

compound 11 in a second fraction, as a white amorphous powder (0.18

mmol, 110 mg, 59%). Compound 11: ¹H NMR (400 MHz, CDCl

3

):

δ(ppm) 9.71 (s, 1H, CHO), 8.42–8.39 (m, 1H, Har-Phthal), 8.28 (d, J = 6.6

Hz, 1H, Har), 8.21–8.19 (m, 1H, Har-Phthal), 7.81–7.72 (m, 2H, 2 × Har-

Phthal), 7.62 (s, 1H, NH), 7.33 (d, J = 2.2 Hz, 1H, Har), 7.21 (dd, J = 8.8

washed with brine, dried over Na

desired compound 14 as a white solid (0.09 mmol, 49 mg, 60%). H

NMR (400 MHz, DMSO‑d

): δ(ppm) 9.97 (s, 1H, NH), 8.31–8.26 (m, 1H,

ar-Phthal), 8.05–8.00 (m, 1H, Har-Phthal), 7.91–7.82 (m, 2H, Har-Phthal),

2 4

SO and concentrated to obtain the

1

6

Hz, J = 2.2 Hz, 1H, Har), 4.55 (s, 2H, CH

2

), 4.27 (t, J = 7.1 Hz, 2H,

S), 2.81 (t, J = 7.2

CONH), 1.77–1.65 (m, 4H,

H

3

4

3

NCH

Hz, 2H, CH

× CH ), 1.42–1.34 (s, 6H, 3 × CH

δ(ppm) 197.7 (CHO), 171.2 (CONH), 159.3 (C

51.3 (CIV-C=N), 141.0 (CIV-C=N), 133.4 (CIV), 133.3 (CHar-Phthal), 131.9

CHar-Phthal), 128.9 (CIV), 128.6 (CHar), 127.8 (CIV), 127.7 (CHar-Phthal),

27.0 (CHar), 125.3 (CHar-Phthal), 122.5 (CHar), 42.9 (NCH ), 39.3

NMe), 37.6 (NCH CH ), 37.2 (SCH ), 33.0 (CH ), 29.9 (CH ), 29.3

CH ), 28.8 (CH ), 28.6 (CH ), 28.3 (CH ), 25.1 (CH ). Two quaternary

33Cl

2

), 3.75 (s, 3H, NMe), 3.21 (t, J = 7.1 Hz, 2H, CH

2

7.75 (d, J = 8.8 Hz, 1H, Har), 7.62 (d, J = 2.4 Hz, 1H, Har), 7.38 (dd, J

4

3

2

CO), 2.39 (t, J = 7.4 Hz, 2H, CH

2

= 8.8 Hz, J = 2.4 Hz, 1H, Har), 4.60 (s, 2H, CH

2

), 4.26 (t, J = 7.3 Hz,

1

3

2

). C NMR (100 MHz, CDCl

3

):

2H, NCH

2

), 4.14 (s, 2H, SCH

2

), 3.65 (s, 3H, NMe), 2.70 (t, J = 7.3 Hz,

–

13

–

): δ(ppm) 171.8 (C O),

O), 151.7 (CIV-C=N),

2H, NCH

166.6 (C

2

CH

2

). C NMR (100 MHz, DMSO‑d

6

–

1

–

O), 158.4 (C

O), 152.7 (CIV-C=N), 149.2 (CIV-C=N), 141.4

(

(CIV-C=N), 133.8 (CIV), 133.1 (CHar-Phthal), 131.9 (CHar-Phthal), 129.4

1

2

(CIV), 128.9 (CHar), 128.8 (CIV), 127.7 (CHar), 127.2 (CIV), 126.7 (CIV),

(

2

126.3 (CHar), 126.1 (CHar-Phthal), 125.7 (CHar-Phthal), 40.2 (NCH ), 38.7

+

2

+

2

(NMe), 37.2 (SCH

for C23 Cl

2

), 33.6 (NCH

2

+

CH

2

), 28.7 (CH

2

). HRMS (ESI ): calcd

carbon atoms are missing. HRMS (ESI ): calcd for C29

H

1

2

N

6

O

3

S

H

20

O

4

N

6

NaS [M+Na] m/z: 569.0536, found 569.0531.

+

[

M+H] m/z 615.1712, found 615.1719. Compound 12: H NMR (400

MHz, CDCl

): δ(ppm) 8.30–8.27 (m, 1H, Har-Phthal), 8.25 (br s, 1H, NH),

.81–7.79 (m, 1H, Har), 7.72–7.63 (m, 3H, 3 × Har-Phthal), 7.33 (d, J =

.4 Hz, 1H, Har), 7.19 (dd, J = 8.8 Hz, J = 2.3 Hz, 1H, Har), 4.42 (s, 2H,

), 3.67 (s, 3H, NMe), 3.07 (t, J = 7.2 Hz, 2H, CH S), 2.38 (t, J = 7.3

Hz, 2H, CH ), 1.38–1.24 (s, 6H, 3 ×

CONH), 1.72–1.60 (m, 4H, 2 × CH

). C NMR (100 MHz, CDCl ): δ(ppm) 171.6 (CONH), 159.6 (C

57.3 (CIV-C=N), 142.22 (CIV-C=N), 133.4 (CIV), 133.2 (CHar-Phthal), 131.7

CHar-Phthal), 129.2 (CIV), 128.8 (CHar), 127.9 (CIV), 127.8 (CHar-Phthal),

3

4.1.9. 4-((4-(3-((tert-butyldimethylsilyl)oxy)propyl)-5-mercapto-4H-

7

2

1,2,4-triazol-3-yl)methyl)-2-methylphthalazin-1(2H)-one (17)

To a suspension of hydrazide 3 (1 eq, 1.29 mmol, 300 mg) in EtOH

(0.065 mol.L ¹) was added 15 (1.28 eq, 1.64 mmol, 380 mg) and Et

ꢀ

CH

2

3

N

2

(4.5 eq, 5.81 mmol, 0.81 mL). The resulting mixture was heated at reﬂux

for 14 h. The resulting solution was concentrated, the residue was sol-

ubilized in AcOEt and washed with brine. The organic layer was dried

1

3

–

O),

CH

2

3

1

(

2 4

over Na SO , ﬁltered and concentrated. The crude was puriﬁed by col-

1

3

2

27.1 (CHar), 125.1 (CHar-Phthal), 123.5 (CIV), 122.7 (CHar), 39.5 (NMe),

7.8 (SCH ), 32.7 (CH ), 31.7 (CH ), 29.5 (CH ), 28.9 (CH ), 28.7 (CH ),

8.3 (CH

umn chromatography on silica gel (cyclohexane/AcOEt, 50:50) to afford

1

2

the thiol 17 as a white solid (1.01 mmol, 450 mg, 80%). H NMR (300

2

), 25.3 (CH

2

). Two quaternary carbon atoms are missing.

MHz, CDCl

3

): δ(ppm) 8.54–8.43 (m, 1H, Har-Phthal), 7.90–7.72 (m, 3H, 3

), 4.23 (t, J = 7.1 Hz, 2H, NCH ), 3.80 (s,

O), 2.05 (m, 2H, NCH CH ), 0.83

). C NMR (75 MHz, CDCl ):

O), 139.9 (2 × CIV-C=N), 133.3 (CHar-

+

35

S [M+H]⁺m/z 559.1450, found

HRMS (ESI ): calcd for C26

H

29Cl

2

N

6

O

2

× Har-Phthal), 4.42 (s, 2H, CH

2

5

59.1464.

3H, NMe), 3.68 (t, J = 5.5 Hz, 2H, CH

2

1

3

(

s, 9H, 3 × CH3-tBu), 0.04 (s, 6H, 2 × SiCH

3

–

4

.1.7. N-(2,4-dichlorophenyl)-2-((4-(3-hydroxypropyl)-5-((3-methyl-4-

δ(ppm) 167.8 (CIV-C=N), 159.5 (C

Phthal), 132.0 (CHar-Phthal), 128.9 (CIV), 128.1 (CIV), 127.6 (CHar-Phthal),

oxo-3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)

acetamide (13)

2 2

124.5 (CHar-Phthal), 59.7 (CH O), 41.8 (NCH ), 39.6 (NMe), 30.7

To a solution of aldehyde 10 (1 eq, 0.15 mmol, 80 mg) in ethanol

(NCH

SiCH

found 446.2046.

2

CH

2

), 29.4 (CH

2

), 25.9 (3 × CH3-tBu), 18.3 (CIV), ꢀ 5.2 (2 ×

0.03 mol.L ¹) at 0 C was added sodium borohydride (0.5 eq, 0.085

ꢀ

◦

). HRMS (ESI ): calcd for C21

+

H

SSi [M+H] m/z 446.2036;

+

(

3

31

N

5

O

2

◦

mmol, 3.2 mg). After stirring an hour at 0 C, the mixture was quenched

by addition of a saturated solution of ammonium chloride. The volatiles

were removed and the crude was diluted in DCM, washed with brine,

4.1.10. 4-((4-(3-(dimethylamino)propyl)-5-mercapto-4H-1,2,4-triazol-3-

yl)methyl)-2-methylphthalazin-1(2H)-one (18)

2 4

dried over Na SO and concentrated. The crude product was puriﬁed by

ﬂash column chromatography on silica gel (DCM/MeOH, 100:0 to 95:5)

To a suspension of hydrazide 3 (1 eq, 0.41 mmol, 95.5 mg) in EtOH

(0.41 mol.L ¹) was added 16 (1.02 eq, 0.457 mmol, 65.9 mg) and Et

ꢀ

to afford the desired alcohol 13 as a white powder (0.110 mmol, 60.9

3

N

mg, 67%). ¹H NMR (400 MHz, DMSO‑d

): δ(ppm) 9.97 (s, 1H, NH),

.31–8.27 (m, 1H, Har-Phthal), 8.05–8.01 (m, 1H, Har-Phthal), 7.92–7.83

(4.5 eq, 1.85 mmol, 0.26 mL). The resulting mixture was heated to reﬂux

30 h. The mixture was cooled to room temperature and concentrated

under reduced pressure. The residue (brown solid) was solubilized in

6

8

3

4

(

m, 2H, Har-Phthal), 7.79 (d, J = 8.8 Hz, 1H, Har), 7.63 (d, J = 2.4 Hz,

3

4

3

1

H, Har), 7.39 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H, Har), 4.68 (t, J = 4.9 Hz,

DCM, washed with brine and the organic layer was dried over Na

ﬁltered and concentrated. The crude was puriﬁed by column chroma-

tography on silica gel (DCM/MeOH-Et N (5%), 85:15) to afford the

corresponding thiol 18 as a brown sticky solid (0.17 mmol, 60.0 mg,

2 4

SO ,

3

1

H, OH), 4.55 (s, 2H, CH

2

), 4.16 (s, 2H, SCH

2

), 4.09 (t, J = 7.4 Hz, 2H,

NCH

2

), 3.67 (s, 3H, NMe), 3.45–3.39 (m, 2H, CH

2

OH), 1.82–1.74 (m,

): δ(ppm) 166.6 (C O),

3

1

3

–

2

H, NCH

2

CH

2

). C NMR (100 MHz, DMSO‑d

6

–

1

58.3 (C

–

O), 152.7 (CIV-C=N), 149.2 (CIV-C=N), 141.3 (CIV-C=N), 133.8

54%). H NMR (300 MHz, CDCl

7.99–7.60 (m, 3H, 3 × Har-Phthal), 4.47 (s, 2H, CH

NCH

CH NMe

(75 MHz, CDCl

3

): δ(ppm) 8.60–8.37 (m, 1H, Har-Phthal),

(

C

IV), 133.1 (CHar-Phthal), 131.8 (CHar-Phthal), 129.3 (CIV), 128.9 (CHar),

2

), 4.22–4.04 (m, 2H,

1

28.8 (CIV), 127.6 (CHar), 127.1 (CIV), 126.5 (CIV), 126.0 (CHar and CHar-

2

), 3.80 (s, 3H, NMe), 2.88 (s, 1H, SH), 2.33 (t, J = 6.7 Hz, 2H,

1

3

Phthal), 125.7 (CHar-Phthal), 57.5 (CH

SCH ), 32.1 (NCH CH ), 28.6 (CH

Cl

2

OH), 41.2 (NCH

2

), 38.8 (NMe), 36.8

2

), 2.22 (s, 6H, NMe

2

), 2.00 (p, J = 6.9 Hz, 2H, CH

2

). C NMR

–

O), 149.2 (CIV-C=N),

+

(

2

+

2

). HRMS (ESI ) calcd for

3

): δ(ppm) 167.9 (C-SH), 159.5 (C

C

23

H

23

O

3

N

6

2

S [M+H] m/z: 533.0924, found 533.0919.

140.1 (CIV-C=N), 133.3 (CHar-Phthal), 132.0 (CHar-Phthal), 128.9 (CIV),

28.0 (CHar-Phthal/CIV), 127.5 (CHar-Phthal/CIV), 124.6 (CHar-Phthal/CIV),

5.9 (CH NMe ), 46.0 (NMe), 45.1 (NMe), 42.6 (NCH ), 39.6 (NMe

-

1

5

2

1

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

+

Phthal), 29.8 (CH

2

), 25.6 (CH

2

). HRMS (ESI ): calcd for C17

H

23

N

6

OS

wt% in pyridine (75 eq, 16.40 mmol). The resulting colorless solution

was stirred 16 h at room temperature. The reaction mixture was

[

M+H]⁺m/z 359.1644; found 359.1654.

quenched with saturated aqueous NaHCO

3

solution and extracted with

4

SO ,

4

.1.11. 8-((4-(3-((tert-butyldimethylsilyl)oxy)propyl)-5-((3-methyl-4-

AcOEt. The organic layer was washed with brine, dried over Na

2

oxo-3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)-N-

2,4-dichlorophenyl)octanamide (19)

To a suspension of mercapto-triazole 17 (1 eq, 0.65 mmol, 291 mg)

was added bromoamide 7 (1.2 eq, 0.78 mmol, 287 mg) and K CO (1.7

ﬁltered and concentrated under reduced pressure. The crude was puri-

ﬁed by column chromatography on silica gel (DCM/MeOH, 95:5) to

(

afford the corresponding alcohol 21 as a white amorphous solid (0.19

1

2

3

mmol, 120 mg, 88%). H NMR (500 MHz, CDCl

3

): δ(ppm) 8.48–8.39 (m,

ꢀ

1

eq, 1.11 mmol, 153 mg) in DMF (0.065 mol.L ). After stirring 24 h at

room temperature, the mixture was quenched with a saturated aqueous

1H, Har-Phthal), 8.33 (d, J = 8.9 Hz, 1H, Har), 8.25 (d, J = 7.5 Hz, 1H, Har-

Phthal), 7.83–7.73 (m, 2H, 2 × Har-Phthal), 7.59 (s, 1H, NH), 7.37 (d, J =

NH

DCM. The organic layers were dried over Na

4

Cl solution. Brine was added and the product was extracted with

2.4 Hz, 1H, Har), 7.22 (m, 1H, Har), 4.52 (s, 2H, CH

2

), 4.11 (t, J = 7.3 Hz,

OH), 3.23 (t, J =

CONH), 1.81 (m, 6H,

). C NMR (75 MHz, CDCl ): δ(ppm) δ 171.4

–

O), 151.9 (CIV-C=N), 151.7 (CIV-C=N), 141.4 (CIV-

2

SO , ﬁltered and concen-

4

2H, NCH

7.3 Hz, 2H, CH

CH ), 1.37 (s, 6H, CH

(CONH), 159.6 (C

2

), 3.81 (s, 3H, NMe), 3.71–3.60 (m, 2H, CH

S), 2.41 (t, J = 7.5 Hz, 2H, CH

2

trated under reduced pressure. The resulting yellow syrup was puriﬁed

by column chromatography on silica gel (DCM/AcOEt, 50:50) to afford

2

1

3

2

3

1

the product 19 as a colorless oil (0.48 mmol, 350 mg, 73%). H NMR

(

500 MHz, CDCl

3

): δ(ppm) 8.44 (dd, J = 7.9, 1.0 Hz, 1H, Har-Phthal), 8.34

Phthal), 133.5 (CIV-NHCO), 133.4 (CHar-Phthal), 131.9 (CHar-Phthal), 129.2

(CIV-Cl), 129.1 (CIV), 128.8 (CHar), 128.1 (CIV), 128.0 (CHar), 127.1

(CHar-Phthal), 125.7 (CHar-Phthal), 123.4 (CIV-Cl), 122.6 (CHar), 58.9

q, J = 9.0 Hz, 1H, Har), 8.24 (d, J = 7.5 Hz, 1H, Har-Phthal), 7.83–7.76

m, 1H, Har-Phthal), 7.77–7.73 (m, 1H, Har-Phthal), 7.59 (s, 1H, NH), 7.36

d, J = 2.4 Hz, 1H, Har), 7.24 (dd, J = 8.9, 2.4 Hz, 1H, Har), 4.51 (s, 2H,

(CH

32.2 (CH

25.3 (CH

2

OH), 41.2 (NCH

2

), 39.4 (NMe), 37.9 (CH

2

CONH), 33.1 (CH

2

S),

),

CH

Hz, 2H, CH

CH

2

), 4.09 (t, J = 7.4 Hz, 2H, NCH

2

), 3.79 (s, 3H, NMe), 3.62 (t, J = 5.6

S), 2.40 (t, J = 7.5 Hz, 2H,

), 1.77–1.68 (m, 4H, 2 × CH ),

), 0.88 (s, 9H, 3 × CH3-tBu), 0.06 (s, 6H, 2 ×

). C NMR (125 MHz, CDCl ): δ(ppm) 171.4 (CONH), 159.6

2

), 30.1 (CH

2

), 29.5 (CH

2

), 29.0 (CH

2

), 28.7 (CH

2

), 28.4 (CH

2

+

S [M+H]⁺m/z

2

O), 3.22 (d, J = 7.4 Hz, 2H, CH

2

). HRMS (ESI ): calcd for C29

H

35Cl N O

2 6 3

2

CONH), 1.84–1.78 (m, 2H, CH

2

617.1868; found 617.1868.

1

.47–1.32 (m, 6H, 3 × CH

2

1

3

SiCH

3

4.1.14. 3-(3-((8-((2,4-dichlorophenyl)amino)-8-oxooctyl)thio)-5-((3-

methyl-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-4-yl)

propanoic acid (22)

–

(

C

O), 152.0 (CIV-C=N), 151.6 (CIV-C=N), 141.4 (CIV-Phthal), 133.6 (CIV-

NHCO), 133.4 (CHar-Phthal), 131.8 (CHar-Phthal), 129.3 (CIV), 129.1 (CIV-Cl),

28.8 (CHar), 128.1 (CIV), 128.0 (CHar), 127.1 (CHar-Phthal), 125.6 (CHar-

Phthal), 123.3 (CIV-Cl), 122.5 (CHar), 59.5 (CH O), 41.4 (NCH ), 39.4

S), 32.7 (CH ), 29.8 (CH ), 29.5

1

TEMPO (0.26 eq, 0.074 mmol, 11.64 mg) and BAIB (2.4 eq, 0.688

mmol, 221 mg) were added to a solution of alcohol 21 (1 eq, 0.286

2

ꢀ 1

(

NMe), 37.9 (CH

2

CONH), 33.0 (CH

), 28.8 (CH ), 28.5 (CH

2

mmol, 177 mg) in DCM/H

atmosphere. After completion the mixture was quenched with aqueous

solution of Na (10%) and extracted with AcOEt. The organic layer

was washed with brine, dried over Na SO , ﬁltered and concentrated

2

O (1:1 mixture; 0.009 mol.L ) under inert

CH

2

), 29.1 (CH

2

), 26.0 (3 × CH3-tBu), 25.4

+

), 18.3 (CIV), ꢀ 5.2 (2 × SiCH

3

). HRMS (ESI ): calcd for C35

H

49

2 2 3

S O

+

Cl

2

N

6

O

3

SSi [M+H] m/z 731.2734; found 731.2733.

2

4

under reduced pressure. The crude was puriﬁed by column chroma-

tography on silica gel (DCM/MeOH-AcOH (3%), 90:10) to afford the

4

.1.12. N-(2,4-dichlorophenyl)-8-((4-(3-(dimethylamino)propyl)-5-((3-

1

methyl-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)

product 22 as a white syrup (0.190 mmol, 120 mg, 66%). H NMR (500

thio)octanamide (20)

MHz, CDCl

Hz, 1H, Har), 8.18 (d, J = 7.5 Hz, 1H, Har-Phthal), 7.89–7.68 (m, 2H, 2 ×

ar-Phthal), 7.63 (s, 1H, NH), 7.37 (d, J = 2.4 Hz, 1H, Har), 7.24–7.21 (m,

1H, Har), 4.60 (s, 2H, CH ), 3.78 (s, 3H,

), 4.27 (t, J = 7.2 Hz, 2H, NCH

S), 2.72 (t, J = 7.2 Hz, 2H,

CONH), 1.82–1.59 (m, 4H, 2 ×

). C NMR (125 MHz, CDCl ): δ(ppm)

O), 152.2 (CIV-C=N), 151.7

3

): δ(ppm) 8.43 (d, J = 7.0 Hz, 1H, Har-Phthal), 8.31 (d, J = 8.7

To a suspension of mercapto-triazole 18 (1 eq, 0.446 mmol, 160 mg)

was added bromoamide 7 (1.8 eq, 0.803 mmol, 295 mg) and K CO (2

2

3

H

eq, 0.892 mmol, 123 mg) in DMF until dissolution. After stirring at room

temperature to complete conversion, the resulting clear yellow solution

2

NMe), 3.25 (t, J = 7.2 Hz, 2H, CH

2

was quenched with a saturated aqueous NH

added and the organic layer was washed with brine, dried over Na

and concentrated under reduced pressure. The crude was puriﬁed by

column chromatography on silica gel (DCM/MeOH-Et N (5%), 90:10) to

afford the product 20 as a white solid (0.20 mmol, 288 mg, 45%). H

NMR (300 MHz, CDCl

4

Cl solution. AcOEt was

CH

2

COOH), 2.41 (t, J = 7.6 Hz, 2H, CH

2

1

3

2

SO

4

2

), 1.45–1.30 (m, 6H, 3 × CH

2

3

–

172.9 (COOH), 171.7 (CONH), 159.7 (C

3

(CIV-C=N), 141.4 (CIV), 133.5 (CIV-NHCO), 133.4 (CHar-Phthal), 131.9 (CHar-

Phthal), 129.2 (CIV-Cl), 129.1 (CIV), 128.8 (CHar), 128.1 (CIV), 128.0

(CHar), 127.2 (CHar), 125.4 (CHar-Phthal), 123.5 (CIV-Cl), 122.8 (CHar),

1

3

): δ(ppm) 8.43 (m, 1H, Har-Phthal), 8.33 (d, J = 8.9

Hz, 1H, Har), 8.28–8.17 (m, 1H, Har-Phthal), 7.84–7.69 (m, 2H, 2 × Har-

Phthal), 7.60 (s, 1H, NH), 7.36 (d, J = 2.4 Hz, 1H, Har), 7.23 (m, 1H, Har),

2 2 2

40.2 (NCH ), 39.5 (NMe), 37.9 (CH CONH), 34.1 (CH COOH), 33.3

(CH

2

S), 29.8 (CH

2

), 29.4 (CH

2

), 28.9 (CH

31Cl

2

), 28.7 (CH

2

), 28.4 (CH

2

), 25.3

+

4

.53 (s, 2H, CH

2

), 3.99 (m, 2H, NCH

2

), 3.81 (s, 3H, NMe), 3.22 (m, 2H,

2

). HRMS (ESI ): calcd for C29

H

2

N

6

O

4

S [M+H] m/z 631.1660;

CH

×

2

S), 2.40 (t, J = 7.5 Hz, 2H, CH

NMe ), 1.73 (m, 6H, 3 × CH

), 2.18 (s, 6H, 3 × CH

). C NMR (75 MHz, CDCl ): δ(ppm) 171.4 (CONH), 159.6

2

CONH), 2.23 (t, J = 6.7 Hz, 2H,

found 631.1661.

2

1

2

), 1.39 (m, 6H, 3

3

CH

–

2

3

4.1.15. Ethyl 2-(3-(2-(1,3-dioxolan-2-yl)ethyl)-4-oxo-3,4-

(

C

–

O), 151.9 (CIV-C=N), 151.7 (CIV-C=N), 141.4 (CIV-C=N), 133.5 (CIV-

dihydrophthalazin-1-yl)acetate (26)

◦

NH), 133.4 (CHar-Phthal), 131.9 (CHar-Phthal), 129.2 (CIV-Phthal), 129.0 (CIV-

Cl), 128.8 (CHar), 128.0 (CIV-Phthal), 128.0 (CHar), 127.1 (CHar-Phthal),

To a solution of 24 (1 eq, 8.6 mmol, 2 g) in DMF at 40 C was added

K

2

CO

3

(2 eq, 17 mmol, 2.3 g) under inert atmosphere. The mixture was

◦

1

25.6 (CHar-Phthal), 123.2 (CIV-Cl), 122.5 (CHar), 56.1 (CH

NMe ), 42.3 (NCH ), 39.5 (NMe ), 37.9 (CH CONH), 33.1 (CH

), 28.8 (CH ), 28.5 (CH

39Cl

2

NMe

S), 30.2

2 2

), 27.6 (CH ), 25.4

2

), 45.4

stirred at 40 C and the bromoacetal 25 (1.5 eq, 12.9 mmol, 2.3 g) was

added. The mixture was stirred overnight, quenched with water and

extracted twice with DCM. The organic layer was washed with brine,

(

2

l

2

CH

2

), 29.5 (CH

2

), 29.1 (CH

+

2

). HRMS (ESI ): calcd for C31

H

2

N

7

O

2

S [M+H]⁺m/z 644.2339;

2 4

dried over Na SO , ﬁltrered and concentrated under reduced pressure.

found 644.2341.

The crude was puriﬁed by column chromatography on silica gel (DCM/

Isopropanol, 100:0 to 95:5) to afford the desired compound 26 as a

1

4

.1.13. N-(2,4-dichlorophenyl)-8-((4-(3-hydroxypropyl)-5-((3-methyl-4-

yellow solid (6.41 mmol, 2.13 g, 75%). H NMR (300 MHz, CDCl

3

):

3

4

oxo-3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)

octanamide (21)

δ(ppm) 8.46 (dd, J = 6.6 Hz, J = 1.7 Hz, 1H, Har-Phthal), 7.83–7.71 (m,

3 4

2H, Har-Phthal), 7.68 (dd, J = 6.6 Hz, J = 1.7 Hz, 1H, Har-Phthal), 5.03 (t,

3

To a solution of the silylated alcohol 19 (1 eq, 0.219 mmol, 160 mg)

J = 4.6 Hz, 1H, CH), 4.37 (t, J = 7.3 Hz, 2H, NCH

2

), 4.18 (q, J = 7.1

ꢀ 1

in distilled THF (0.015 mol.L ) was carefully added a solution of HF 70

Hz, 2H, CH

2

CH

3

), 4.01–3.82 (m, 6H, CH , 2 × CH2-diox), 2.27–2.18 (m,

2

1

2

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

2

H, NCH

2

CH

2

), 1.23 (t, ³J = 7.1 Hz, 3H, CH

3

). ¹³C NMR (75 MHz,

found: 597.0882.

–

CDCl

3

): δ(ppm) 169.8 (C

O), 159.3 (C

O), 140.4 (CIV-C=N), 133.0

(

CHar-Phthal), 131.5 (CHar-Phthal), 129.4 (CIV), 128.2 (CIV), 127.5 (CHar-

4.1.18. N-(2,4-dichlorophenyl)-2-((4-methyl-5-((4-oxo-3-(3-oxopropyl)-

3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)acetamide

(29)

Phthal), 124.5 (CHar-Phthal), 102.8 (CH), 65.1 (2 × CH2-diox), 61.5

(

CH

2

CH

3

), 46.6 (NCH

2

), 39.2 (CH

2

), 32.8 (NCH

2

CH

2

), 14.3 (CH

3

).

+

[M+H]⁺m/z 333.1445; found

HRMS (ESI ) calcd for C17

H

21

N

2

O

5

To a solution of ketal 28 (1 eq, 0.18 mmol, 105 mg) in acetone/water

ꢀ

1

3

33.1447.

mixture (9:1, 0.01 mmol ) at room temperature was added para-tol-

◦

uenesulfonic acid (10 eq). The mixture was stirred overnight at 60 C.

4

1

.1.16. 2-(2-(1,3-dioxolan-2-yl)ethyl)-4-((5-mercapto-4-methyl-4H-

,2,4-triazol-3-yl)methyl)phthalazin-1(2H)-one (27)

After completion of the reaction, the mixture was quenched by addition

of a saturated aqueous NaHCO

with DCM and the organic layers were washed with brine, dried over

Na SO and concentrated. The crude product was puriﬁed by ﬂash

3

solution. The product was extracted

To a solution of ester 26 (1 eq, 1.1 mmol, 362 mg) in ethanol (0.4

ꢀ

mol.L ¹) at room temperature was slowly added hydrazine mono-

2

4

hydrate (6 eq, 6.54 mmol, 320

μ

L). The mixture was reﬂuxed for 4 h.

column chromatography on silica gel (DCM/MeOH, 97:3) to afford the

1

After completion of the reaction, the solution was allowed to cool to

room temperature and the precipitate was ﬁltered, washed with diethyl

desired aldehyde 29 (0.15 mmol, 77 mg, 81%). H NMR (300 MHz,

4

CHCl

3

): δ(ppm) 10.04 (s, 1H, NH), 9.77 (t, J = 1.5 Hz, 1H, CHO),

3

ether and dried. The white solid hydrazide was used in the next step

8.45–8.40 (m, 1H, Har-Phthal), 8.24 (d, J = 8.9 Hz, 1H, Har), 8.21–8.16

1

4

without further puriﬁcation. H NMR (300 MHz, DMSO‑d

6

): δ(ppm)

(m, 1H, Har-Phthal), 7.87–7.76 (m, 2H, Har-Phthal), 7.31 (d, J = 2.4 Hz, 1H,

3

4

3

9

.34 (s, 1H, NH); 8.28 (d, J = 7.6 Hz, 1H, Har-Phthal); 7.96–7.80 (m, 3H,

H

ar), 7.19 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H, Har), 4.53 (t, J = 6.4 Hz, 2H,

3

H

ar-Phthal); 4.93 (t, J = 4.5 Hz, 1H, CH); 4.40–4.10 (m, 4H, NCH

2

, NH

2

);

2 2 2

NCH ), 4.48 (s, 2H, CH ), 4.05 (s, 2H, SCH ), 3.53 (s, 3H, NMe), 2.91 (td,

4

3

13

3

.94–3.73 (m, 6H, 3 × CH

2

); 2.11–2.00 (m, 2H, NCH

2

CH

2

). HRMS

J = 1.5 Hz, J = 6.4 Hz, 2H, CH

2 3

COH). C NMR (75 MHz, CHCl ):

+

(

ESI ) calcd for C15

H

16

N

2

NaO

3

[M+Na] m/z 319.1401; found

δ(ppm) 199.9 (CHO), 167.1 (CONH), 159.2 (CONPhthal), 152.6 (CIV-C=N),

152.1 (CIV-C=N), 141.0 (CIV-C=N), 134.0 (CIV), 133.8 (CHar-Phthal), 132.3

(CHar-Phthal), 129.5 (CIV), 129.1 (CHar), 128.8 (CIV), 128.0 (CIV), 127.6

(CHar), 127.5 (CHar), 125.2 (CHar), 124.7 (CIV), 123.2 (CHar), 45.0

3

0

19.1402. To a suspension of the hydrazide previously obtained (1 eq,

ꢀ 1

.98 mmol, 312 mg) in ethanol (0.33 mol.L ) at room temperature

under inert atmosphere were added successively methylisothiocyanate

5 eq, 4.9 mmol, 395 L) followed by triethylamine (5 eq, 4.9 mmol, 689

L). The mixture was reﬂuxed overnight under argon atmosphere. After

(

μ

(NCH

2

), 42.4 (CH

2

COH), 36.1 (SCH

2

), 30.8 (NMe), 30.0 (CH

2

). HRMS

+

NaS [M+Na]⁺m/z 553.0592, found:

μ

(ESI ): calcd for C23

2 6 3

H20Cl N O

completion of the reaction, the solution was allowed to cool to room

temperature and the precipitate was ﬁltered, washed with diethyl ether

553.0610.

and dried. The thiol 27 was obtained as a solid and used without further

4.1.19. N-(2,4-dichlorophenyl)-2-((5-((3-(3-hydroxypropyl)-4-oxo-3,4-

dihydrophthalazin-1- yl)methyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)

acetamide (30)

1

puriﬁcation (0.87 mmol, 326 mg, 89%). H NMR (300 MHz, DMSO‑d

6

):

4

3

δ(ppm) 13.56 (bs, 1H, SH), 8.30 (dd, J = 1.3 Hz, J = 7.6 Hz, 1H, Har-

Phthal), 8.07–8.00 (m, 1H, Har-Phthal), 7.99–7.85 (m, 2H, Har-Phthal), 4.86

To a solution of the aldehyde 29 (1 eq, 0.15 mmol, 80 mg) in ethanol

t, ³J = 4.5 Hz, 1H, CH), 4.52 (s, 2H, CH

), 4.17 (t, J = 7.3 Hz, 2H,

3

at 0 C was added sodium borohydride (2 eq, 0.3 mmol, 12 mg). After

◦

(

2

◦

NCH

2

), 3.91–3.71 (2 m, 4H, 2 × CH

2

), 3.46 (s, 3H, NMe), 2.06–1.95 (m,

): δ(ppm) 167.0 (CIVSH),

O), 149.5 (CIV-C=N), 140.5 (CIV-C=N), 133.2 (CHar-Phthal),

stirring one hour at 0 C, the mixture was quenched by addition of a

1

3

2

1

H, NCH

2

CH

2

). C NMR (75 MHz, DMSO‑d

6

saturated aqueous NH

4

Cl solution. The volatiles were removed and the

SO and

–

58.0 (C

–

crude was diluted in DCM, washed with brine, dried over Na

2

4

31.9 (CHar-Phthal), 128.5 (CIV), 127.2 (CIV), 126.2 (CHar-Phthal), 125.4

concentrated. The crude product was puriﬁed by ﬂash column chro-

matography on silica gel (DCM/MeOH, 100:0 to 97:3) to afford the

(

CHar-Phthal), 101.7 (CH), 64.3 (2 × CH2-diox), 45.6 (NCH

2

), 32.1

1

NCH

2

CH

2

), 30.1 (NMe), 28.9 (CH

2

). HRMS (ESI + ) calcd for

desired alcohol 30 as a white powder (0.12 mmol, 64 mg, 80%). H NMR

C

17

H

19

5

N NaO

3

S [M+Na]⁺m/z 396.1101; found 396.1091.

(300 MHz, CDCl

3

): δ(ppm) 10.02 (s, 1H, NH), 8.48–8.42 (m, 1H, Har-

Phthal), 8.28 (m, 2H, Har and Har-Phthal), 7.89–7.76 (m, 2H, Har-Phthal),

4

3

4

.1.17. 2-((5-((3-(2-(1,3-dioxolan-2-yl)ethyl)-4-oxo-3,4-

7.30 (d, J = 2.3 Hz, 1H, Har), 7.19 (dd, J = 8.9 Hz, J = 2.3 Hz, 1H,

3

dihydrophthalazin-1-yl)methyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)-N-

H

ar), 4.52 (s, 2H, CH

SCH

), 3.60–3.51 (m, 5H, NMe and CH

NMR (100 MHz, CDCl ): δ(ppm) 167.0 (CONH), 160.1 (CONPhthal),

2

), 4.35 (t, J = 6.1 Hz, 2H, NCH

2

), 4.05 (s, 2H,

1

3

(

2,4-dichlorophenyl)acetamide (28)

2

OH), 1.98 (m, 2H, CH

2

).

C

To a solution of thiol 27 (1 eq, 0.83 mmol, 310 mg) in DMF (0.06

3

mol.L ¹) under argon atmosphere was added K

ꢀ

(1.5 eq, 1.24 mmol,

152.7 (CIV-C=N),152.0 (CIV-C=N), 141.5 (CIVC=N), 134.0 (CIV), 133.8

(CHarPhthal), 132.3 (CHarPhthal), 129.6 (CIV), 129.0 (CHar), 128.8 (CIV),

127.8 (CIV), 127.6 (CHar), 127.5 (CHar), 125.2 (CHar), 124.7 (CIV), 123.2

2

CO

3

1

72 mg). The mixture was stirred for 15 min. at room temperature

before a solution of -bromoamide 6 (1.1 eq, 0.91 mmol, 258 mg) in

α

ꢀ 1

DMF (0.91 mol.L ) was added. After completion of the reaction, a

saturated solution of NH Cl was added. The mixture was extracted twice

with AcOEt. The organic layer was washed with brine, dried over MgSO

(CHar), 58.7 (CH

(NMe), 30.1 (CH

z 533.0929, found: 533.0932.

2

OH), 47.8 (CH

2

N), 36.1 (SCH

2

), 31.7 (CH

2

), 30.8

+

S [M+H]⁺m/

4

2

). HRMS (ESI ) calcd for C23

H

23

O

3

6

N Cl

2

4

and concentrated. The crude product was puriﬁed by ﬂash column

chromatography on silica gel (DCM/MeOH, 97:3) to afford the desired

4.1.20. 3-(4-((5-((2-((2,4-dichlorophenyl)amino)-2-oxoethyl)thio)-4-

methyl-4H-1,2,4-triazol-3-yl)methyl)-1-oxophthalazin-2(1H)-yl)propanoic

acid (31)

1

product 28 as a white powder (0.74 mmol, 425 mg, 89%). H NMR (300

MHz, CDCl

3

): δ(ppm) 10.08 (s, 1H, NH), 8.47–8.41 (m, 1H, Har-Phthal),

8

.26–8.18 (m, 2H, Har and Har-Phthal), 7.85–7.73 (m, 2H, Har-Phthal), 7.31

To a solution of aldehyde 29 (1 eq, 0.36 mmol, 192 mg) in a mixture

4

3

4

ꢀ 1

(

d, J = 2.4 Hz, 1H, Har), 7.19 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H, Har),

of tert-butanol/water (3:1, 0.02 mol.L ) at room temperature was

3

4

.97 (t, J = 7.3 Hz, 1H, CH), 4.51 (s, 2H, CH

2

), 4.34 (t, J = 7.3 Hz, 2H,

added 2-methyl-2-butene (50 eq, 18.3 mmol, 1.27 g), followed by

NCH

2

), 4.05 (s, 2H, SCH

2

), 3.97–3.79 (m, 4H, OCH

2

CH

2

), 3.56 (s, 3H,

NaH

2

PO

4

(10 eq, 3.65 mmol, 569 mg). The mixture was stirred 20 min.

(4.5 eq, 1.64 mmol, 148 mg) was added. The mixture was

1

3

NMe), 2.21–2.13 (m, 2H, CH

2

). C NMR (75 MHz, CDCl

3

): δ(ppm)

before NaClO

2

1

67.2 (CONH), 159.1 (CONPhthal), 152.9 (CIV-C=N), 152.1 (CIV-C=N),

stirred 2 h. at room temperature and ethyl acetate was added followed

by an aqueous 1 N HCl solution. The aqueous layer was extracted with

ethyl acetate, the organic layers were combined and washed with brine,

40.5 (CIV-C=N), 134.0 (CIV), 133.5 (CHar-Phthal), 132.0 (CHar-Phthal),

29.5 (CIV), 129.1 (CHar), 128.9 (CIV), 128.2 (CIV), 127.6 (CHar), 127.5

(

CHar), 125.1 (CHar), 124.7 (CIV), 123.2 (CHar), 102.6 (C

–

O), 65.1 (2 ×

dried over Na

tained pure as a white powder (0.31, 167 mg, 85%). H NMR (400 MHz,

DMSO‑d ): δ(ppm) 12.32 (bs, 1H, CO H), 9.97 (s, 1H, NH), 8.31–8.27

2 4

SO and concentrated. The carboxylic acid 31 was ob-

1

CH

2

O), 46.5 (NCH

2

), 36.0 (SCH

2

), 32.9 (CH

2

), 30.9 (NMe), 30.2 (CH

2

).

+

NaS [M+Na]⁺m/z 597.0855,

HRMS (ESI ) calcd for C25

H

24

O

N

4 6

Cl

2

6

2

1

3

J. Smadja et al.

m, 1H, Har-Phthal), 8.13–8.09 (m, 1H, Har-Phthal), 7.94–7.85 (m, 2H, Har

Bioorganic & Medicinal Chemistry 39 (2021) 116161

◦

(

stirred at 40 C overnight then quenched with water and extracted with

4

AcOEt. The organic layer was washed with brine, dried over Na SO ,

3

4

and Har-Phthal), 7.88 (d, J = 8.8 Hz, 1H, Har), 7.63 (d, J = 2.4 Hz, 1H,

2

3

4

H

ar), 7.39 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H, Har), 4.55 (s, 2H, CH

2

), 4.28

), 3.58 (s, 3H, NMe), 2.68 (t,

H). ¹³C NMR (100 MHz, DMSO‑d

): δ(ppm)

ﬁltrered and concentrated under reduced pressure. The crude was pu-

riﬁed by column chromatography on silica gel (DCM/MeOH, 98:2 to

3

(

3

t, J = 7.1 Hz, 2H, NCH

2

), 4.09 (s, 2H, SCH

2

J = 7.1 Hz, 2H, CH

72.4 (CO

2

CO

2

6

94:6) to afford the desired amine 36 as an oil (1.89 mmol, 0.6 g, 75%).

1

2

H), 166.8 (CONH), 158.2 (CONPhthal), 152.9 (CIV-C=N), 149.4

H NMR (400 MHz, CDCl

3

): δ(ppm) 8.48–8.45 (m, 1H, CHar), 7.81–7.73

(

C

IV-C=N), 141.3 (CIV-C=N), 133.8 (CIV), 133.3 (CIV), 132.0 (CHar-Phthal),

(m, 2H, 2 × CHar), 7. 71–7.68 (m, 1H, CHar), 4.27 (t, J = 7.2 Hz, 2H,

NCH CH ), 3.96 (s, 2H, CH CO Et), 2.41,

(t, 2H, J = 7.4 Hz, 2H, CH ), 2.25 (s, 6H, NMe ), 2.07–1.99 (m,

NCH CH ), 1.24 (t, 3H, CH ). C NMR (100 MHz, CDCl ): δ(ppm)

169.7 (COOEt), 159.2 (C

29.2 (CIV), 128.0 (CIV), 127.3 (CHar), 124.3 (CHar), 61.3 (CH

1

29.4 (CIV), 129.0 (CHar), 128.6 (CIV), 127.7 (CIV), 127.3 (CHar), 126.6

2

), 4.19 (q, J = 7.1 Hz, 2H, CH

2

3

2

(

C

IV), 126.3 (CHar-Phthal), 126.2 (CHar), 125.8 (CHar), 46.1 (CH

2

N), 36.9

2

NMe

2

+

13

SCH

2

), 32.5 (CH

2

CO

2

H), 30.6 (NMe), 28.9 (CH

2

). HRMS (ESI ) calcd

2

CH

3

NaS [M+Na]⁺m/z 569.0542, found: 569.0524.

–

O), 140.2 (CIV), 132.9 (CHar), 131.3 (CHar),

O), 56.8

for C23

H20Cl

2

N

6

O

4

1

2

4

.1.21. N-(2,4-dichlorophenyl)-2-((5-((3-(3-(dimethylamino)propyl)-4-

(Me

(CH

2

NCH

2

), 49.3 (NCH

CH

2

), 45.3 (2 × CH

3 2

), 39.0 (CH COOEt), 26.5

+

[M+H]⁺

oxo-3,4-dihydrophthalazin-1-yl)methyl)-4-methyl-4H-1,2,4-triazol-3-yl)

thio)acetamide (32)

2

), 14.1 (CH

3

2

O). HRMS (ESI ): calcd for C17

H

24

N

3

O

3

m/z 318.1818; found 318.1821.

To a solution of aldehyde 29 (1 eq, 0.15 mmol, 80 mg) in MeOH (2

mL) at room temperature was added dimethylamine (1.1 eq, 0.16 mmol,

4.1.24. 2-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-((5-mercapto-4-

methyl-4H-1,2,4-triazol-3-yl)methyl)phthalazin-1(2H)-one (37)

To a solution of ester 35 (1 eq, 0.98 mmol, 0.4 g) in ethanol (0.2 mol.

2

1

μ

L) followed by AcOH (1 eq.). The mixture was stirred at room

temperature for 40 min before NaBH(OAc) (1.2 eq, 0.18 mmol, 40 mg)

was added. After completion, the solution was quenched with saturated

aqueous NaHCO solution and extracted with DCM, washed with brine,

dried over Na SO and concentrated. The crude was diluted with Et O,

ﬁltered and concentrated to afford the product 32 as a white powder

3

ꢀ 1

L

) at room temperature was added hydrazine monohydrate (6 eq, 6

◦

3

mmol, 300

μL). The mixture was stirred at 42 C during 24 h then a

2

4

2

second portion of hydrazine monohydrate was added (6 eq, 6 mmol,

300 L) and the mixture was reﬂuxed overnight. After completion of the

μ

1

(

0.05 mmol, 28 mg, 33%). H NMR (300 MHz, CHCl

3

): δ(ppm) 10.02 (s,

reaction, the solution was allowed to cool to room temperature and the

precipitate was ﬁltered, washed with ethylacetate and dried. The solid

1

H, NH), 8.39 (d, J = 7.9 Hz, 1H, Har-Phthal), 8.18–8.14 (m, 2H, Har and

H

ar-Phthal), 7.78–7.73 (m, 2H, Har-Phthal), 7.27–7.25 (m, 1H, Har), 7.14

hydrazide (0.82 mmol, 0.32 g, 84%) was used in the next step without

1

(

dd, 1H, J = 8.8, J = 2.1 Hz, Har), 4.47 (s, 2H, CH

2

), 4.18 (t, 2H, J = 7.3

further puriﬁcation. H NMR (400 MHz, DMSO‑d

6

): δ(ppm) 9.33 (s, 1H,

Hz, CH

2

NMe

2

), 4.01 (s, 2H, SCH

2

), 3.54 (s, 3H, NMe), 2.41–2.36 (m, 2H,

NH), 8.29–8.27 (m, 1H, CHar), 7.92–7.83 (m, 3H, CHar), 4.25 (s, 2H,

NH ), 3.78 (s, 2H, CH

6.1 Hz, 2H, CH

6H, 2 × SiCH

1

3

CH

2

), 2.22 (s, 6H, NMe

2

), 1.98–1.91 (m, 2H, CH

2

). C NMR (75 MHz,

2

), 4.17 (t, J = 7.1 Hz, 2H, NCH

2

CO), 3.67 (t, J =

CDCl

3

): δ(ppm) 167.1 (CONH), 159.1 (CONPhthal), 152.8 (CIV-C=N),

51.9 (CIV-C=N), 140.7 (CIV-C=N), 133.9 (CIV), 133.5 (CHar-Phthal), 132.0

2

O), 1.93 (m, 2H, CH

2

), 0.85 (s, 9H, 3 × CH3-tBu), 0.02 (s,

+

1

3

). HRMS (ESI ): calcd for C19

H

31

N

4

O

3

Si [M+H] m/z

(

CHar-Phthal), 129.4 (CIV), 129.0 (CHar), 128.7 (CIV), 128.1 (CIV), 127.5

CHar), 127.3 (CHar-Phthal), 125.1 (CHar-Phthal), 124.6 (CIV), 123.1 (CHar),

391.2165; found 391.2166. Under inert atmosphere, to a solution of the

hydrazide previously obtained (1 eq, 0.394 mmol, 154 mg), methyl

(

ꢀ 1

5

3

6.5 (CH

0.0 (CH

2

NMe

2

), 49.3 (CH

2

N), 45.0 (NMe

2

), 36.0 (SCH

2

), 30.8 (NMe),

2 7 4

28Cl N O S

isothiocyanate (5.5 eq, 2.17 mmol, 158 mg) in ethanol (0.033 mol.L

were added TEA (5 eq, 1.97 mmol, 0.275 mL). The mixture was reﬂuxed

3 h 30 then concentrated under reduced pressure. Et O was added to the

resulting residue followed by evaporation (3 times). The solid was then

triturated in an Et O/cyclohexane mixture. The thiol 37 was obtained by

ﬁltration as a white solid (0.359 mmol, 160 mg, 91%). H NMR (400

MHz, DMSO‑d

): δ(ppm) 13.50 (s, 1H, SH), 8.49–8.21 (m, 1H, CHar),

8.05–8.00 (m, 1H, CHar), 7.97–7.83 (m, 2H, 2 × CHar), 4.50 (s, 2H, CH ),

4.14 (t, J = 7.1 Hz, 2H, NCH ), 3.63 (t, J = 6.1 Hz, 2H, CH O), 3.47 (s,

3H, NMe), 1.89 (m, 2H, CH

), 0.82 (s, 9H, 3 × CH3-tBu), ꢀ 0.02 (s, 6H, 2

). C NMR (100 MHz, DMSO‑d ): δ(ppm) 167.0 (CIV-SH), 158.1

O), 149.4 (CIV-C=N), 140.4 (CIV), 133.2 (CHar), 131.9 (CHar), 128.5

(CIV), 127.3 (CIV), 126.2 (CHar), 125.5 (CHar), 60.2 (CH O), 47.4

)

2

), 26.5 (CH

2

). HRMS (ESI + ) calcd for C25

H

[

M+H]⁺m/z 560.1402, found 560.1402.

2

4

.1.22. Ethyl2-(3-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-oxo-3,4-

2

1

dihydrophthalazin-1-yl)acetate (35)

To a solution of 24 (1 eq, 5 mmol, 1.16 g) in DMF (30 mL) was added

6

NaH (1.5 eq, 0.3 g of a 60% suspension in oil) under argon, and after gas

2

completion the bromoalkyle 33 (2 eq, 10 mmol, 2.53 g) in DMF solution

2

◦

(

2 mL) was added. The mixture was stirred at 40 C during 48 h. then

2

1

3

quenched with water and extracted with AcOEt. The organic layer was

washed with brine, dried over Na SO , ﬁltrered and concentrated under

× SiCH

3

6

–

2

4

(C

–

reduced pressure. The crude was puriﬁed by column chromatography on

2

silica gel (cyclohexane/AcOEt, 100:0 to 70:30) to afford the desired

(NCH

2

), 31.1 (CH

2

), 30.1 (NMe), 28.9 (CH

2

), 25.7 (3 × CH3-tBu), 17.8

1

+

SSi [M+H]⁺

ester 35 as an oil (2.95 mmol, 1.2 g, 60%). H NMR (300 MHz, CDCl

3

):

(CIV), ꢀ 5.4 (2 × SiCH

3

). HRMS (ESI ): calcd for C21

H

32

N

5

O

2

δ(ppm) 8.48–8.45 (m, 1H, Har-Phthal), 7.81–7.73 (m, 2H, Har-Phthal),

m/z 446.2038; found 446.2046.

3

7

.69–7.67 (m, 1H, Har-Phthal), 4.30 (t, J = 7.2 Hz, 2H, NCH

2

), 4.18 (q, J

3

=

7.1 Hz, 2H, CH

2

CH

3

), 3.96 (s, 2H, CH

2

CO

2

Et)

,

3.73 (t, J = 6.3 Hz, 2H,

4.1.25. 2-(3-(dimethylamino)propyl)-4-((5-mercapto-4-methyl-4H-1,2,4-

triazol-3-yl)methyl)phthalazin-1(2H)-one (38)

3

CH

2

OSi), 2.11–2.02 (m, 2H, NCH

2

CH

2

), 1.24 (t, J = 7.1 Hz, 3H, CH

3

),

). C NMR (75 MHz,

O), 140.1 (CIV-C=N), 132.9

1

3

0

.89 (s, 9H, 3 × CH3-tBu), ꢀ 0.04 (s, 6H, 2 × SiCH

3

To a solution of the ester 36 (1 eq, 1.57 mmol, 0.5 g) in ethanol (0.2

–

ꢀ 1

CDCl

3

): δ(ppm) 169.8 (C

O), 159.2 (C

mol.L ) at room temperature was added hydrazine monohydrate (8 eq,

(

CHar-Phthal), 131.3 (CHar-Phthal), 129.2 (CIV), 128.1 (CIV), 127.3 (CHar-

12.56 mmol, 620

μL). The mixture was reﬂuxed overnight. After

Phthal), 124.3 (CHar-Phthal), 61.4 (CH

2

CH

3

), 60.71 (CH

2

O), 48.6 (NCH

2

),

completion of the reaction, the solution was concentrated under reduced

pressure. The crude was diluted with DCM, the organic layer was

3

9.0 (CH

5.4 (2 × SiCH

05.2210, found 405.2199.

2

), 31.6 (NCH

2

CH

2

), 25.9 (3 × CH3-tBu), 18.3 (CIV), 14.1 (CH

3

+

Si [M+H]⁺m/z

ꢀ

3

). HRMS (ESI ) calcd for C21

H

33

2

N O

4

2 4

washed with water, dried over Na SO , ﬁltered and concentrated under

4

reduced pressure. The solid hydrazide (0.99 mmol, 0.3 g, 63%) was

1

directly used in the next step without further puriﬁcation. H NMR (400

4

.1.23. Ethyl 2-(3-(3-(dimethylamino)propyl)-4-oxo-3,4-

MHz, DMSO‑d

7.93–7.91 (m, 2H, 2 × CHar), 7.87–7.83 (m, 1H, CHar), 4.25 (s, 2H,

NH ), 3.79 (s, 2H, CH

6

): δ(ppm) 9.32 (s, 1H, NH), 8.30–8.27 (m, 1H, CHar),

dihydrophthalazin-1-yl)acetate (36)

To a solution of amino mesylate 34 in its HCl form (2 eq, 5 mmol,

.08 g) in suspension in DMF (8 mL) was added NaH (3 eq, 0.6 g of a

0% suspension in oil) under argon, and after gas completion the ester

4 (1 eq, 2.5 mmol, 0.58 g) in DMF (8 mL) was added. The mixture was

2

), 4.13 (t, J = 7.3 Hz, 2H, NCH

2

CO), 2.27 (t, J =

1

6

2

6.9 Hz, 2H, CH

2

NMe

2

), 2.13 (s, 6H, NMe

2

), 1.89–1.82 (m, 2H, CH

2

).

+

HRMS (ESI ): calcd for C15

H

22

N

5

O

2

[M+H] m/z 304.1773; found

304.1783. Under inert atmosphere, to a solution of this hydrazide

1

4

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

previously obtained (1 eq, 0.375 mmol, 114 mg) and methyl isothio-

Phthal), 123.3 (CIV-Cl), 122.6 (CHar), 56.9 (CH

(NMe ), 37.9 (CH CONH), 33.1 (CH S), 30.7 (NMe), 29.9 (CH

), 28.8 (CH ), 28.5 (CH

SCl

2

NMe

2

), 49.3 (NCH

2

), 45.2

), 29.5

), 25.4 (CH ).

ꢀ

1

cyanate (6 eq, 2.25 mmol, 165 mg) in ethanol (0.031 mol.L ) were

added Et N (5 eq, 1.88 mmol, 0.261 mL). The mixture was reﬂuxed for 3

h 30, then concentrated under reduced pressure. The resulting yellow

crude was taken up in Et O, the precipitate was ﬁltered and washed with

Et O. The thiol 38 was recovered as a white solid (0.335 mmol, 120 mg,

9%). ¹H NMR (400 MHz, CDCl

): δ(ppm) 8.51–8.33 (m, 1H, CHar),

),

2

3

(CH

2

), 29.0 (CH

2

), 26.5 (CH

2

+

HRMS (ESI ): calcd for C31

644.2341.

H

40

N

7

O

2

[M+H] m/z 644.2343; found

2

8

7

4

3

4.1.28. N-(2,4-dichlorophenyl)-8-((5-((3-(3-hydroxypropyl)-4-oxo-3,4-

dihydrophthalazin-1-yl)methyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)

octanamide (41)

.99–7.86 (m, 1H, CHar), 7.85–7.63 (m, 2H, 2 × CHar), 4.35 (s, 2H, CH

2

.21 (t, J = 7.2 Hz, 2H, NCH

NMe ), 2.29 (s, 6H, NMe

MHz, CDCl

2

), 3.54 (s, 3H, NMe), 2.60–2.38 (m, 2H,

13

CH

2

), 2.11–1.78 (m, 2H, CH

2

). C NMR (100

To a solution of the silylated alcohol 39 (1 eq, 0.672 mmol, 480 mg)

–

ꢀ 1

3

): δ(ppm) 168.5 (CIV-SH), 159.2 (C

–

O), 148.9 (CIV-triazole),

in distilled THF (0.034 mol.L ) was carefully added a solution of HF

1

39.8 (CIV), 133.4 (CHar-Phthal), 132.0 (CHar-Phthal), 128.7 (CIV), 128.2

IV), 127.6 (CHar-Phthal), 124.4 (CHar-Phthal), 56.5 (CH NMe ), 49.4

), 26.3 (CH ). HRMS

(70 wt% in pyridine, 0.9 mL). The resulting colorless solution was stirred

4 h at room temperature. The reaction mixture was quenched with

(

C

2

NCH

2

), 44.9 (NMe

2

), 31.1 (NMe), 30.2 (CH

2

saturated solution of NaHCO

3

until neutral pH and extracted with DCM.

SO , ﬁltered

+

OS [M+H]⁺m/z 359.1646; found 359.1654.

ESI ): calcd for C17

H

23

N

6

The organic layer was washed with brine, dried over Na

2

4

and concentrated under reduced pressure to afford the corresponding

1

4

.1.26. 8-((5-((3-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-oxo-3,4-

alcohol 41 as a white solid (0.648 mmol, 400 mg, 98%). H NMR (400

dihydrophthalazin-1-yl)methyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)-N-

MHz, CDCl

3

): δ(ppm) 8.45 (dd, J = 7.7, 1.7 Hz, 1H, Har-Phthal), 8.33 (d, J

(

2,4-dichlorophenyl)octanamide (39)

= 8.8 Hz, 1H, Har), 8.26 (d, J = 7.8 Hz, 1H, Har-Phthal), 7.88–7.75 (m, 2H,

2 × Har-Phthal), 7.61 (s, 1H, NH), 7.37 (d, J = 2.4 Hz, 1H, Har), 7.25–7.21

The mercapto-triazole 37 (1 eq, 0.269 mmol, 120 mg), bromoamide

7

(1.2 eq, 0.323 mmol, 118 mg) and K

2

CO

3

(1.5 eq, 0.403 mmol, 56 mg)

(m, 1H, Har), 4.55 (s, 2H, CH

2

), 4.36 (t, J = 6.1 Hz, 2H, NCH

2

),

S), 2.41 (t,

), 1.82–1.68 (m, 4H, 2 ×

). C NMR (100 MHz, CDCl ): δ(ppm) 171.2

O), 152.3 (CIV), 151.6 (CIV-S), 141.6 (CIV), 133.7

were dissolved in DMF and stirred overnight. The mixture was quenched

with water and extracted with AcOEt. The organic layer was washed

3.62–3.45 (m, 5H, CH

J = 7.5 Hz, 2H, CH CONH), 1.99 (m, 2H, CH

CH

), 1.38 (s, 6H, 3 × CH

(CONH), 160.0 (C

2

OH, NMe), 3.22 (t, J = 7.3 Hz, 2H, CH

2

1

3

with brine, dried over Na

SO

2 4

, ﬁltered and concentrated under reduced

2

3

–

pressure. The resulting crude was puriﬁed by column chromatography

on silica gel (DCM/MeOH, 85:15). The desired product 39 was obtained

(CHar-Phthal), 133.4 (CIV-CONH), 132.1 (CHar-Phthal), 129.0 (CIV), 128.8

(CIV), 128.7 (CHar), 127.9 (CHar), 127.6 (CIV), 127.3 (CHar-Phthal), 125.3

1

as a colorless syrup (0.191 mmol, 140 mg, 71%). H NMR (400 MHz,

CDCl

3

): δ(ppm) 8.47–8.39 (m, 1H, Har-Phthal), 8.33 (d, J = 8.9 Hz, 1H,

ar), 8.30–8.23 (m, 1H, Har-Phthal), 7.83–7.70 (m, 2H, 2 × Har-Phthal),

.60 (s, 1H, NH), 7.36 (d, J = 2.4 Hz, 1H, Har), 7.23 (dd, J = 8.9, 2.4 Hz,

H, Har), 4.48 (s, 2H, CH ), 3.73 (t, J =

), 4.28 (t, J = 7.3 Hz, 2H, NCH

.1 Hz, 2H, CH S),

O), 3.54 (s, 3H, NMe), 3.19 (t, J = 7.4 Hz, 2H, CH

CONH), 2.14–1.96 (m, 2H, CH ), 1.80–1.65

), 0.89 (s, 9H, 3 × CH3-tBu),

). C NMR (100 MHz, CDCl ): δ(ppm) 171.4

(CHar-Phthal), 122.4 (CHar), 122.4 (CIV), 58.4 (CH

(CH CONH), 32.9 (CH ), 31.6 (CIV), 30.6 (NMe), 29.9 (CH

28.9 (CH ), 28.6 (CH

2

O), 47.6 (NCH

2

), 37.7

),

H

2

), 29.3 (CH

2

7

1

6

2

), 28.3 (CH

2

), 25.2 (CH

2

). One quaternary carbon

+

[M+H]⁺m/z

2

atom is missing. HRMS (ESI ): calcd for C29

H

35 6 3 2

N O SCl

2

617.1868; found 617.1880.

.40 (t, J = 7.5 Hz, 2H, CH

2

(

m, 4H, 2 × CH

2

), 1.48–1.30 (m, 6H, 3 × CH

2

4.1.29. N-(2,4-dichlorophenyl)-8-((4-methyl-5-((4-oxo-3-(3-oxopropyl)-

3,4-dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)octanamide

(42)

1

3

0

.04 (s, 6H, 2 × SiCH

3

–

(

CONH), 159.3 (C O), 152.1 (CIV), 152.0 (CIV-S), 140.8 (CIV), 133.6

–

C

IV-CONH), 133.4 (CHar-Phthal), 131.9 (CHar-Phthal), 129.1 (CIV), 128.9

IV-Cl), 128.8 (CHar), 128.3 (CIV), 128.0 (CHar-Phthal), 127.3 (CHar-Phthal),

To a solution of the alcohol 41 (1 eq, 0.162 mmol, 100 mg) in DCM

(0.008 mol.L ¹) was added Dess Martin periodinane (DMP) (2 eq, 0.323

ꢀ

C

1

25.5 (CHar-Phthal), 123.3 (CIV-Cl), 122.6 (CHar), 60.8 (CH

NCH ), 37.9 (CH CONH), 33.0 (CH ), 31.9 (CIV), 30.7 (NMe), 30.4

CH ), 29.5 (CH ), 29.0 (CH ), 28.8 (CH

2

O), 48.8

mmol, 140 mg). The resulting white suspension was allowed to stir at

1

(

2

room temperature for 20 h. Incomplete conversion was observed ( H

2

), 28.5 (CH

2

), 26.0 (3 × CH3-

NMR), and an additional portion of DMP (2 eq, 0.323 mmol, 140 mg)

was added and DMSO (20 mL) to increase solubility. Upon full conver-

sion (40 h), the reaction mixture was quenched with saturated solutions

+

tBu), 25.4 (CH

2

), 18.4 (CIV-Si), ꢀ 5.2 (2 × SiCH

3

). HRMS (ESI ): calcd for

C

35

H

49

6

N O

3

SSiCl

2

[M+H]⁺m/z 731.2731; found 731.2733.

of Na

2

S

2

O

3

(10 mL) and NaHCO

3

(10 mL). The organic layer was washed

SO , ﬁltered

4

.1.27. N-(2,4-dichlorophenyl)-8-((5-((3-(3-(dimethylamino)propyl)-4-

with brine and water. The organic layer was dried over Na

2

4

oxo-3,4-dihydrophthalazin-1-yl)methyl)-4-methyl-4H-1,2,4-triazol-3-yl)

thio)octanamide (40)

and concentrated under reduced pressure to afford the corresponding

1

aldehyde 42 as a white solid (0.120 mmol, 74 mg, 74%). H NMR (400

The mercapto-triazole 38 (1 eq, 0.181 mmol, 65 mg), bromoamide 7

MHz, CDCl

ar-Phthal), 8.33 (d, J = 8.9 Hz, 1H, Har), 8.27–8.18 (m, 1H, Har-Phthal),

7.89–7.73 (m, 2H, 2 × Har-Phthal), 7.61 (s, 1H, NH), 7.37 (d, J = 2.4 Hz,

1H, Har), 7.28–7.19 (m, 1H, Har), 4.55 (t, J = 6.5 Hz, 3H, NCH ), 4.50 (s,

2H, CH S), 2.94 (td, J =

), 3.52 (s, 3H, NMe), 3.22 (t, J = 7.3 Hz, 2H, CH

6.5, 1.6 Hz, 2H, CH CONH), 1.74 (m,

CHO), 2.41 (t, J = 7.5 Hz, 2H, CH

4H, 2 × CH ), 1.49–1.33 (m, 6H, 3 × CH ). C NMR (100 MHz, CDCl ):

δ(ppm) 200.0 (COH), 171.4 (CONH), 159.3 (C O), 152.2 (CIV-triazole),

3

): δ(ppm) 9.81 (t, J = 1.6 Hz, 1H, Hald), 8.48–8.38 (m, 1H,

(

1.2 eq, 0.217 mmol, 80 mg) and K

2

CO

3

(1.5 eq, 0.272 mmol, 37 mg)

H

were dissolved in DMF and stirred at room temperature overnight. The

resulting yellow solution was quenched with water and extracted with

2

AcOEt. The organic layer was washed with brine, dried over Na

2 4

SO ,

2

ﬁltered and concentrated under reduced pressure. The resulting crude

was puriﬁed by column chromatography on silica gel (DCM/MeOH,

2

1

3

2

3

–

8

5

5:15) to give the desired product 40 as a white powder (0.085 mmol,

1

5 mg, 47%). H NMR (400 MHz, CDCl

3

): δ(ppm) 8.48–8.40 (m, 1H, Har-

151.7 (CIV-triazole), 141.4 (CIV), 133.8 (CHar-Phthal), 133.6 (CIV-COHN),

132.2 (CHar-Phthal), 129.1 (CIV-Cl), 128.9 (CIV), 128.8 (2 × CHar), 128.0

(CIV), 127.3 (CHar-Phthal), 125.5 (CHar-Phthal), 123.3 (CIV-Cl), 122.6 (CHar),

Phthal), 8.33 (d, J = 8.9 Hz, 1H, Har), 8.24 (dd, J = 7.5, 1.2 Hz, 1H, Har-

Phthal), 7.86–7.71 (m, 2H, 2 × Har-Phthal), 7.60 (s, 1H, NH), 7.37 (d, J =

2

.4 Hz, 1H, Har), 7.24 (dd, J = 8.9, 2.4 Hz, 1H, Har), 4.49 (s, 2H, CH

2

),

45.1 (NCH

2

), 42.5 (CH

2

CHO), 37.9 (CH

2

CONH), 33.1 (CH S), 30.7

2

4

.25 (t, J = 7.2 Hz, 2H, NCH

2

), 3.55 (s, 3H, NMe), 3.19 (t, J = 7.3 Hz, 2H,

), 2.41 (t, J = 7.5 Hz, 2H, CH CONH), 2.34

), 1.78–1.68 (m, 4H, 2 ×

). C NMR (100 MHz, CDCl ): δ(ppm)

(NMe), 30.2 (CH

2

), 29.5 (CH

2

), 29.0 (CH

2

), 28.8 (CH ), 28.4 (CH ), 25.4

2 2

+

[M+H]⁺m/z 615.1712;

CH

s, 6H, NMe

2

S), 2.50 (s, 2H, CH NMe

2

(CH

2

). HRMS (ESI ): calcd for C29

H

33

N

6

O

3

SCl

2

(

2

), 2.08 (d, J = 8.5 Hz, 2H, CH

2

found 615.1719.

1

3

CH

2

), 1.50–1.32 (m, 6H, 3 × CH

2

3

–

1

71.4 (CONH), 159.3 (C

–

O), 152.1 (CIV), 151.9 (CIV-C=N), 141.2 (CIV),

33.6 (CHar-Phthal, CIV-NH), 132.0 (CHar-Phthal), 129.1 (CIV), 128.9 (CIV-Cl),

28.8 (CHar), 128.2 (CIV), 128.0 (CHar), 127.3 (CHar-Phthal), 125.4 (CHar-

1

5

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

4

.1.30. 3-(4-((5-((8-((2,4-dichlorophenyl)amino)-8-oxooctyl)thio)-4-

0.109 mg) were added

.

The mixture was then stirred for 48 h, then

methyl-4H-1,2,4-triazol-3-yl)methyl)-1-oxophthalazin-2(1H)-yl)propanoic

acid (43)

quenched with a saturated aqueous NH

4

Cl solution and extracted with

and

DCM. The organic layer was washed with brine, dried over MgSO

4

To a solution of aldehyde 42 (1 eq, 0.113 mmol, 70 mg) in t-BuOH/

concentrated under reduced pressure. The crude was puriﬁed by column

chromatography on silica gel (DCM/MeOH, 95:5) to afford the desired

ꢀ 1

THF 1:1 (0.011 mol.L ) was added DMSO, 2-methyl-2-buten (1 mL)

and water (5 mL). Then an aqueous solution of NaHPO (15 eq) and

NaClO (9 eq, 1.02 mmol, 92.6 mg) were added. The solution was stirred

at room temperature during 16 h and diluted with a saturated aqueous

NH Cl solution (30 mL), then extracted with DCM. The combined

organic layers were washed with brine, water, dried over Na SO

1

4

product 46 as a white powder (0.34 mmol, 190 mg, 49%). H NMR (300

2

6

MHz, DMSO‑d ): δ(ppm) 12.55 (s, 1H, NHPhthal); 9.52 (s, 1H, NH);

8.27–8.24 (m, 1H, Har-Phthal); 8.07–8.04 (m, 1H, Har); 7.95–7.85(m, 2H,

4

H

ar-Phthal); 7.68 (d, J = 8.7 Hz, 1H, Har-Phthal); 7.63 (d, J = 2.4 Hz, m, 1H,

ar); 7.6–7.40 (m, 1H, Har); 4.51 (s, 2H, CH ); 3.47 (s, 3H, NMe); 3.07 (t,

); 2.36 (t, 2H, J = 7.2 Hz, CH CO); 1.64–1.54 (m,

2

4

,

2

ﬁltered, and concentrated under reduced pressure. The crude was pu-

riﬁed by column chromatography on silical gel (DCM/MeOH, 96:4) to

J = 7.2 Hz, 2H, SCH

4H, 2 × CH ); 1.35–1.28 (m, 6H, 3 × CH

–

DMSO‑d ): δ(ppm) 171.7 (CONH), 159.4 (C O), 152.7 (CIV-C=N), 151.6

(CIV-C=N), 141.6 (CIV-Phthal), 134.2 (CIV-NHCO), 133.4 (CHar-Phthal), 131.7

(CHar-Phthal), 129.2 (CIV), 129.1 (CIV-Cl), 128.8 (CHar), 128.1 (CIV), 127.7

(CHar), 127.5 (CHar-Phthal), 125.9 (CHar-Phthal), 123.3 (CIV-Cl), 125.6

2

1

3

2

). C NMR (75 MHz,

afford the carboxylic acid 43 as a white powder (0.043 mmol, 27 mg,

6

1

3

8

7%). H NMR (400 MHz, CDCl

3

): δ(ppm) 8.47–8.36 (m, 1H, Har-Phthal),

.30 (d, J = 9.0 Hz, 1H, Har), 8.16–8.09 (m, 1H, Har-Phthal), 7.84–7.72

(

m, 2H, 2 × Har-Phthal), 7.65 (s, 1H, NH), 7.36 (d, J = 2.4 Hz, 1H, Har),

7

3

.23 (dd, J = 8.9, 2.4 Hz, 1H, Har), 4.54–4.34 (m, 4H, CH

.52 (s, 3H, NMe), 3.17 (t, J = 7.3 Hz, 2H, CH S), 2.82 (t, J = 6.7 Hz, 2H,

COOH), 2.42 (t, J = 7.5 Hz, 2H, CH CONH), 1.70 (m, 4H, 2 × CH ),

). C NMR (100 MHz, CDCl ): δ(ppm) 173.9

O), 152.3 (CIV-C=N), 151.9 (CIV-C=N),

2

and NCH

2

),

(CHar), 39.5 (NMe), 32.5 (CH

2

CONH), 30.2 (CH

2

S), 29.1 (CH ), 28.6

2

(CH ), 28.4 (CH ), 28.2 (CH ), 27.8 (CH

2

2 2

), 25.0 (CH ). Four quaternary

CH

2

carbon atoms are missing and the carbon atom of NMe is in the solvent

1

3

+

S [M+H]⁺m/z 559.1447;

1

.47–1.30 (m, 6H, 3 × CH

2

3

signal. HRMS (ESI ): calcd for C26

found 559.1450.

2 6 2

H29Cl N O

–

(

COOH), 171.7 (CONH), 159.2 (C

1

41.1 (CIV-Phthal), 133.7 (CHar-Phthal), 133.5 (CIV), 132.1 (CHar-Phthal),

29.3 (CIV-Cl), 128.9 (CIV), 128.9 (CIV), 128.8 (CHar), 127.9 (CHar), 127.4

4.1.34. N-(4-benzoylphenyl)-8-iodooctanamide (48)

(

CHar-Phthal), 125.1 (CHar-Phthal), 123.5 (CIV-Cl), 122.8 (CHar), 46.7

NCH ), 37.8 (CH CONH), 33.2 (CH S), 33.0 (CH COOH), 30.8 (NMe),

0.5 (CH ), 28.9 (CH ), 28.3 (CH

To a solution of octanoic acid (1 eq, 1.12 mmol, 250 mg) in DCM

(0.11 mmol.mL ¹) was added oxalyl chloride (2 eq, 2.24 mmol, 0.197

ꢀ

(

2

◦

3

2

), 29.8 (CH

2

), 29.4 (CH

32Cl

2

), 25.3 (CH

2

).

mL) with some drops of DMF at 0 C. The resulting clear solution was

ꢀ

S [Mꢀ H]^ꢀm/z 629.1492; found

HRMS (ESI ): calcd for C29

H

2

N

6

O

4

stirred until completion of the reaction. The mixture was concentrated

under reduced pressure and a solution of 4-amino benzophenone (1.2

6

29.1505.

3

eq, 1.34 mmol, 265 mg), Et N (2 eq, 2.24 mmol, 0.312 mL) and DMAP

4

.1.31. 2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetohydrazide (44)

To a suspension of 23 (1 eq, 3.44 g, 15.7 mmol) in EtOH was added

(0.05 eq, 0.056 mmol, 7 mg) in DCM (5 mL) was added slowly and then

stirred at room temperature. The mixture was diluted with DCM and

hydrazine monohydrate (1 eq, 15.7 mmol, 775

μ

L) at room temperature.

2 4

washed with water and brine. The organic layer was dried over Na SO ,

The solution was stirred for 45 min. then a second portion of hydrazine

was added (4 eq, 62.8 mmol, 3.1 mL) and the mixture was reﬂuxed

during 14 h. The mixture was then cooled to room temperature and the

ﬁltered and concentrated under reduced pressure. The crude was puri-

ﬁed by column chromatography on silica gel (DCM/AcOEt, 50:50) to

afford the N-(4-benzoylphenyl)-8-bromooctanamide as

a

white-

yellowish solid (0.831 mmol, 327 mg, 74%). H NMR (300 MHz,

CDCl

1

2

solid was ﬁltrated and washed with Et O to afford the desired hydrazide

1

4

4 as a white powder (12.9 mmol, 2.81 g, 82%). H NMR (300 MHz,

3

): δ(ppm) 7.85–7.79 (m, 2H, 2 × CHar), 7.79–7.71 (m, 2H, 2 ×

DMSO‑d

6

): δ(ppm) 12.75 (bs, 1H, NH), 9.36 (br, 1H, NH), 8.24 (m, 1H,

), 3.75 (s, 2H,

CHar), 7.68–7.62 (m, 2H, 2 × CHar), 7.62–7.53 (m, 1H, CHar), 7.53–7.44

(m, 2H, 2 × CHar), 7.39 (s, 1H, NH), 3.41 (t, J = 6.8 Hz, 2H, CH2-Br),

H

ar-Phthal), 7.91–7.84 (m, 3H, Har-Phthal), 4.28 (bs, 2H, NH

2

). ¹³C NMR (75 MHz, DMSO‑d

COPhthal), 141.9 (CIV), 133.3 (CHar-Phthal), 131.5 (CHar-Phthal), 129.7

–

O), 159.5

2.51–2.35 (m, 2H, CH2-CONH), 1.92–1.81 (m, 2H, CH

), 1.81–1.66 (m,

CH

6

): δ(ppm) 167.9 (C

2

+

(

2H, CH

2

), 1.49–1.37 (m, 6H, 3 × CH

2

). HRMS (ESI ): calcd for

+

C

IV), 127.5 (CIV), 125.8 (CHar-Phthal), 125.6 (CHar-Phthal), 37.6 (CH

2

).

C

21

H

25NO

2

Br [M+H] m/z 402.1059; found 402.1069. NaI (2.25 eq,

ꢀ

[Mꢀ H]^ꢀm/z 217.0734; found

HRMS (ESI ): calcd for C10

H

9

N

4

O

2

14.54 mmol, 2.18 g) was added to a solution of bromo compound pre-

ꢀ 1

2

17.0726.

viously obtained (1 eq, 6.46 mmol, 2.60 g) in acetone (0.7 mol.L ). The

solution was allowed to reﬂux during 2 h then concentrated. The residue

was solubilized in DCM and NaI/NaBr salts were ﬁltered off. The organic

layer was then concentrated under reduced pressure to afford the ex-

4

.1.32. 4-((5-mercapto-4-methyl-4H-1,2,4-triazol-3-yl)methyl)

phthalazin-1(2H)-one (45)

To a suspension of hydrazide 44 (1 eq, 2.2 mmol, 0.5 g) in EtOH were

added MeNCS (5 eq, 11.4 mmol, 0.84 g), and the mixture was heated at

reﬂux during 3 h. Then DBU (1 eq, 2.2 mmol, 0.35 g) was added and

heating was maintained during another 3 h. The reaction was cooled to

room temperature and the precipitate was ﬁltered and washed with

pected iodo compound 48 as a yellow viscous oil (6.45 mmol, 2.90 g,

1

quant.). H NMR (300 MHz, CDCl

3

): δ (ppm) 8.30 (s, 1H, NH), 7.81–7.72

(m, 4H, 4 × CHar-CO), 7.68 (d, J = 8.8 Hz, 2H, 2 × CHar), 7.61–7.52 (m,

1H, CHar), 7.50–7.37 (m, 2H, 2 × CHar), 3.14 (t, J = 7.0 Hz, 2H, CH2-I),

2.38 (t, J = 7.5 Hz, 2H, CH2-CONH), 1.82–1.73 (m, 2H, CH

2

), 1.70 (m, 2H,

): δ (ppm) 196.1

–

O), 172.2 (CONH), 142.5 (CIV-NHCO), 137.8 (CIV-CO), 132.7 (CIV-CO),

EtOH and Et

mmol, 2.81 g, 69%). H NMR (300 MHz, DMSO‑d

H, SH); 12.58 (s, 1H, NH); 8.27 (m, 1H, Har-Phthal); 8.0–7.85(m, 3H, Har-

Phthal) 4.51(s, 2H, CH ); 3.42 (s, 3H, CH ):

). ¹³C NMR (75 MHz, DMSO‑d

δ(ppm) 166.9 (CIV); 159.3 (C O); 149.6 (CIV); 140.9 (CIV); 133.6 (CHar-

2

O to afford the desired thiol 45 as a white powder (12.9

CH

2

), 1.34 (m 6H, 3 × CH

). ¹³C NMR (75 MHz, CDCl

2 3

1

6

): δ(ppm) 13.58 (s,

(C

1

132.4 (CHar), 131.7 (2 × CHar), 129.9 (2 × CHar), 128.4 (2 × CHar),

2

3

6

118.9 (2 × CHar), 37.7 (CH2-CONH), 33.4 (CH

2

), 30.3 (CH

+

2

), 29.1 (CH

2

),

–

28.3 (CH

2

), 25.4 (CH

2

), 7.3 (CH2-I). HRMS (ESI ): calcd for C21

H

25NO I

2

Phthal); 131.9 (CHar-Phthal); 129.1 (CIV); 127.6 (CIV); 125.9 (CHar-Phthal);

[M+H]⁺m/z 450.0922; found 450.0930.

+

1

25.5 (CHar-Phthal); 30.1 (CH

3

); 28.1 (CH

2

). HRMS (ESI ): calcd for

C

12

H

12

N

5

OS [M+H]⁺m/z 274.0763; found 274.0752.

4.1.35. N-(4-benzoylphenyl)-8-((4-methyl-5-((3-methyl-4-oxo-3,4-

dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)octanamide

4

.1.33. N-(2,4-dichlorophenyl)-8-((4-methyl-5-((4-oxo-3,4-

dihydrophthalazin-1-yl)methyl)-4H-1,2,4-triazol-3-yl)thio)octanamide

46)

To a solution of thiol 45 (1 eq, 0.7 mmol, 0.2 g), in dry DMF, bro-

moamide 7 (1 eq, 0.7 mmol, 0.255 mg) and K CO (1.2 eq, 0.8 mmol,

(49)

In a reactor tube was added K

2 3

CO (2.2 eq, 0.43 mmol, 59 mg) to a

(

solution of thiol 47 (1 eq, 0.19 mmol, 56 mg) and iodo compound 48

ꢀ 1

(1.3 eq, 0.253 mmol, 114 mg) in DMF (0.05 mol.L ). The solution was

◦

2

3

allowed to stir at 40 C during 1 h30, then at room temperature during

1

6

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

1

2 h. Upon full conversion the mixture was diluted in AcOEt (15 mL),

Part of the work was ﬁnancially supported by PIRAMID consortium

(ﬁnancial support from R ´e gion Bretagne-Pays de Loire, http://piramid-

research.fr) with a grant for PhD Jimmy Smadja. Most of the co-authors

are members of the consortium PIRAMID (e.g. J. Lebreton, E Mortier, D.

Dubreuil, A. Qu ´e m ´e ner and M. Math ´e -Allainmat).

washed with water and brine, dried over Na SO , ﬁltered and concen-

2

4

trated under reduced pressure. The resulting residue was puriﬁed by

column chromatography on silica gel (DCM/MeOH, 100:0 to 95:5) to

afford the corresponding compound 49 as white powder (0.197 mmol,

1

8

20 mg, 99%.). H NMR (400 MHz, CDCl

3

): δ(ppm) 9.02 (s, 1H, NH),

.55–8.31 (m, 1H, CHar-Phthal), 8.22–8.08 (m, 1H, CHar-Phthal), 7.81–7.68

Acknowledgments

(

m, 8H, 2 × CHar-Phthal, 3 × CHar), 7.60–7.51 (m, 1H, CHar), 7.49–7.40

m, 2H, 2 × CHar), 4.48 (s, 2H, CH ), 3.77 (s, 3H, NMe-Phthal), 3.58 (s,

H, NMe-Triazole), 3.12 (t, J = 7.3 Hz, 2H, CH2-S), 2.40–2.29 (m, 2H, CH2-

), 1.36 (m, 2H, CH ), 1.26

): δ(ppm) 195.9 (CO-benzo),

O), 152.2 (CIV), 152.1 (CIV), 142.9 (CIV-NHCO),

(

2

This work was supported by INSERM, CNRS, University of Nantes

and Region Pays de Loire (PIRAMID, http://piramid-research.fr ). J.S.

was supported by a fellowship from R ´e gion Pays de Loire (PIRAMID).

We thank CHEM-Symbiose (https://pf-chemsymbiose.univ-nantes.fr/)

and IMPACT (http://www.crcina.org/plateformes/impact) facilities for

technical assistance.

3

´

CONH), 1.66 (td, J = 7.4, 5.1 Hz, 4H, 2 × CH

2

1

3

(

m, 4H, 2 × CH

2

). C NMR (100 MHz, CDCl

3

1

72.5 (CONH), 159.5 (C^–

–

40.7 (CIV-S), 138.1 (CIV-CO), 133.4 (CHar-Phthal), 132.5 (CHar-Phthal),

32.3 (CIV-CO), 132.1 (CHar), 131.6 (2 × CHar), 129.9 (2 × CHar), 129.0

IV), 128.4 (2 × CHar), 128.0 (CIV), 127.2 (CHar-Phthal), 125.1 (CHar-

(

C

Appendix A. Supplementary material

Phthal), 118.9 (2 × CHar-Phthal), 39.6 (NMe-Phthal), 37.6 (CH2-CONH), 33.1

(

CH2-S), 30.9 (NMe-Triazole), 30.0 (CH

2

), 29.3 (CH

2

), 28.7 (CH

2

), 28.2

₁_H_N_M_R_a_n_d13C NMR spectra of the compounds 1-2, 8-14, 19-22,

+

CH

2

), 27.9 (CH

2

), 25.2 (CH

2

). HRMS (ESI ): calcd for C34

H

37

6

N O

3

S

2

1

8-32, 39-43, 46 and 49, molecular properties of compounds 1, 2, 2A,

B, 2C, 49, 12, 46, 11, 41,42, and SPR sensorgrams of compounds 2, 11,

2, 41, 42, 46 and 49 are provided in Supplementary Material. Sup-

+

[

M+H] m/z 609.2650; found 609.2648.

4

.2. Biological assays

plementary data to this article can be found online at https://doi.

org/10.1016/j.bmc.2021.116161 .

.2.1. Cell lines, recombinant proteins and antibodies

The 32Dβ and NK-92 cell lines were used in that study and cultured

References

as described previously.³

7,38

Recombinant human IL-15, recombinant

murine IL-3 were obtained from Peprotech, Inc. and recombinant human

1

2

Grabstein KH, Eisenman J, Shanebeck K, et al. Cloning of a T Cell Growth Factor That

Interacts with the Beta Chain of the Interleukin-2 Receptor. Science. 1994;264:

965–968. https://doi.org/10.1126/science.8178155 .

IL-2 from Chiron. RLI, an IL-15 agonist was produced as described

_p_r_e_v_i_o_u_s_l_y_.₃9

Burton JD, Bamford RN, Peters C, et al. A Lymphokine, Provisionally Designated

Interleukin T and Produced by a Human Adult T-Cell Leukemia Line, Stimulates T-

Cell Proliferation and the Induction of Lymphokine-Activated Killer Cells. Proc Natl

Acad Sci U S A. 1994;91:4935–4939. https://doi.org/10.1073/pnas.91.11.4935 .

Waldmann TA, Miljkovic MD, Conlon KC. Interleukin-15 (Dys) Regulation of

Lymphoid Homeostasis: Implications for Therapy of Autoimmunity and Cancer. J Exp

Med. 2020;217. https://doi.org/10.1084/jem.20191062 .

4

.2.2. Proliferation assays

3

9

The proliferation response of 32Dβ cells to RLI, was assessed by

3

Alamar blue reduction assay (AbDSerotec). Cells were starved in the

4

culture medium without cytokine for 4 h. Cells (1x10 ) were cultured for

4

5

Fehniger TA, Caligiuri MA. Interleukin 15: Biology and Relevance to Human Disease.

Blood. 2001;97:14–32. https://doi.org/10.1182/blood.V97.1.14 .

2

.5 days in the medium supplemented with 100 pM RLI or 1.5 nM IL-2,

previously preincubated for 30 min with increasing concentrations of

compounds. Cell proliferation was revealed by adding Alamar blue (10

µL) to each well and, after a 6 h-incubation period at 37 ^◦C, by

measuring the emitted ﬂuorescence at 590 nm under excitation at 560

nm using an EnSpire Multimode Plate Reader (Perkin Elmer).

Waldmann TA. The Biology of Interleukin-2 and Interleukin-15: Implications for

Cancer Therapy and Vaccine Design. Nat Rev Immunol. 2006;6:595–601. https://doi.

org/10.1038/nri1901 .

6

7

Miyazaki T, Kawahara A, Fujii H, et al. Functional Activation of Jak1 and Jak3 by

Selective Association with IL-2 Receptor Subunits. Science. 1994;266:1045–1047.

https://doi.org/10.1126/science.7973659.

Lin JX, Migone TS, Tsang M, et al. The Role of Shared Receptor Motifs and Common

Stat Proteins in the Generation of Cytokine Pleiotropy and Redundancy by IL-2, IL-4,

IL-7, IL-13, and IL-15. Immunity. 1995;2:331–339. https://doi.org/10.1016/1074-

4

.2.3. P-Stat5 assays

Exponentially-growing NK-92 cells were washed and serum-starved

7

613 (95)90141-8 .

5

overnight to reduce basal phosphorylation. Cells (2x10 ) were then

8

9

Ruchatz H, Leung BP, Wei XQ, McInnes IB, Liew FY. Soluble IL-15 Receptor Alpha-

Chain Administration Prevents Murine Collagen-Induced Arthritis: A Role for IL-15

in Development of Antigen-Induced Immunopathology. J Immunol Baltim Md 1950 .

◦

stimulated at 37 C for 1 h with ﬁxed concentrations of IL-15 or IL-2 that

have been preincubated for 30 min with increasing concentrations of

chemical compounds. At the end of the stimulation, cells were lysed and

Stat5 phosphorylation was measured according to the manufacturer’s

instructions using p-Stat5 AlphaScreen Sureﬁre kit (Perkin Elmer Life

Sciences). The signal in the wells was then detected using an EnSpire

Multimode Plate Reader (Perkin Elmer).

1

998;160:5654 –5660 .

Ferrari-Lacraz S, Zanelli E, Neuberg M, et al. Targeting IL-15 Receptor-Bearing Cells

with an Antagonist Mutant IL-15/Fc Protein Prevents Disease Development and

Progression in Murine Collagen-Induced Arthritis. J Immunol Baltim Md 1950. 2004;

1

73:5818–5826. https://doi.org/10.4049/jimmunol.173.9.5818 .

10 Wang D, Deng X, Leng X, Mao X. Interleukin-15 Receptor-Directed Immunotoxins

Atteunuate Disease Severity in Rat Adjuvant Arthritis. Mol Immunol. 2010;47:

1

535 –1543. https://doi.org/10.1016/j.molimm.2010.01.023 .

1

2

Mitra S, Ring AM, Amarnath S, et al. Interleukin-2 Activity Can Be Fine-Tuned with

Engineered Receptor Signaling Clamps. Immunity . 2015;42:826 –838, 10.1016/j.

immuni.2015.04.018.

4

.2.4. SPR experiment

The SPR experiments were performed at 25 C on a Biacore T200

◦

Nata T, Basheer A, Cocchi F, et al. Targeting the Binding Interface on a Shared

Receptor Subunit of a Cytokine Family Enables the Inhibition of Multiple Member

Cytokines with Selectable Target Spectrum. J Biol Chem. 2015;290:22338–22351.

https://doi.org/10.1074/jbc.M115.661074.

Zhang M, Griner LAM, Ju W, et al. Selective Targeting of JAK/STAT Signaling Is

Potentiated by Bcl-XL Blockade in IL-2 –Dependent Adult T-Cell Leukemia. Proc Natl

Acad Sci. 2015;112:12480–12485. https://doi.org/10.1073/pnas.1516208112.

biosensor (GE Healthcare, Chalfont St Giles, UK). Recombinant IL-15

was covalently immobilized to CM5 sensor chips using the amine

coupling method in accordance with the manufacturer’s instructions,

and the binding of compounds at increasing concentrations, prepared in

HBSEP/DMSO (0.1%) was monitored. The Biacore T200 Evaluation 3.0

software (GE Healthcare) was used to run experiments and for data

analysis.

1

3

14 Smith XG, Bolton EM, Ruchatz H, Wei X, Liew FY, Bradley JA. Selective Blockade of

IL-15 by Soluble IL-15 Receptor

J Immunol. 2000;165:3444–3450. https://doi.org/10.4049/jimmunol.165.6.3444 .

Bouchaud G, Gehrke S, Krieg C, et al. Epidermal IL-15R Acts as an Endogenous

Antagonist of Psoriasiform Inﬂammation in Mouse and Man. J Exp Med. 2013;210:

α-Chain Enhances Cardiac Allograft Survival.

1

5

6

α

Declaration of Competing Interest

2

105–2117. https://doi.org/10.1084/jem.20130291 .

1

Rückert R, Brandt K, Braun A, et al. Blocking IL-15 Prevents the Induction of

Allergen-Speciﬁc T Cells and Allergic Inﬂammation In Vivo. J Immunol. 2005;174:

5507–5515. https://doi.org/10.4049/jimmunol.174.9.5507 .

The authors declare the following ﬁnancial interests/personal re-

lationships which may be considered as potential competing interests:

1

7

J. Smadja et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116161

1

7

8

Villadsen LS, Schuurman J, Beurskens F, et al. Resolution of Psoriasis upon Blockade

of IL-15 Biological Activity in a Xenograft Mouse Model. J Clin Invest. 2003;112:

29 Qu ´e m ´e ner A, Maillasson M, Arzel L, et al. Discovery of a Small-Molecule Inhibitor of

Interleukin 15: Pharmacophore-Based Virtual Screening and Hit Optimization. J Med

Chem. 2017;60:6249–6272. https://doi.org/10.1021/acs.jmedchem.7b00485 .

30 Krauth F, Ihling CH, Rüttinger HH, Sinz A. Heterobifunctional Isotope-Labeled

Amine-Reactive Photo-Cross-Linker for Structural Investigation of Proteins by

Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight and

Electrospray Ionization LTQ-Orbitrap Mass Spectrometry. Rapid Commun Mass

Spectrom. 2009;23:2811–2818. https://doi.org/10.1002/rcm.4188.

31 Dalcanale E, Montanari F. Selective Oxidation of Aldehydes to Carboxylic Acids with

Sodium Chlorite-Hydrogen Peroxide. J Org Chem. 1986;51:567–569. https://doi.org/

10.1021/jo00354a037.

32 Wong R, Dolman SJ. Isothiocyanates from Tosyl Chloride Mediated Decomposition of

in Situ Generated Dithiocarbamic Acid Salts. J Org Chem. 2007;72:3969–3971.

https://doi.org/10.1021/jo070246n .

33 Park S, Hayes BL, Marankan F, et al. Regioselective Covalent Modiﬁcation of

Hemoglobin in Search of Antisickling Agents. J Med Chem. 2003;46:936–953.

https://doi.org/10.1021/jm020361k .

1

571–1580. https://doi.org/10.1172/JCI200318986.

Malamut G, El Machhour R, Montcuquet N, et al. IL-15 Triggers an Antiapoptotic

Pathway in Human Intraepithelial Lymphocytes That Is a Potential New Target in

Celiac Disease-Associated Inﬂammation and Lymphomagenesis. J Clin Invest. 2010;

1

20:2131–2143. https://doi.org/10.1172/JCI41344 .

1

9

Waldmann TA. Targeting the Interleukin-15/Interleukin-15 Receptor System in

Inﬂammatory Autoimmune Diseases. Arthritis Res Ther. 2004;6:174–177. https://doi.

org/10.1186/ar1202 .

2

0

1

Waldmann TA. Targeting the Interleukin-15 System in Rheumatoid Arthritis. Arthritis

Rheum . 2005;52:2585 –2588. https://doi.org/10.1002/art.21363 .

Tinubu SA, Hakimi J, Kondas JA, et al. Humanized Antibody Directed to the IL-2

Receptor Beta-Chain Prolongs Primate Cardiac Allograft Survival. J Immunol Baltim

Md 1950 . 1994;153:4330 –4338 .

2

Raimundo BC, Oslob JD, Braisted AC, et al. Integrating Fragment Assembly and

Biophysical Methods in the Chemical Advancement of Small-Molecule Antagonists of

IL-2: An Approach for Inhibiting Protein ꢀ Protein Interactions. J Med Chem. 2004;47:

34 Fourmaintraux E, Depreux P, Lesieur I. Synthesis of New Phthalazinyl Compounds as

Potential Inhibitors of Aldose Reductase and Sorbitol Dehydrogenase. Heterocycl

Commun. 1999;5:213–216. https://doi.org/10.1515/HC.1999.5.3.213.

3

111–3130. https://doi.org/10.1021/jm049967u .

2

3

4

5

6

7

8

Waal ND, Yang W, Oslob JD, et al. Identiﬁcation of Nonpeptidic Small-Molecule

′

Inhibitors of Interleukin-2. Bioorg Med Chem Lett. 2005;15:983–987. https://doi.org/

35 Hampton SE, Baraga n˜ a B, Schipani A, et al. Design, Synthesis, and Evaluation of 5 -

1

0.1016/j.bmcl.2004.12.045 .

Diphenyl Nucleoside Analogues as Inhibitors of the Plasmodium Falciparum

DUTPase. ChemMedChem. 2011;6:1816–1831. https://doi.org/10.1002/

cmdc.201100255.

Leimbacher M, Zhang Y, Mannocci L, et al. Discovery of Small-Molecule Interleukin-

2

7

Inhibitors from a DNA-Encoded Chemical Library. Chem – Eur J. 2012;18:

729–7737. https://doi.org/10.1002/chem.201200952.

36 Heymans F, Borrel MC, Broquet C, Lefort J, Godfroid JJ. Structure-Activity

Relationship in PAF-Acether. 2. RAC-1-O-Octadecyl-2-O-Acetyl-3-O-[.Gamma.-

(Dimethylamino)Propyl]Glycerol. J Med Chem 1985;28:1094–6. https://doi.org/

10.1021/jm00146a019.

Kalsoom S, Rashid U, Shaukat A, et al. L. In Vitro and in Silico Exploration of IL-2

Inhibition by Small Drug-like Molecules. Med Chem Res. 2013;22:5739–5751.

https://doi.org/10.1007/s00044-013-0564-x .

Halim, S. A.; Abdalla, O. M.; Mesaik, M. A.; Wadood, A.; ul-Haq, Z.; Kontoyianni, M.

Identiﬁcation of Novel Interleukin-2 Inhibitors through Computational Approaches.

Mol Divers 2013, 17 (2), 345–355. https://doi.org/10.1007/s11030-013-9431-4.

37 Vincent M, Bessard A, Cochonneau D, et al. Tumor Targeting of the IL-15

Superagonist RLI by an Anti-GD2 Antibody Strongly Enhances Its Antitumor Potency.

Int J Cancer. 2013;133:757–765. https://doi.org/10.1002/ijc.28059 .

˙

´

Zyzynska-Granica B, Trzaskowski B, Niewieczerzał S, et al. Pharmacophore Guided

Discovery of Small-Molecule Interleukin 15 Inhibitors. Eur J Med Chem. 2017;136:

38 Tamzalit F, Barbieux I, Plet A, et al. IL-15.IL-15R Complex Shedding Following

α

Trans-Presentation Is Essential for the Survival of IL-15 Responding NK and T Cells.

Proc Natl Acad Sci USA. 2014;111:8565–8570. https://doi.org/10.1073/

pnas.1405514111.

5

43–547. https://doi.org/10.1016/j.ejmech.2017.05.034 .

˙

´

Zyzynska-Granica B, Trzaskowski B, Dutkiewicz M, et al. The Anti-Inﬂammatory

Potential of Cefazolin as Common Gamma Chain Cytokine Inhibitor. Sci Rep. 2020;

39 Mortier E, Qu ´e m ´e ner A, Vusio P, et al. Soluble Interleukin-15 Receptor Alpha (IL-15R

Alpha)-Sushi as a Selective and Potent Agonist of IL-15 Action through IL-15R Beta/

Gamma. Hyperagonist IL-15 x IL-15R Alpha Fusion Proteins. J Biol Chem. 2006;281:

1

0. https://doi.org/10.1038/s41598-020-59798-3.

1

612–1619. https://doi.org/10.1074/jbc.M508624200 .

1

8

Article Doi

Rational modification, synthesis and biological evaluation of N-substituted phthalazinone derivatives designed to target interleukine-15 protein

DOI: 10.1016/j.bmc.2021.116161

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2021.116161

Full text of DOI:10.1016/j.bmc.2021.116161

Products guided by the article

R&D Labs maybe for 25947-18-6

Relevant to this article

Hot Product