Substituted arylsulphonamides as inhibitors of perforin-mediated lysis.

The structure-activity relationships for a series of arylsulphonamide-based inhibitors of the pore-forming protein perforin have been explored. Perforin is a key component of the human immune response, however inappropriate activity has also been implicated in certain auto-immune and therapy-induced conditions such as allograft rejection and graft versus host disease. Since perforin is expressed exclusively by cells of the immune system, inhibition of this protein would be a highly selective strategy for the immunosuppressive treatment of these disorders. Compounds from this series were demonstrated to be potent inhibitors of the lytic action of both isolated recombinant perforin and perforin secreted by natural killer cells in vitro. Several potent and soluble examples were assessed for in vivo pharmacokinetic properties and found to be suitable for progression to an in vivo model of transplant rejection.

Introduction

Perforin is a 67 kDa, calcium-dependent glycoprotein expressed by only the natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) of the mammalian immune system [1], [2]. These “killer lymphocytes” utilise the pore-forming ability of perforin as a critical component of the granule exocytosis pathway; the principal mechanism used by NK and CTL cells for tumour immunosurveillance and as a defence against viral infection and intracellular pathogens [3]. Identification of a target cell by an effector cell results in the formation of an immune synapse whereupon CTL (or NK) secretory granules polarise to the site of contact. These granules contain both perforin and a group of pro-apoptotic serine proteases known as granzymes, and upon fusion with the CTL plasma membrane, release their luminal contents into the synapse [2], [4]. Perforin performs a key role in this process because entry of the granzymes required for cell death into the target cell cytosol is solely dependent on its presence [1], [5].

Although perforin is synthesized and secreted into the immune synapse as a monomer, it rapidly binds to the target cell membrane through its calcium-dependent C2 domain [6], [7] and oligomerises into large transmembrane pores composed of approximately 24 perforin monomers. This process was elucidated using a combination of the perforin X-ray crystal structure and cryoelectron microscopy to reconstruct an entire perforin pore [8]. Electron microscopy, X-ray crystallography and functional studies have also shown that the process involves electrostatic interactions which include a salt bridge formed between R213 on the ‘front’ surface of one monomer interacting with E343 on the ‘back’ surface of the adjacent monomer [9]. Similarly, mutational studies reveal that D191, which is immediately adjacent to R213, makes interactions that are key to oligomerisation and that substitution with a bulky hydrophobic residue (D191V) abrogates this process [9].

Until recently, the precise mechanism of granzyme entry into the target cell was debated, but it is beyond any doubt that the pore-forming activity of perforin is indispensable. In essence, secreted perforin forms large (18 nm diameter) transmembrane pores on the surface of the target cell, through which the granzyme monomers (4 nm diameter) diffuse into the cytosol [10], [11]. Once internalised the granzymes cleave key substrates to initiate rapid apoptotic death [5], [10], [11], [12], [13]. Unlike the granzymes, which are encoded by many genes and are, therefore, subject to considerable redundancy of function, the gene encoding perforin (PRF1) is present as a single copy in all mammals. Gene-targeting studies in mice [1] and naturally occurring disease-causing mutations in humans [14], [15] confirm that perforin deficiency cannot be compensated by any other protein. This makes perforin an ideal target for therapeutic intervention.

While perforin is a key component of the immune response, inappropriate activity has also been implicated in a number of human immunopathologies and therapy-induced conditions. These include cerebral malaria, insulin-dependent diabetes, juvenile idiopathic arthritis and postviral myocarditis [16], [17], [18], as well as therapy-induced conditions such as allograft rejection and graft versus host disease [19], [20], [21]. Our current goal is to seek small molecule inhibitors of perforin as potential immunosuppressive agents for the treatment of autoimmune diseases and other conditions characterised by dysfunction of this pathway. This should be a highly selective strategy since perforin is expressed exclusively by CTL and NK cells, in contrast to approaches using conventional immunosuppression treatments which indiscriminately depress immune function [22], [23], [24].

Based on an initial hit from a mass screen [25], we have previously designed and optimised inhibitors of perforin that can; (i) block recombinant purified perforin, (ii) block perforin delivered by intact NK cells and, (iii) withstand incubation in serum (e.g. 1; Fig. 1) [26], [27], [28], [29], [30].

Fig. 1

Perforin inhibitors and PI3Kα clinical candidate GSK2126458.

While these compounds appeared highly promising, replacement of the 2-thioxoimidazolidinone moiety that contained a potential Michael acceptor and showed variable toxicity toward perforin-producing NK cells proved problematic. This issue was only overcome when we amalgamated our own finding that an aryl sulphonamide could act as a bioisosteric replacement [30] with a strategy implemented by GSK workers, where a thiazolidinedione was replaced with a pyridyl-linked benzenesulphonamide to give 2 [31]. This approach resulted in a new series of benzenesulphonamide-based perforin inhibitors, exemplified by 3, which were potent, soluble and essentially non-toxic toward NK cells [32]. In the following report we extend our study to explore whether it is possible to further modulate activity and physicochemical properties through variation of the sulphonamide linker, linker position, and substitution on the central pyridine ring and terminal benzene (Fig. 2).

Fig. 2

Variations on 3 to give new benzenesulphonamide analogues.

Results and discussion

Chemistry

The majority of the target compounds were constructed from right to left starting with our previously published key iodide 75 [32] (Scheme 1). Suzuki reaction of 75 with a variety of commercially available aminopyridine boronates under standard conditions gave the required amine intermediates 76–79 which were subsequently reacted with a range of substituted aryl sulphonyl chlorides. The 5-amino-3-pyridine derivative 78 [32] in particular was employed in the preparation of all compounds in Table 3. One exception was where the central pyridine ring was replaced with a benzene; in this case the Suzuki step was carried out with 2-methyl-5-nitrobenzeneboronic acid, the nitro compound (80) hydrogenated to give the amine (81), which was then reacted with either 2,4-difluorobenzenesulphonyl chloride or 2-pyridinesulphonyl chloride to afford 13 and 15 respectively. Finally, amido-linked compound 8 was prepared by reaction of 78 with 2,4-difluorobenzoic acid chloride.

Scheme 1

Reagents and conditions: (i) Boronate, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux; (ii) H2, 60 psi, 1:1:1 EtOH/EtOAc/THF, RT, 3 h; (iii) 2,4-Difluorobenzenesulfonyl chloride or pyridine-2-sulfonyl chloride, pyridine, 0–45 °C; (iv) 2,4-Difluorobenzenoic acid chloride, pyridine, 0–45 °C, 16 h.

Table 3

Modulation of activity through substitution on the sulphonamide.

Compound	R1	Inhibition of Jurkat Cell Lysis IC50 (μM)
21	benzene	8.46
22	2-F-benzene	2.03
23	3-F-benzene	∼20
24	4-F-benzene	9.65
3a	2,4-diF-benzene	1.17
25	3,4-diF-benzene	18.6
26	2,4,6-triF-benzene	1.76
27	2-Cl-benzene	4.01
28	3-Cl-benzene	2.42
29	4-Cl-benzene	5.39
30	3,4-diCl-benzene	1.32
31	2,4-diCl-benzene	15.0
32	2-Br-benzene	2.66
33	3-Br-benzene	2.11
34	4-Br-benzene	8.77
35	2,4-diBr-benzene	2.87
36	4-I-benzene	5.00
37	2-OCH3-benzene	14.8
38	3-OCH3-benzene	2.56
39	4-OCH3-benzene	6.27
40	3,4-diOCH3-benzene	13.8
41	2-OCF3-benzene	17.7
42	3-OCF3-benzene	1.42
43	4-OCF3-benzene	>20
44	2-CF3-benzene	1.45
45	3-CF3-benzene	1.22
46	4-CF3-benzene	>20
47	3,5-diCF3-benzene	>20
48	2-CN-benzene	9.17
49	3-CN-benzene	>20
50	4-CN-benzene	5.17
51	2-COOCH3-benzene	2.90
52	3-COOCH3-benzene	7.77
53	4-COOCH3-benzene	8.16
54	4-COOCH2CH3-benzene	19.4
55	4-COOH-benzene	0.75
56	2-SO2CH3	6.80
57	4-SO2CH3	>20
58	2-NO2-benzene	6.65
59	4-NO2-benzene	2.74
60	2-F,3-Cl-benzene	13.0
61	2-CH3-4-F-benzene	5.53
62	3-CF3-4-F-benzene	3.75
63	3-Cl, 4-CH3-benzene	8.14
64	2-Cl, 4-CF3-benzene	>20
65	3-CF3-5-Br-benzene	>20
66	2-pyridyl	1.07
67	3-pyridyl	15.1
68	2-thiophenyl	1.07
69	3-thiophenyl	12.5
70	Image 5	3.05
71	Image 6	7.10
72	Image 7	19.1
73	Image 8	>20
74	Image 9	11.6

Included as a comparator; see Ref. [32].

In a smaller number of cases, mostly those examples with substitution on the central pyridine ring, the target compounds were effectively synthesized from two halves; the fully elaborated left-hand side benzenesulphonamide subunit (e.g 82–85) and key iodide 75 as the right-hand side (Scheme 2). The intermediate bromides 82–85 and 91–93 were prepared from a variety of commercially available aryl sulphonyl chlorides and substituted 3-aminopyridines (or anilines) under standard conditions. In the case of 85, the sulphonamide NH was methylated with NaH and MeI in DMF to give 86, and for 91–93 where protection of this NH was required for the subsequent coupling to be successful, the alkylation was carried out with (chloromethoxy)ethane to give 94–96. All bromides were converted to the corresponding boronates 87–90 and 97–99 under palladium-catalysed conditions using bis(pinacolato)diboron and KOAc in DMSO, then finally reacted in a Suzuki step with iodide 75 to introduce the thiophene-N-methylisoindolinone subunit (6, 10–12, 17, 18, 20). Where required (for 17, 18, 20), deprotection was carried out under acidic conditions.

Scheme 2

Reagents and conditions: (i) a. NaH, DMF, 0° C, 0.5 h, b. MeI or (chloromethoxy)-ethane, DMF, 0° C-RT, 1.5 h; (ii) Bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, DMSO, 90 °C, 3 h.; (iii) a. 75, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux, b. Deprotection if required: 3 M HCl/1,4-dioxane (1:1), 1 h.

A limited number of “reverse” sulphonamides were also prepared (Scheme 3). In the case of target compound 7, intermediate bromide 100 was prepared from 2,4-difluoroaniline and 5-bromopyridine-3-sulphonyl chloride. Protection of the sulphonamide was not required and conversion to the boronate 101 and subsequent Suzuki coupling with 75 to give 7 proceeded smoothly. Likewise, bromide 102 was prepared from 2-aminopyridine 3-bromo-4-methylbenzenesulfonyl chloride, converted to the boronate 107 and therein coupled with 75 to afford the final product 16. For bromides 103 and 104, protection of the sulphonamide NH (105, 106) was required for the sequential borylation (108, 109) and Suzuki reactions to proceed in good yield, and afforded targets 14 and 19 respectively in good yield.

Scheme 3

Reagents and conditions: (i) a. NaH, DMF, 0 °C, 0.5 h, b. (Chloromethoxy)ethane, DMF, 0 °C-RT, 1.5 h; (ii) Bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, DMSO, 90 °C, 3 h.; (iii) a. 75, Pd(dppf)Cl2, EtOH/toluene, 2 M Na2CO3, reflux, b. Deprotection if required: 3 M HCl/1,4-dioxane (1:1), 1 h.

Inhibition of recombinant perforin-mediated lysis

In our initial report describing the discovery of benzenesulphonamide-based inhibitors of perforin [32], analogue design focussed on exploration of the thiophene and N-methylisoindolinone subunits which comprise the right-hand side of the molecule. For the current study we sought to further optimise potency and physicochemical characteristics through manipulation of the central pyridine and sulphonamide linker, as well as employing a wide range of substituents on the left-hand benzene (or aryl) ring.

Table 1 shows a group of compounds that explore the effect of changing the position of the sulphonamide link to the pyridine, the location of the pyridine nitrogen, and modification/replacement of the sulphonamide itself.

Table 1

Variation of the sulphonamide linker and position.

Compound	X (linker)	Linker Position	Y	Z	Inhibition of Jurkat Cell Lysis IC50 (μM)
3a	SO2NH	5	CH	N	1.17
4	SO2NH	4	C	N	>20
5	SO2NH	5	N	CH	4.70
6	SO2NCH3	5	CH	N	16.1
7b	NHSO2	5	CH	N	2.24
8	CONH	5	CH	N	>20

Included as a comparator; see Ref. [32].

Reverse sulphonamide.

By moving the sulphonamide link from the 5-position (3) to the 4-position (4), activity is abolished. If the sulphonamide is retained in the preferred 5-position and the pyridine nitrogen moved to the 4-position (5), activity is lost 4-fold compared to the lead, 3 (IC50s = 4.70 and 1.17 μM respectively). The requirement for a free NH in the sulphonamide was probed through methylation of 3 to give N-methyl compound 6. It appears likely that this acidic hydrogen is required for interaction with the target protein since a 14-fold drop in activity to IC50 = 16.1 μM was observed. A “reverse” sulphonamide (7) is however still acceptable, with less than a 2-fold reduction in activity (IC50 = 2.24 μM). Finally, replacement of the sulphonamide with a carboxamide (8) results in complete loss of potency, further supporting our hypothesis that the sulphonamide NH is required in the linker for optimal activity.

The effect of variation about the central pyridine ring was investigated next (Table 2). Direct replacement of the pyridine (3) with a benzene (9) resulted in a loss of activity from IC50 = 1.17 μM to 5.74 μM. Introduction of a 2-fluoro- (10) or 2-chloro-substituent (11) to the pyridine gave similar activity to 3 (IC50s = 1.03 and 1.99 μM respectively), while the electron-donating 2-methoxy-substituent (12) gave an analogue which was somewhat poorer (IC50 = 3.56 μM). Benzene-linked compounds (13–19) were then explored to determine if substitution on the benzene ring could improve activity. Introduction of a single methyl group on 9 to give 13 resulted in complete loss of potency, however at this point we reasoned that the increased lipophilicity and reduced solubility associated with the presence of two benzene rings (9 and 13) could be improved if we effectively moved the central pyridine of 3 to the other side of the sulphonamide link (2-pyridylsulphonamide compounds 14–20). This enabled us to explore a small number of compounds containing substitution on the central benzene ring, without further detriment to the overall physicochemical properties. Although far from a comprehensive list, the introduction of methyl (15; IC50 = 15.4 μM), fluoro (17; >20 μM), trifluoromethoxy (18; >20 μM) and methoxy (19; 9.68 μM) groups proved unsuccessful. The effect of reversing the sulphonamide orientation is consistent with Table 1, appearing to be slightly detrimental, although we only had a single matched pair for this comparison (15 vs 16; IC50s = 15.4 vs > 20 μM). Lastly, compound 20 contains pyridine rings on either side of the sulphonamide link. While the solubility was significantly improved, the resulting activity (IC50 = 4.10 μM) was still no improvement over the lead 3.

Table 2

Variation of the pyridine ring.

Compound	R1	R2	Z	R3	Inhibition of Jurkat Cell Lysis IC50 (μM)
3a	2,4-diF-benzene	H	N	H	1.17
9	2,4-diF-benzene	H	CH	H	5.74
10	2,4-diF-benzene	F	N	H	1.99
11	2,4-diF-benzene	Cl	N	H	1.03
12	2,4-diF-benzene	OCH3	N	H	3.56
13	2,4-diF-benzene	H	CH	CH3	>20
14b	2-pyridyl	H	CH	H	8.92
15	2-pyridyl	H	CH	CH3	15.4
16b	2-pyridyl	H	CH	CH3	>20
17	2-pyridyl	H	CH	F	>20
18	2-pyridyl	OCF3	CH	H	>20
19b	2-pyridyl	OCH3	CH	H	9.68
20	2-pyridyl	F	N	H	4.10

Included as a comparator; see Ref. [32].

Reverse sulphonamide.

Table 1, Table 2 are limited to either 2,4-difluorobenzene- or 2-pyridyl-sulphonamides. Thus it remained for us to investigate the effect of alternative substituents on the left-hand side of the molecule to determine if we could optimise potency and physicochemical properties further. A total of 52 new substituted benzene or aryl sulphonamides are shown in Table 3.

Clearly substitution on the benzene ring is required, with unsubstituted analogue 21 (IC50 = 8.46 μM) suffering a 7-fold loss in activity. While 2,4-difluoro-compound (3) was the original lead substitution pattern, it can be separated into the constituent mono-fluoro- analogues 22–24. This reveals that the contribution of the 2- position is most important (IC50 = 2.03 μM), followed by 4- (9.65 μM), and lastly the 3-position which is detrimental to activity (>20 μM). Accordingly, the 3,4-difluoro-substituted compound 25 shows poor potency (18.6 μM) while that of the 2,4,6-trifluoro- analogue 26 is excellent (1.76 μM). This positional effect however, is not apparent in the corresponding chloro- compounds (27–29; IC50s 2.11–8.77 μM) where the SAR between 2-, 3- and 4- is relatively flat. This may help explain why the most potent compound of the set is the 3,4-dichloro- 30 (1.32 μM) and not the 2,4-dichloro- target 31 (15.0 μM) as might be expected from the fluorinated series of analogues. The bromo- (32–35; IC50s 2.11–8.77 μM) and iodo-substituted (36; 5.00 μM) compounds also displayed flat SAR, perhaps because all these halogens are less electron-withdrawing than fluorine, a concept that we explore in more detail below. A different trend was observed when the electron-donating substituents methoxy (37–40) and trifluoromethoxy (41–43) were employed; here the meta- position was favoured over ortho- and para-. The two meta-substituted examples 38 (2.56 μM) and 42 (1.42 μM) showed significantly better activity in comparison to other positional isomers. We then surveyed a variety of electron-withdrawing substituents by preparing compounds 44–59. As a group, these broadly paralleled the fluorine-substituted compounds discussed above, where the ortho- or para- positional isomers showed superior potency over the corresponding meta- examples. More specifically, for the trifluoromethyl- (44–47), ester- (51–54) and sulphonyl- (56, 57) substituted compounds, the ortho isomer was best (IC50s = 1.45, 2.90, 6.80 μM respectively), while for the cyano- (48–50), carboxylic acid (55) and nitro- (58, 59) examples the para isomer showed high potency (5.17, 0.75 and 2.74 μM respectively). Compound 55 was particularly noteworthy, being one of the few sub-micromolar inhibitors of perforin identified to date. This subset of results is consistent with an inductive effect being exerted by electron-withdrawing substituents on the benzene ring and through to the sulphonamide, enhancing interactions with the protein and resulting in improved activity.

Hybrid compounds 60–65 were also prepared to investigate whether the effects of individual substituents could be combined. The resulting activities were neither synergistic nor additive, bringing no further gain to the overall potency. A set of four compounds (66–69) with a heterocycle (pyridine or thiophene) linked to the sulphonamide were also prepared. The preference for the heteroatom to be located directly next to the sulphonamide bond was clear with the 2-pyridyl and 2-thiophenyl compounds 66 and 68 (both IC50s = 1.07 μM) far superior to the corresponding 3-linked isomers 67 and 69 (15.13 and 12.51 μM respectively). Finally, a set of compounds containing a variety of substituted heterocycles were prepared (70–74), but with the exception of the 4-oxazole 70 (IC50 = 3.05 μM), none showed much promise.

Advanced assessment of selected compounds

Having shown that a range of benzenesulphonamides block lysis by recombinant perforin, a subset of promising examples was identified to test for inhibitory effect on the lytic action of whole NK cells. Compounds were selected based on potency, and included several for which the Jurkat IC50s were >20 μM, to further validate our use of this higher throughput screen as our primary assay. The inhibitors were co-incubated with KHYG1 human NK cells in medium for 30 min at room temperature, 51Cr-labelled target cells were added, and the resulting level of chromium release used to determine residual lytic activity and thus degree of inhibition. The use of whole NK cells to deliver perforin provides a more realistic model of conditions in vivo compared to isolated recombinant protein which acts indiscriminately. Recognition of a+ target cell, formation of a synaptic cleft, and release of the granular contents into the cavity between effector and target are all required elements for lysis to occur. Confirmation that the observed level of inhibition is due to blocking the activity of perforin rather than non-specific killing of the effector cell was also sought by measuring the viability of the NK cells 24 h later. Our lead compound for the current work and most potent compound from our previous study [32], 2,4-difluorobenzene 3, is included as a reference point (Table 4). One notable omission from this table is the potent 4-carboxylic acid-substituted compound 55 as this was toxic to the NK cells and therefore the degree of inhibition was unable to be determined.

Table 4

Capacity of selected compounds to inhibit perforin delivered by KHYG1 NK cells.

Compound	Jurkat IC50 (μM)a	KHYG1 Inhibition(% at 10 μM)b	KHYG1 Viability(% at 10 μM)c
3	1.17	67.5 ± 17.5	100
10	1.99	92.5 ± 1.8	92.0 ± 3.0
11	1.03	95.0 ± 2.5	94.0 ± 5.3
13	>20	50.3 ± 8.5	91.7 ± 10.0
16	>20	0	96.1 ± 6.0
17	>20	0	91.4 ± 6.5
26	1.76	81.3 ± 6.3	95.1 ± 2.1
44	1.45	85.0 ± 5.6	100
45	1.22	88.8 ± 1.9	100
47	>20	0	90.2 ± 3.4
49	>20	83.7 ± 3.2	94.4 ± 7.1
50	5.17	75.0 ± 7.8	99.3 ± 0.3
57	>20	0	90.0 ± 7.8
58	6.65	95.6 ± 1.9	98.7 ± 1.5
59	2.74	77.5 ± 5.6	85.0 ± 12.0

Data given for compounds as determined by the Jurkat assay.

Inhibition by compound (10 μM) of the perforin-induced lysis of 51Cr-labelled K562 leukemia target cells when co-incubated with KHYG1 human NK cells. Percent inhibition calculated compared to untreated control (n = 4).

Viability of KHYG1 NK cells after 24 h by Trypan blue exclusion assay (n = 3). See Experimental section for further details.

All of the compounds with Jurkat IC50s < 10 μM showed excellent suppression of NK-cell mediated killing of labelled target cells (68–96% at 10 μM), however this activity did not correlate exactly with their potency against isolated recombinant protein. This finding may reflect the varying ability of the respective inhibitors to access perforin located in a synaptic cleft within a complex biological milieu. Two examples with halogen on the central pyridine ring (10, 11) and the 2-nitrobenzene compound 58 were particularly effective in blocking NK cell action (93, 95 and 96% inhibition respectively). Results from the set of compounds with IC50s > 20 μM broadly validated our use of this assay as a primary screen, with four of six examples showing no potency in either assay (16, 17, 47, 57). Compound 13 demonstrated 50% inhibition at 10 μM, perhaps not surprisingly given that the only structural change from 3 was the replacement of a bridging pyridine with a benzene ring. The one unexpected outlier was the 3-cyano compound 49 which, although it had poor activity against isolated recombinant perforin, had excellent activity against perforin produced by whole NK cells. The NK cells also retained excellent viability across all examples, consistent with our previous findings for this series [32] and in contrast to earlier reported classes [26], [27], [28].

Preliminary physicochemical data was collected on the same (active) subset of compounds in order to assess their potential for progression to in vivo pharmacokinetic (PK) studies (Table 5). Following conversion to the corresponding sodium salts the solubility varied widely, with the 2,4,6-trifluorobenzene (26) and 4-cyanobenzene (50) analogues being highly soluble, while the presence of 2-fluoropyridine (10), 2-nitrobenzene or the more lipophilic trifluoromethylbenzene group (44, 45) had a negative impact on solubility. All examples tested showed good stability in aqueous solution over 24 h, however results were more varied in the presence of human, rat and mouse microsomes. While 10, 11, and 58 showed acceptable stability (>70% parent after 30 min) across all three species, the remaining compounds (3, 26, 45, 50, 59 and especially 44) showed moderate to poor stability with human microsomes. This data in combination with poor solubility resulted in the elimination of 44 and 45 from consideration for the in vivo PK studies reported in section 2.4 below.

Table 5

Physicochemical properties of selected compounds.

Compound	Solubilitya(μg/mL)	cLogPb	Stability in Solutionc(%)	Microsome Stability (%)d
				Mouse	Rat	Human
3e	1080	2.70 ± 0.72	82	92	99	58
10	428	2.34 ± 0.79	100	96	100	94
11	4027	3.21 ± 0.73	100	97	99	95
26	10674	2.65 ± 0.77	88	100	83	63
44	88	2.97 ± 0.65	92	84	49	14
45	574	3.13 ± 0.65	–	97	80	57
50	12900	2.17 ± 0.64	85	87	64	52
58	671	1.83 ± 0.61	100	100	89	73
59	1680	2.36 ± 0.61	–	85	72	39

Solubility of the sodium salt in water at room temperature; conversion to salt as described in experimental section 4.1.

cLogP calculated using ACD/PhysChem software v12.5.

Percentage of parent compound (as sodium salt) remaining after 24 h at 20 °C in water.

Percentage of parent compound (as sodium salt) remaining after exposure to mouse, rat or human microsomes for 30 min.

Data for compound 3 from Ref. [32]. See Supplementary Material for further details of assay conditions.

In vivo pharmacokinetics

The in vivo PK parameters were measured for seven compounds selected on the basis of the in vitro assessment described above. Plasma pharmacokinetics were determined in male CD-1 mice for compounds 3, 10, 11, 26, 50, 58 and 59 (Table 6). Blood samples were collected at 5–8 time-points after dosing the compounds at 10 mg/kg in a solution of 20% hydroxypropyl-β-cyclodextrin by intraperitoneal (IP) injection. For analysis of the samples, chromatographic conditions were optimised by HPLC for each compound of interest and an internal standard. A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was then developed and validated for quantitation of each analyte.

Table 6

In vivo Pharmacokinetics of selected compounds.a

Compound	T1/2(h)	Cmax(μmol/L)	AUC0-∞(μmol/L*h)
3	12.0	10	220
10	6.6	236	2885
11	9.5	149	2364
26	4.5	124	1019
50	3.3	87	642
58	2.5	105	415
59	2.2	64	383

Pharmacokinetic parameters derived from the plasma-concentration time profiles for each compound following a 10 mg/kg i.p. dose. The results were processed using a noncompartment model approach using Phoenix WinNonlin 6.2 (Pharsight Corporation, St. Louis, MO). The derived parameters are: maximum plasma concentration (Cmax), the area under the curve (AUC) and plasma half-life (T1/2). See Supplementary Material for further details of assay conditions.

Compound 3 possessed the best half-life (12 h) but showed much lower Cmax and AUC than other examples. While 10, 11 and 26 had shorter half-lives (4.5–9.5 h) they also showed the highest Cmax (236, 149 and 124 μmol/L) and exposures (AUC = 2885, 2364 and 1019 μmol/L*h respectively). 4-Cyanobenzene (50) and both nitrobenzene isomers (58, 59), had even shorter half-lives, however the maximum concentration and overall exposure reached was still superior to the compound with the overall longest half-life (3).

Conclusions

Between our previous report [32] and the current study, we have explored in detail how changes made on a benzenesulphonamide-based template affects perforin inhibitory activity. Analysis of the resulting SAR shows that although a range of variations were explored throughout the molecule, the isoindolinone, thiophene, pyridine and sulphonamide link of 3 are probably close to optimal, while there is some tolerance for substitution on the central pyridine ring and the terminal benzene ring. A smaller panel of compounds was selected based on the following criteria; in vitro potency against isolated recombinant perforin and whole human NK cells, lack of toxicity against NK cells, solubility and stability (aqueous and microsomal). For this group the in vivo PK parameters were assessed to select potential candidates for evaluation in a mouse model of transplant rejection. We sought acceptable half-life (potentially impacts dosing frequency), Cmax and AUC to ensure sufficient exposure to maximise our chances of achieving efficacy. Other key parameters taken into consideration for selection of the final in vivo candidates were in vitro potency and solubility. While the in vivo mouse efficacy studies are carried out using IP dosing, ultimately this product would be administered to human patients intravenously, meaning that sufficient solubility in a formulation close to physiological pH is crucial.

Given the above criteria, one example from the panel that appears to possess a suitable balance of properties is the 2-chloro pyridine 11. This compound shows excellent potency (without toxicity) in vitro, good solubility, stability across all three species of microsomes and acceptable PK properties. Compounds 58 and 3 are also worth consideration; 58 because it is the most potent compound of the set, and 3 based on a combination of potency, solubility and half-life. The next step will be to conduct studies to determine if these compounds are capable of preventing transplant rejection in a mouse model.

Experimental

Elemental analyses were performed by the Microchemical Laboratory, University of Otago, Dunedin, NZ; values are indicated by the symbols of the elements and were within ±0.4% of the theoretical values. Melting points were determined using an Electrothermal Model 9200 and are as read. Several compounds were examined as sharp-melting solvates, on which elemental analyses were determined. NMR spectra were measured on a Bruker Advance 400 MHz spectrometer and referenced to Me4Si. Mass spectra were recorded either on a Varian VG 7070 spectrometer at nominal 5000 resolution or a Finnigan MAT 900Q spectrometer. All final compound purities were determined to be >95% by HPLC on an Alltech Alltima C18 column (3.2 × 150 mm, 5 μm) eluting with 5–80% MeCN/45 mM NH4HCO3.

General procedure A: 5-(5-(6-Aminopyridin-3-yl)thiophen-2-yl)-2-methylisoindolin-1-one (76)

Iodide 75 [32] (500 mg, 1.41 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (465 mg, 2.11 mmol) were dissolved in a mixture of toluene (8 mL) and EtOH (4 mL). A solution of 2 M Na2CO3 (2 mL) and Pd(dppf)Cl2.CH2Cl2 (57 mg, 0.07 mmol) were added and the entire mixture heated at reflux under N2 for 2 h. Upon cooling, the desired product precipitated from the reaction mixture, was isolated by filtration, and washed with H2O, CH2Cl2 and MeOH. No further purification was required, giving 76 as a green-yellow solid (326 mg, 72%); mp (MeOH/CH2Cl2) 252–254 °C. 1H NMR [400 MHz, (CD3)2SO] δ 8.28 (dd, J = 2.6, 0.6 Hz, 1 H), 7.86 (s, 1 H), 7.76 (dd, J = 8.0, 1.5 Hz, 1 H), 7.70 (dd, J = 8.6, 2.6 Hz, 1 H), 7.67 (d, J = 7.9 Hz, 1 H), 7.62 (d, J = 3.8 Hz, 1 H), 6.35 (d, J = 3.8 Hz, 1 H), 6.51 (dd, J = 8.6, 0.6 Hz, 1 H), 6.25 (br s, 2 H) 4.49 (s, 2 H), 3.08 (s, 3 H). HRMS (ESI+) calcd for C18H16N3OS 322.1009 (MH+), found 322.1007.

General procedure B: 2,4-Difluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-2-yl)benzenesulfonamide (4)

To a solution of 76 (120 mg, 0.37 mmol) in dry pyridine (12 mL) under N2 at RT, was added 2,4-difluorobenzenesulphonyl chloride (159 mg, 0.74 mmol) in CH2Cl2 (1.5 mL) dropwise over 5 min. The mixture was stirred at 45 °C under N2 for 16 , and the solvent then removed under reduced pressure. The reaction was quenched with a little water and the resulting solid collected by filtration and washed with water and Et2O. Purification was carried out by trituration with hot CH2Cl2/MeOH solution to give 4 as a pale brown solid (65%); mp (CH2Cl2/MeOH) 269–272 °C. 1H NMR [400 MHz, (CD3)2SO] δ 12.00 (bs, 1 H), 8.39 (bs, 1 H), 7.99–8.10 (m, 2 H), 7.89 (s, 1 H), 7.78 (dd, J = 8.0, 1.3 Hz, 1 H), 7.69 (d, J = 4.2 Hz, 1 H), 7.68 (d, J = 8.1 Hz, 1 H), 7.57 (d, J = 3.9 Hz, 1 H), 7.44–7.53 (m, 1 H), 7.26–7.33 (m, 1 H), 7.16–7.26 (m, 1 H) 4.50 (s, 2 H), 3.07 (s, 3 H). HRMS (ESI+) calcd for C24H18N3O3S2F2 498.0752 (MH+), found 498.0746. Anal. C, H, N. In some cases a bis-sulphonamide was also formed; here a second step was introduced where the crude product was treated with a 1:1 mixture of 1,4-dioxane and 2 M NaOH. The mono-sulphonamide resulting from subsequent acidification of the reaction mixture was isolated by filtration, washed well with water, and dried. Purification was carried out by flash column chromatography (MeOH/CH2Cl2 gradient).

5-(5-(2-Aminopyridin-4-yl)thiophen-2-yl)-2-methylisoindolin-1-one (77)

Reaction of 75 with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine according to general procedure A gave 77 as a green-yellow solid (72%); mp (CH2Cl2/MeOH) 302–306 °C. 1H NMR [400 MHz, (CD3)2SO] δ 7.90–7.97 (m, 2 H), 7.82 (d, J = 7.1 Hz, 1 H), 7.67–7.73 (m, 2 H), 7.65 (d, J = 3.4 Hz, 1 H), 6.83 (d, J = 4.0 Hz, 1 H), 6.69 (s, 1 H), 6.05 (br s, 2 H) 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (ESI+) calcd for C18H16N3OS 322.1009 (MH+), found 322.1002.

2,4-Difluoro-N-(4-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-2-yl)benzenesulfonamide (5)

Amine 77 was reacted with 2,4-difluorobenzenesulfonyl chloride according to general procedure B to give 5 as a pale brown solid (26%); mp (CH2Cl2/MeOH) 235–239 °C. 1H NMR [400 MHz, (CD3)2SO] δ 13.27 (br s, 1 H), 7.99–8.09 (m, 2 H), 7.85–7.98 (m, 3 H), 7.80 (d, J = 3.8 Hz, 1 H), 7.72 (d, J = 7.9 Hz, 1 H), 7.38–7.51 (m, 2 H), 7.19–7.32 (m, 2 H) 4.53 (s, 2 H), 3.09 (s, 3 H). HRMS (ESI+) calcd for C24H18N3O3S2F2 498.0752 (MH+), found 498.0754. Anal. C, H, N.

N-(5-Bromopyridin-3-yl)-2,4-difluoro-N-methylbenzenesulphonamide (86)

2,4-Difluorobenzenesulfonyl chloride and 5-bromopyridin-3-amine were reacted according to general procedure B. Without further purification, the resulting crude sulphonamide 85 (540 mg, 15.5 mmol) was dissolved in dry DMF (20 mL) and cooled to 0 °C. NaH (41 mg, 17.0 mmol) was added and the mixture stirred for 0.5 , gradually being allowed to return to R.T. Methyl iodide (241 mg, 17.0 mmol) was then added dropwise and stirring continued for 1.5 h. After quenching with water the mixture was extracted with CH2Cl2, dried with MgSO4 and evaporated to give a solid which was purified by flash column chromatography (1–3% MeOH/CH2Cl2 as eluant), yielding 86 as a brown solid (320 mg, 57%). 1H NMR [400 MHz, (CD3)2SO] δ 8.65 (d, J = 2.0 Hz, 1 H), 8.51 (d, J = 2.2 Hz, 1 H), 8.04 (t, J = 2.2 Hz, 1 H), 7.72–7.83 (m, 1 H), 7.57–7.68 (m, 1 H), 7.32 (dt, J = 8.2, 2.4 Hz, 1 H), 3.26 (s, 3 H). LRMS (APCI+) calcd for C12H9BrF2N2O2S 364 (MH+), found 364.

General procedure C: 2,4-Difluoro-N-methyl-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-benzenesulphonamide (6)

Bromide 86 (300 mg, 0.83 mmol), bis(pinacolato)diboron (232 mg, 0.91 mmol), KOAc (243 mg, 2.48 mmol) and Pd(dppf)Cl2.CH2Cl2 (34 mg, 0.04 mmol) were weighed into a flask, DMSO (5 mL) added, and the entire mixture heated and stirred under N2 for 4 h. Upon cooling, the reaction was diluted with CH2Cl2 (25 mL) and filtered through a pad of Celite®, washing well with additional CH2Cl2. The filtrate and combined washings (ca 80 mL) were washed with water (3 × 40 mL), brine (50 mL), dried (Na2SO4) and filtered. Removal of the solvent under reduced pressure gave the crude boronate 90 which was coupled directly to 75 according to general procedure A, giving 6 as a cream solid (220 mg, 52%); mp (CH2Cl2/MeOH) 200–202 °C. 1H NMR [400 MHz, (CD3)2SO] δ 8.87 (d, J = 2.0 Hz, 1 H), 8.43 (d, J = 2.3 Hz, 1 H), 7.96 (t, J = 2.2 Hz, 1 H), 7.94 (s, 1 H), 7.83 (dd, J = 7.8, 1.4 Hz, 1 H), 7.73–7.81 (m, 3 H), 7.71 (d, J = 7.9 Hz, 1 H), 7.63 (m, 1 H), 7.32 (dt, J = 8.1, 2.0 Hz, 1 H), 4.52 (s, 2 H), 3.34 (s, 3 H), 3.09 (s, 3 H). Anal. C, H, N.

5-Bromo-N-(2,4-difluorophenyl)pyridine-3-sulphonamide (100)

2,4-Difluoroaniline and 5-bromopyridine-3-sulphonyl chloride were reacted according to general procedure B. The crude product was recrystallised from 5% MeOH/CH2Cl2 and hexanes, and triturated with EtOAc to give 100 as an ivory solid (370 mg, 56%). 1H NMR [400 MHz, (CD3)2SO] δ 10.52 (br s, 1 H), 9.20 (d, J = 2.2 Hz, 1 H), 8.77 (d, J = 2.0 Hz, 1 H), 8.26 (t, J = 2.1 Hz, 1 H), 7.22–7.35 (m, 2 H), 7.02–7.15 (m, 1 H). LRMS (APCI+) calcd for C11H7BrF2N2O2S 350 (MH+), found 350.

N-(2,4-Difluorophenyl)-5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridine-3-sulphonamide (7)

Bromide 100 was reacted with bis(pinacolato)diboron according to general procedure C and the crude boronate 101 subsequently coupled to 75 according to general procedure A, to give 7 as a yellow solid (23%); mp (CH2Cl2/MeOH) 221–224 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.50 (br s, 1 H), 9.23 (d, J = 2.2 Hz, 1 H), 8.71 (d, J = 2.1 Hz, 1 H), 8.21 (t, J = 2.2 Hz, 1 H), 7.96 (s, 1 H), 7.81–7.88 (m, 2 H), 7.78 (d, J = 3.9 Hz, 1 H), 7.72 (d, J = 8.0 Hz, 1 H), 7.23–7.35 (m, 2 H), 7.09 (ddt, J = 9.2, 1.4 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

2,4-Difluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzamide (8)

To 2,4-difluorobenzoic acid (99 mg, 0.62 mmol) in dry CH2Cl2 (2 mL) was added oxalyl chloride (193 mg, 1.52 mmol) and 1 drop of dry DMF. The whole mixture was refluxed for 2 , cooled to RT and concentrated in vacuo to give 2,4-difluorobenzoic acid chloride. To amine 78 [32] (100 mg, 0.31 mmol) in dry pyridine (10 mL) at 0 °C under N2 was added the acid chloride (110 mg, 0.62 mmol) in dry CH2Cl2 (2 mL) dropwise over 4 min. The mixture was then left to stir at 45 °C for 16 , quenched with H2O and concentrated in vacuo. The residue was taken up in citric acid, sonicated for 5 min, and the precipitate formed was filtered and washed thoroughly with H2O, MeOH, diethyl ether and dried on to silica gel. The crude material was chromatographed (1–3% MeOH/CH2Cl2) to give 8 as a yellow solid (52 mg, 36%); mp 267–269 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.76 (br s, 1 H), 8.77 (t, J = 2.9 Hz, 2 H), 8.52 (t, J = 2.0 Hz, 1 H), 7.96 (s, 1 H), 7.81–7.88 (m, 2 H), 7.75 (d, J = 3.9 Hz, 1 H), 7.71 (d, J = 3.8 Hz, 1 H), 7.70 (s, 1 H), 7.49 (dt, J = 9.4, 2.5 Hz, 1 H), 7.28 (dt, J = 8.6, 2.2 Hz, 1 H) 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI+) calcd for C25H17N3O2F2S 462.5 (MH+), found 462.8. Anal. C, H, N.

5-(5-(3-Aminophenyl)thiophen-2-yl)-2-methylisoindolin-1-one (79)

Iodide 75 was reacted with (3-aminophenyl)boronic acid according to general procedure A to give 79 as a green solid (69%); mp (CH2Cl2/MeOH) 244–247 °C. 1H NMR [400 MHz, (CD3)2SO] δ 7.89 (s, 1 H), 7.77 (dd, J = 7.9, 1.3 Hz, 1 H), 7.68 (d, J = 8.0 Hz, 1 H), 7.63 (d, J = 3.8 Hz, 1 H), 7.41 (d, J = 3.8 Hz, 1 H), 7.08 (t, J = 7.72 Hz, 1 H), 6.82–6.91 (m, 2 H), 6.55 (dd, J = 7.9, 1.3 Hz, 1 H), 5.25 (br s, 2 H), 4.50 (s, 2 H), 3.08 (s, 3 H).

2,4-Difluoro-N-(3-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)phenyl)benzene sulphonamide (9)

Amine 79 was reacted with 2,4-difluorobenzenesulphonyl chloride according to general procedure B to give 9 as a yellow solid (62%); mp (CH2Cl2/MeOH) 260–262 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.84 (br s, 1 H), 7.93–8.02 (m, 1 H), 7.91 (s, 1 H), 7.81 (dd, J = 8.0, 1.3 Hz, 1 H), 7.70 (d, J = 8.0 Hz, 1 H), 7.67 (d, J = 3.8 Hz, 1 H), 7.55 (dt, J = 8.5, 2.5 Hz, 1 H), 7.47 (d, J = 3.8 Hz, 1 H), 7.38–7.45 (m, 2 H), 7.25–7.36 (m, 2 H), 7.05 (d, J = 7.4 Hz, 1 H), 4.51 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

N-(5-Bromo-2-fluoropyridin-3-yl)-2,4-difluorobenzenesulphonamide (82)

5-Bromo-2-fluoropyridin-3-amine was reacted with 2,4-difluorobenzenesulphonyl chloride according to general procedure B to give 82 as a brown solid (17%). 1H NMR [400 MHz, (CD3)2SO] δ 11.13 (br s, 1 H), 8.18 (s, 1 H), 8.01 (dd, J = 8.6, 2.3 Hz, 1 H), 7.81–7.92 (m, 1 H), 7.53–7.64 (m, 1 H), 7.22–7.32 (m, 1 H). LRMS (APCI+) calcd for C11H6BrF3N2O2S 368 (MH+), found 368.

2,4-Difluoro-N-(2-fluoro-5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulphonamide (10)

Bromide 82 was reacted with bis(pinacolato)diboron according to general procedure C and the crude boronate 87 subsequently coupled to 75 according to general procedure A to give 10 as a pale green solid (32%); mp (MeOH/CH2Cl2) 237–239 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.04 (br s, 1 H), 8.42 (s, 1 H), 8.06 (dd, J = 9.1, 2.3 Hz, 1 H), 7.94 (s, 1 H), 7.84–7.92 (m, 1 H), 7.82 (dd, J = 7.9, 1.5 Hz, 1 H), 7.74 (d, J = 3.9 Hz, 1 H), 7.72 (d, J = 8.0 Hz, 1 H), 7.68 (d, J = 3.9 Hz, 1 H), 7.57–7.65 (m, 1 H), 7.28 (dt, J = 8.9, 2.4 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). In this case the product was dissolved in 1,4-dioxane and the sodium salt precipitated by slow addition of 2 M NaOH to give a light-green solid (89%). 1H NMR [400 MHz, (CD3)2SO] δ 7.91 (s, 1 H), 7.83–7.90 (m, 1 H), 7.79 (dd, J = 7.9, 1.5 Hz, 1 H), 7.71 (d, J = 2.3 Hz, 1 H), 7.67 (d, J = 7.3 Hz, 1 H), 7.60–7.65 (m, 2 H), 7.33 (d, J = 3.8 Hz, 1 H), 7.22 (dt, J = 9.7, 2.5 Hz, 1 H), 7.11 (dt, J = 8.3, 2.5 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.

N-(5-Bromo-2-chloropyridin-3-yl)-2,4-difluorobenzenesulphonamide (83)

5-Bromo-2-chloropyridin-3-amine was reacted with 2,4-difluorobenzenesulphonyl chloride according to general procedure B, giving 83 as an off-white solid (32%). 1H NMR [400 MHz, (CD3)2SO] δ 11.03 (br s, 1 H), 8.47 (d, J = 2.3 Hz, 1 H), 8.04 (d, J = 2.3 Hz, 1 H), 7.75–7.85 (m, 1 H), 7.53–7.63 (m, 1 H), 7.20–7.29 (m, 1 H). LRMS (APCI+) calcd for C11H6BrF2ClN2O2S 385 (MH+), found 385.

2,4-Difluoro-N-(2-chloro-5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulphonamide (11)

Bromide 83 was reacted with bis(pinacolato)diboron according to general procedure C and the crude boronate 88 subsequently coupled to 75 according to general procedure A to give 11 as a yellow solid (12%); mp (CH2Cl2/MeOH) 238–240 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.92 (br s, 1 H), 8.68 (d, J = 1.8 Hz, 1 H), 8.02 (d, J = 2.4 Hz, 1 H), 7.96 (s, 1 H), 7.84 (dd, J = 8.2, 1.8 Hz, 1 H), 7.74–7.85 (m, 3 H), 7.71 (d, J = 8.0 Hz, 1 H), 7.55–7.65 (m, 1 H), 7.26 (dt, J = 8.5, 2.0 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

N-(5-Bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulphonamide (84)

5-Bromo-2-methoxypyridin-3-amine was reacted with 2,4-difluorobenzenesulphonyl chloride according to general procedure B, giving 84 as an ivory solid (45%). 1H NMR [400 MHz, (CD3)2SO] δ 10.44 (br s, 1 H), 8.12 (d, J = 2.3 Hz, 1 H), 7.72–7.81 (m, 1 H), 7.75 (d, J = 2.3 Hz, 1 H), 7.52–7.61 (m, 1 H), 7.18–7.27 (m, 1 H), 3.61 (s, 3 H). LRMS (APCI+) calcd for C12H9BrF2N2O3S 380 (MH+), found 380.

2,4-Difluoro-N-(2-methoxy-5-(5-(2-methyl-1-oxoisoindolin-5-yl)-thiophen-2-yl)pyridin-3-yl)benzenesulphonamide (12)

Bromide 84 was reacted with bis(pinacolato)diboron and the crude boronate 89 subsequently coupled to 75 according to general procedure A, to give 12 as a yellow powder (42%); mp (MeOH/CH2Cl2) 224–227 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.35 (br s, 1 H), 8.35 (s, 1 H), 7.92 (s, 1 H), 7.85 (d, J = 2.2 Hz, 1 H), 7.82 (dd, J = 8.0, 1.5 Hz, 1 H), 7.74–7.83 (m, 1 H), 7.70 (d, J = 7.3 Hz, 1 H), 7.69 (d, J = 4.0 Hz, 1 H), 7.51–7.61 (m, 1 H), 7.54 (d, J = 3.7 Hz, 1 H), 7.22 (dt, J = 8.3, 2.3 Hz, 1 H), 4.51 (s, 2 H), 3.66 (s, 3 H), 3.09 (s, 3 H). Anal. C, H, N.

2-Methyl-5-(5-(2-methyl-5-nitrophenyl)thiophen-2-yl)isoindolin-1-one (80)

Iodide 75 was reacted with (2-methyl-5-nitrophenyl)boronic acid according to general procedure A, followed by flash column chromatography (CH2Cl2/MeOH 98:2 as eluant) to give 80 as a yellow solid (80%). 1H NMR [400 MHz, CDCl3] δ 8.32 (d, J = 2.5 Hz, 1 H), 8.11 (dd, J = 8.4, 2.5 Hz, 1 H), 7.87 (d, J = 7.9 Hz, 1 H), 7.74 (dd, J = 7.9, 1.5 Hz, 1 H), 7.69 (dd, J = 1.4, 0.7 Hz, 1 H), 7.46 (d, J = 8.4 Hz, 1 H), 7.42 (d, J = 3.8 Hz, 1 H), 7.16 (d, J = 3.8 Hz, 1 H), 4.43 (s, 2 H), 3.23 (s, 3 H), 2.59 (s, 3 H). LRMS (APCI+) calcd for C20H17N2O3S 365 (MH+), found 365.

2,4-Difluoro-N-(4-methyl-3-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)phenyl)benzenesulfonamide (13)

A 200 mL Parr hydrogenation vessel was charged with 80 (388 mg, 1.06 mmol) which was dissolved in a 1:1:1 mixture of ethanol, EtOAc and THF (90 mL). The mixture was agitated under 60 psi hydrogen at RT for 3 , before being filtered through Celite®. The solvents were evaporated to give the crude aniline 81 (350 mg) of which a subsample (111 mg, 0.33 mmol) was reacted with 2,4-difluorobenzenesulfonyl chloride according to general procedure B, giving 13 as a cream solid (51 mg, 30%); mp (MeOH/CH2Cl2) 268–271 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.68 (s, 1 H), 7.88–7.95 (m, 2 H), 7.80 (dd, J = 8.0, 1.5 Hz, 1 H), 7.70 (d, J = 8.0 Hz, 1 H), 7.67 (d, J = 3.8 Hz, 1 H), 7.56 (ddd, J = 11.4, 9.2, 2.4 Hz, 1 H), 7.29 (dt, J = 8.5, 8.4, 2.1 Hz, 1 H), 7.18–7.24 (m, 2 H), 7.03 (dd, J = 8.2, 2.4 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3H), 2.33 (s, 3 H). Anal. C, H, N.

General procedure D: 3-Bromo-N-(ethoxymethyl)-N-(pyridin-2-yl)benzenesulfonamide (105)

Reaction of pyridine-2-amine and 3-bromobenzenesulfonyl chloride was carried out according to general procedure B. Without any further purification, to a solution of the crude sulphonamide 103 (500 mg, 1.60 mmol) and (chloromethoxy)ethane (166 mg, 1.76 mmol) in DMF at RT was added NaH (42 mg, 1.76 mmol), then the reaction stirred for 1 h. After quenching with water the mixture was extracted with CH2Cl2, dried with MgSO4 and evaporated to give a solid which was purified by flash chromatography on silica gel (3:1 hexanes/EtOAc) giving 105 as a colourless oil (373 mg, 63%). 1H NMR [400 MHz, CDCl3] δ 8.35 (ddd, J = 4.8, 1.9, 0.8 Hz, 1 H), 7.98 (dd, J = 1.8, 1.8 Hz, 1 H), 7.69–7.74 (m, 3 H), 7.66 (ddd, J = 8.1, 1.9, 1.0 Hz, 1 H), 7.48 (ddd, J = 8.2, 0.8, 0.8 Hz, 1 H), 7.32 (dd, J = 8.0, 8.0 Hz, 1 H), 7.16 (ddd, J = 7.4, 4.9, 1.0 Hz, 1 H), 5.38 (s, 2 H), 3.68 (q, J = 7.1 Hz, 2 H), 1.19 (t, J = 7.1 Hz, 3 H). LRMS (APCI+) calcd for C12H10BrFN2O2S 325 (M-EtO)+, found 325.

General procedure E: sodium ((3-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)phenyl)sulfonyl)(pyridin-2-yl)amide (14)

Bromide 105 was reacted with bis(pinacolato)diboron and the crude boronate 108 subsequently coupled to 75 according to general procedure A. After extraction of the reaction mixture with EtOAc and evaporation, the protected crude intermediate was subjected to a one-pot deprotection and conversion to the sodium salt as follows; the solid was taken up in a 1:1 solution of 3 M HCl and 1,4-dioxane and then heated to reflux for 1 h. Upon cooling the white precipitate, consisting of essentially pure sulfonamide, was filtered and taken up in EtOH. Precipitation of the sodium salt was accomplished by slow addition of 2 M NaOH to give 14 as a yellow solid (68%, over 3 steps); mp (MeOH/CH2Cl2) 302–306 °C. 1H NMR [400 MHz, (CD3)2SO] δ 8.09 (dd, J = 1.6, 1.6 Hz, 1 H), 7.94 (br s, 1 H), 7.87 (ddd, J = 4.9, 2.1, 0.7 Hz, 1 H), 7.83 (dd, J = 8.0, 1.5 Hz, 1 H), 7.68–7.72 (m, 4 H), 7.56 (d, J = 3.9 Hz, 1 H), 7.41 (dd, J = 7.8, 7.8 Hz, 1 H), 7.19 (ddd, J = 8.5, 7.0, 2.2 Hz, 1 H), 6.59 (ddd, J = 8.6, 0.9, 0.9 Hz, 1 H), 6.36 (ddd, J = 7.0, 5.0, 1.0 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.

N-(4-Methyl-3-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)phenyl)pyridine-2-sulfonamide (15)

Amine 81 was reacted with pyridine-2-sulfonyl chloride according to general procedure B to give 15 as a cream solid (66 mg, 42%); mp (MeOH/CH2Cl2) 274–277 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.58 (s, 1 H), 8.74 (ddd, J = 4.65, 1.6, 0.8 Hz, 1 H), 8.08 (dt, J = 7.7, 7.7, 1.7 Hz, 1 H), 7.98 (td, J = 7.9, 1.0, 1.0 Hz, 1 H), 7.90 (s, 1 H), 7.80 (dd, J = 7.9, 1.6 Hz, 1 H), 7.70 (d, J = 8.0 Hz, 1 H), 7.64–7.68 (m, 2 H), 7.26 (d, J = 2.3 Hz, 1 H), 7.16–7.20 (m, 2 H), 7.06 (dd, J = 8.2, 2.3 Hz, 1 H), 4.51 (s, 2 H), 3.09 (s, 3 H), 2.32 (s, 3 H). Anal. C, H, N.

Sodium (4-methyl-3-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)phenylsulfonyl)(pyridin-2-yl)amide (16)

3-Bromo-4-methylbenzenesulfonyl chloride and 2-aminopyridine were reacted according to general procedure B to give 102. This was then reacted with bis(pinacolato)diboron according to general procedure C and the crude boronate 107 was coupled to 75 according to general procedure A to give 16. Conversion to the sodium salt using general procedure E afforded a yellow solid (55%, 4 steps). 1H NMR [400 MHz, (CD3)2SO] δ 7.93 (br s, 1 H), 7.87–7.88 (m, 2 H), 7.83 (dd, J = 8.0, 1.4 Hz, 1 H), 7.68–7.69 (m, 2 H), 7.64 (dd, J = 7.9, 1.8 Hz, 1 H), 7.30 (d, J = 8.0 Hz, 1 H), 7.25 (d, J = 3.8 Hz, 1 H), 7.18 (ddd, J = 8.6, 7.0, 2.2 Hz, 1 H), 6.58 (ddd, J = 8.6, 1.0, 1.0 Hz, 1 H), 6.35 (ddd, J = 7.0, 5.0, 1.0 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H), 2.44 (s, 3 H). Anal. C, H, N.

N-{4-Fluoro-3-[5-(2-methyl-1-oxo-2,3-dihydro-1H-isoindol-5-yl)-2-thienyl]phenyl}-2-pyridinesulfonamide (17)

2-Pyridinesulfonyl chloride and 3-bromo-4-fluoroaniline were reacted according to general procedure B to give 91. Protection of the sulfonamide nitrogen was then carried out according to general procedure D, giving 94 which was purified by column chromatography eluting with hexanes/EtOAc 3:1. The protected sulphonamide was then reacted directly with bis(pinacolato)diboron according to general procedure C and the crude boronate 97 coupled to 75 according to general procedure A. Deprotection according to general procedure E gave 17 (11%, 5 steps) 1H NMR [400 MHz, (CD3)2SO] δ 8.13 (br s, 1 H), 8.01 (br s, 1 H), 7.88–7.97 (m, 3 H), 7.83 (dd, J = 8.0, 1.6 Hz, 1 H), 7.79–7.81 (m, 1 H), 7.75–7.77 (m, 1 H), 7.68–7.73 (m, 3 H), 7.61 (t, J = 7.8 Hz, 1 H), 7.24 (d, J = 8.4 Hz, 1 H), 6.87 (t, J = 6.9 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.

N-(5-Bromo-2-(trifluoromethoxy)phenyl)-N-(ethoxymethyl)pyridine-2-sulfonamide (95)

Pyridine-2-sulfonyl chloride and 5-bromo-2-(trifluoromethoxy)aniline were reacted according to general procedure B to give 92. Protection of the sulfonamide according to general procedure D, gave 95 which was purified by column chromatography eluting with hexanes/EtOAc 3:1 and isolated as a colourless oil (89%, 2 steps).1H NMR [400 MHz, CDCl3] δ 8.75 (ddd, J = 4.8, 1.6, 1.0 Hz, 1H), 7.82–7.90 (m, 2 H), 7.46–7.54 (m, 3 H), 7.10 (dddd, J = 8.9, 1.9, 1.8, 1.8 Hz, 1 H), 5.21 (br s, 2 H), 3.79 (q, J = 7.0 Hz, 2 H), 1.20 (t, J = 7.0 Hz, 3 H). LRMS (APCI+) calcd for C13H10BrF3N2O3S 409 (M-EtO)+, found 409.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)-2-(trifluoromethoxy)phenyl)pyridine-2-sulfonamide (18)

Bromide 95 was reacted with bis(pinacolato)diboron, to give the crude boronate 98 which was subsequently coupled to 75 according to general procedure A. The intermediate from this step was then deprotected according to general procedure E giving 18 as a yellow solid (51%, 3 steps); mp (MeOH/CH2Cl2) 200–201 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.61 (s, 1 H), 8.79 (ddd, J = 4.6, 1.6, 0.8 Hz, 1 H), 8.11 (ddd, J = 7.7, 7.7, 1.7 Hz, 1 H), 7.99 (ddd, J = 7.9, 0.9, 0.9 Hz, 1 H), 7.93–7.95 (m, 1 H), 7.82 (dd, J = 8.0, 1.5 Hz, 1 H), 7.79 (d, J = 2.3 Hz, 1 H), 7.68–7.74 (m, 3 H), 7.61 (dd, J = 8.6, 2.3 Hz, 1 H), 7.53 (d, J = 3.4 Hz, 1 H), 7.39 (dddd, J = 8.5, 1.5, 1.5, 1.5 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

5-Bromo-N-(ethoxymethyl)-2-methoxy-N-(pyridin-2-yl)benzenesulfonamide (106)

Pyridine-2-amine and 5-bromo-2-methoxybenzenesulfonyl chloride were reacted according to general procedure B. Protection of the sulfonamide 104 was then carried out according to general procedure D, giving 106 as a colourless oil (99%, 2 steps). 1H NMR [400 MHz, CDCl3] δ 8.76 (ddd, J = 4.7, 1.6, 0.8 Hz, 1 H), 7.82 (dd, J = 7.7, 1.7 Hz, 1 H), 7.76 (ddd, J = 7.9, 1.1, 1.1 Hz, 1 H), 7.48 (ddd, J = 7.6, 4.8, 1.3 Hz, 1 H), 7.37–7.40 (m, 2 H), 6.64–6.68 (m, 1 H), 5.19 (br s, 2 H), 3.80 (q, J = 7.0 Hz, 2 H), 3.37 (s, 3 H), 1.21 (t, J = 7.0 Hz, 3 H). LRMS (APCI+) calcd for C13H13BrN2O3S 355 (M-EtO)+, found 355.

Sodium ((2-methoxy-5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)phenyl)sulfonyl)(pyridin-2-yl)amide (19)

Bromide 106 was reacted with bis(pinacolato)diboron according to general procedure C, then the crude boronate 109 was subsequently coupled to 75 according to general procedure A. The intermediate from this step was deprotected and converted to the sodium salt according to general procedure E, furnishing 19 as a yellow solid (68%, 3 steps); mp (MeOH/CH2Cl2) 322–325 °C. 1H NMR [400 MHz, (CD3)2SO] δ 8.10 (br d, J = 2.5 Hz, 1 H), 7.89 (br s, 1 H), 7.86 (ddd, J = 4.9, 2.1, 0.8 Hz, 1 H), 7.79 (dd, J = 8.0, 1.5 Hz, 1 H), 7.63–7.68 (m, 3 H), 7.36 (d, J = 3.8 Hz, 1 H), 7.20 (ddd, J = 8.4, 7.0, 2.1 Hz, 1 H), 7.04 (d, J = 8.7 Hz, 1 H), 6.71 (br d, J = 8.4 Hz, 1 H), 6.36 (ddd, J = 6.9, 4.8, 1.0 Hz, 1 H), 4.50 (s, 2 H), 3.74 (s, 3 H), 3.08 (s, 3 H). Anal. C, H, N.

N-(5-Bromo-2-fluoropyridin-3-yl)-N-(ethoxymethyl)pyridine-2-sulfonamide (96)

Pyridine-2-sulfonyl chloride and 5-bromo-2-fluoropyridin-3-amine were reacted according to general procedure B. Intermediate 93 was then protected according to general procedure D to give 96 as a colourless oil (32%, 2 steps). 1H NMR [400 MHz, (CD3)2SO] δ 8.75 (ddd, J = 4.7, 1.6, 0.9 Hz, 1 H), 8.22 (dd, J = 2.4, 1.5 Hz, 1 H), 8.01 (dd, J = 8.1, 2.4 Hz, 1 H), 7.84–7.92 (m, 2 H), 7.54 (ddd, J = 7.4, 4.7, 1.4 Hz, 1 H), 5.21 (s, 2 H), 3.76 (q, J = 7.1 Hz, 2 H), 1.20 (t, J = 7.0 Hz, 3 H). LRMS (APCI+) calcd for C11H8BrFN3O2S 346 (M-EtO)+, found 346.

N-(2-Fluoro-5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)pyridine-2-sulfonamide (20)

Bromide 96 was reacted with bis(pinacolato)diboron to afford crude boronate 99 which was subsequently coupled to 75 according to general procedure A. The intermediate from this step was then deprotected according to general procedure E to give 20 as a white solid (11%, 3 steps); mp (CH2Cl2/MeOH) 279–382 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.9 (br s, 1 H), 8.76 (ddd, J = 4.7, 1.6, 0.9 Hz, 1 H), 8.39 (dd, J = 2.0, 1.3 Hz, 1 H), 8.21 (dd, J = 9.2, 2.4 Hz, 1 H), 8.13 (dd, J = 9.2, 2.4 Hz, 1 H), 8.02 (ddd, J = 7.9, 1.0, 1.0 Hz, 1 H), 7.95 (br s, 1 H), 7.84 (dd, J = 7.9, 1.6 Hz, 1 H), 7.70–7.74 (m, 3 H), 7.65 (J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulphonamide (21)

Amine 78 was reacted with benzenesulphonyl chloride according to general procedure B to give 21 as a yellow solid (64%); mp 300–303 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.77 (br s, 1 H), 8.67 (d, J = 2.0 Hz, 1 H), 8.21 (d, J = 2.4 Hz, 1 H), 7.94 (s, 1 H), 7.75–7.80 (m, 3 H), 7.68–7.76 (m, 3 H), 7.55–7.67 (m, 4 H), 4.52 (s, 2 H), 3.09 (s, 3 H). In this case the product was converted to its sodium salt to give the desired product 21 as a yellow solid (94%). 1H NMR [400 MHz, (CD3)2SO] δ 8.00 (d, J = 2.1 Hz, 1 H), 7.90 (s, 1 H), 7.88 (d, J = 2.4, 1 H), 7.80 (dd, J = 7.9,1.5 Hz, 1 H), 7.71–7.77 (m, J = 8.0, 2 H), 7.68 (d, J = 8.0, 1 H), 7.63 (d, J = 3.8, 1 H), 7.10–7.30 (m, 5 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.

2-Fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (22)

Amine 78 was reacted with 2-fluorobenzenesulphonyl chloride according to general procedure B to give 22 as a beige solid (82%); mp (CH2Cl2/MeOH) 289–292 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.11 (br s, 1 H), 8.67 (d, J = 2.0 Hz, 1 H), 8.25 (d, J = 2.4 Hz, 1 H), 7.95 (dt, J = 7.4, 1.7 Hz, 2 H), 7.83 (dd, J = 8.0, 1.5 Hz, 1 H), 7.68–7.76 (m, 4 H), 7.63 (d, J = 3.8 Hz, 1 H), 7.38–7.49 (m, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H18N3O3FS2 479 (M − H), found 479. Anal. C, H, N.

3-Fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (23)

Amine 78 was reacted with 3-fluorobenzenesulphonyl chloride according to general procedure B to give 23 as a beige solid (56%); mp (CH2Cl2/MeOH) 292–294 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.88 (br s, 1 H), 8.70 (d, J = 2.0 Hz, 1 H), 8.22 (d, J = 2.3 Hz, 1 H), 7.94 (s, 1 H), 7.83 (dd, J = 7.9, 1.5 Hz, 1 H), 7.69–7.75 (m, 3 H), 7.61–7.68 (m, 4 H), 7.50–7.58 (m, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

4-Fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (24)

Amine 78 was reacted with 4-fluorobenzenesulphonyl chloride according to general procedure B to give 24 as a yellow solid (86%); mp (CH2Cl2/MeOH) 272–274 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.79 (br s, 1 H), 8.69 (d, J = 2.0 Hz, 1 H), 8.21 (d, J = 2.3 Hz, 1 H), 7.94 (s, 1 H), 7.85–7.92 (m, 2 H), 7.83 (dd, J = 7.9, 1.5 Hz, 1 H), 7.68–7.75 (m, 3 H), 7.66 (d, J = 3.9 Hz, 1 H), 7.40–7.48 (m, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H). In this case the product was converted to its sodium salt to give the desired product as a yellow solid (90%). 1H NMR [400 MHz, (CD3)2SO] δ 8.03 (d, J = 2.1 Hz, 1 H), 7.90 (s, 1 H), 7.88 (d, J = 2.5 Hz, 1 H), 7.73–7.84 (m, 3 H), 7.68 (d, J = 7.9 Hz, 1 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.37–7.42 (m, 2 H), 7.15–7.23 (m, 2 H), 4.51 (s, 2 H) 3.08 (s, 3 H). Anal. C, H, N.

3,4-Difluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (25)

Amine 78 was reacted with 3,4-difluorobenzenesulphonyl chloride according to general procedure B to give 25 as a yellow solid (18%); mp (CH2Cl2/MeOH) 282–285 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.88 (br s, 1 H), 8.72 (d, J = 1.4 Hz, 1 H), 8.23 (d, J = 2.3 Hz, 1 H), 7.95 (s, 1 H), 7.90 (d, J = 8.3 Hz, 1 H), 7.83 (d, J = 8.0 Hz, 1 H), 7.66–7.76 (m, 6 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

2,4,6-Trifluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (26)

Amine 78 was reacted with 2,4,6-trifluorobenzenesulphonyl chloride according to general procedure B to give 26 as a beige solid (16%); mp (CH2Cl2/MeOH) 272–275 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.43 (br s, 1 H), 8.28 (d, J = 2.3 Hz, 1 H), 7.95 (s, 1 H), 7.83 (dd, J = 7.9, 1.5 Hz, 1 H), 7.79 (t, J = 2.2 Hz, 1 H), 7.73 (d, J = 3.9 Hz, 1 H), 7.71 (d, J = 7.9 Hz, 1 H), 7.65 (d, J = 3.9 Hz, 1 H), 7.47 (br t, J = 9.4 Hz, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

2-Chloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (27)

Amine 78 was reacted with 2-chlorobenzenesulfonyl chloride according to general procedure B to give 27 as a yellow solid (29%); mp (MeOH/CH2Cl2) 299–303 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.14 (br s, 1 H), 8.64 (d, J = 1.9 Hz, 1 H), 8.26 (d, J = 2.3 Hz, 1 H), 8.17 (dd, J = 7.3, 1.2 Hz, 1 H), 7.94 (s, 1 H), 7.83 (dd, J = 7.9, 1.5 Hz, 1 H), 7.55–7.73 (m, 7 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H17ClN3O3S2 495 (M − H), found 495. Anal. C, H, N.

3-Chloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (28)

Amine 78 was reacted with 3-chlorobenzenesulfonyl chloride according to general procedure B to give 28 as an orange solid (49%); mp (MeOH/CH2Cl2) 291–295 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.88 (br s, 1 H), 8.71 (d, J = 2.0 Hz, 1 H), 8.22 (d, J = 2.4 Hz, 1 H), 7.95 (d, J = 0.7 Hz, 1 H), 7.81–7.86 (m, 2 H), 7.69–7.79 (m, 5 H), 7.67 (d, J = 3.9 Hz, 1 H), 7.63 (t, J = 7.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H17ClN3O3S2 495 (M − H), found 495. Anal. C, H, N.

4-Chloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (29)

Amine 78 was reacted with 4-chlorobenzenesulfonyl chloride according to general procedure B to give 29 as a yellow solid (59%); mp (MeOH/CH2Cl2) 281–284 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.84 (br s, 1 H), 8.70 (d, J = 2.0 Hz, 1 H), 8.21 (d, J = 2.3 Hz, 1 H), 7.94 (s, 1 H), 7.79–7.85 (m, 3 H), 7.65–7.74 (m, 6 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H17ClN3O3S2 495 (M − H), found 495. Anal. C, H, N. In this case the product was converted to its sodium salt to give the desired product as a yellow solid (77%), mp (EtOH) 240–244 °C. 1H NMR [400 MHz, (CD3)2SO] δ 8.04 (d, J = 2.1 Hz, 1 H), 7.91 (d, J = 0.8 Hz, 1 H), 7.89 (d, J = 2.4 Hz, 1 H), 7.80 (dd, J = 7.9, 1.5 Hz, 1 H), 7.73 (d, J = 8.6 Hz, 2 H), 7.68 (d, J = 7.8 Hz, 1 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.43 (d, J = 8.6 Hz, 2 H), 7.41 (d, J = 3.8 Hz, 1 H), 7.39 (t, J = 2.3 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.

3,4-Dichloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (30)

Amine 78 was reacted with 3,4-dichlorobenzenesulfonyl chloride according to general procedure B to give 30 as an orange-brown solid (15%); mp (MeOH/CH2Cl2) 256–259 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.92 (br s, 1 H), 8.72 (d, J = 2.0 Hz, 1 H), 8.23 (d, J = 2.4 Hz, 1 H), 8.03 (d, J = 2.2 Hz, 1 H), 7.94 (s, 1 H), 7.89 (d, J = 8.5 Hz, 1 H), 7.83 (dd, J = 8.0, 1.6 Hz, 1 H), 7.70–7.77 (m, 4 H), 7.69 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (ESI−) calcd for C24H16Cl2N3O3S2 528.0016 (M − H), found 528.0048.

2,4-Dichloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (31)

Amine 78 was reacted with 2,4-dichlorobenzenesulphonyl chloride according to general procedure B to give 31 as a yellow solid (55%); mp (CH2Cl2/MeOH) 282–285 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.20 (br s, 1 H), 8.67 (d, J = 2.0 Hz, 1 H), 8.26 (d, J = 2.4 Hz, 1 H), 8.14 (d, J = 8.6 Hz, 1 H), 7.96 (s, 1 H), 7.91 (d, J = 2.0 Hz, 1 H), 7.83 (dd, J = 8.0, 1.5 Hz, 1 H), 7.65–7.74 (m, 4 H), 7.64 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). In this case the product was converted to its sodium salt to give the desired product as a beige solid (89%). 1H NMR [400 MHz, (CD3)2SO] δ 8.08 (d, J = 2.1 Hz, 1 H), 8.01 (d, J = 8.4 Hz, 1 H), 7.88–7.92 (m, 2 H), 7.80 (dd, J = 7.9, 1.5 Hz, 1 H), 7.68 (d, J = 8.0, 1 H), 7.64 (d, J = 3.8, 1 H), 7.54 (d, J = 2.1, 1 H), 7.46 (dd, J = 8.4, 2.2 Hz, 1 H), 7.41 (d, J = 3.8 Hz, 1 H), 7.37 (t, J = 2.3 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.

2-Bromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (32)

Amine 78 was reacted with 2-bromobenzenesulfonyl chloride according to general procedure B to give 32 as an orange solid (30%); mp (MeOH/CH2Cl2) 289–293 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.15 (br s, 1 H), 8.63 (s, 1 H), 8.26 (d, J = 2.3 Hz, 1 H), 8.20 (dd, J = 7.9, 1.7 Hz, 1 H), 7.94 (s, 1 H), 7.80–7.88 (m, 2 H), 7.68–7.74 (m, 2 H), 7.67 (t, J = 2.2 Hz, 1 H), 7.59–7.65 (m, 2 H), 7.54 (dt, J = 7.6, 1.7 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H17BrN3O3S2 539 (M − H), found 539. Anal. C, H, N.

3-Bromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (33)

Amine 78 was reacted with 3-bromobenzenesulfonyl chloride according to general procedure B to give 33 as a yellow-orange solid (30%); mp (MeOH/CH2Cl2) 303–307 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.87 (br s, 1 H), 8.71 (d, J = 1.9 Hz, 1 H), 8.21 (d, J = 2.3 Hz, 1 H), 7.98 (t, J = 1.8 Hz, 1 H), 7.95 (d, J = 0.7 Hz, 1 H), 7.78–7.90 (m, 3 H), 7.69–7.75 (m, 3 H), 7.67 (d, J = 3.9 Hz, 1 H), 7.56 (t, J = 8.0 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (ESI+) calcd for C24H18BrN3O3S2Na 561.9865 (M + Na+), found 561.9862. Anal. C, H, N.

4-Bromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (34)

Amine 78 was reacted with 4-bromobenzenesulfonyl chloride according to general procedure B to give 34 as a yellow solid (60%); mp (MeOH/CH2Cl2) 276–279 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.84 (br s, 1 H), 8.69 (d, J = 2.0 Hz, 1 H), 8.21 (d, J = 2.4 Hz, 1 H), 7.94 (d, J = 0.8 Hz, 1 H), 7.80–7.86 (m, 3 H), 7.69–7.76 (m, 5 H), 7.66 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H17BrN3O3S2 539 (M − H), found 539. Anal. C, H, N.

2,4-Dibromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamides (35)

Amine 78 was reacted with 2,4-dibromobenzenesulfonyl chloride according to general procedure B to give 35 as a cream solid (81%); mp (MeOH/CH2Cl2) 276–279 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.23 (br s, 1 H), 8.66 (d, J = 1.9 Hz, 1 H), 8.25 (d, J = 2.4 Hz, 1 H), 8.16 (d, J = 1.9 Hz, 1 H), 8.08 (d, J = 8.5 Hz, 1 H), 7.94 (d, J = 0.7 Hz, 1 H), 7.81–7.87 (m, 2 H), 7.67–7.74 (m, 2 H), 7.65 (t, J = 2.2 Hz, 1 H), 7.63 (d, J = 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H17Br2N3O3S2 617, 619, 621 (M), found 617, 619, 621. Anal. C, H, N.

4-Iodo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamides (36)

Amine 78 was reacted with 4-iodobenzenesulfonyl chloride according to general procedure B to give 36 as a pale yellow solid (59%); mp (CH2Cl2/Et2O) 295–298 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.80 (br s, 1 H), 8.70 (d, J = 2.0 Hz, 1 H), 8.20 (d, J = 2.4 Hz, 1 H), 7.99 (d, J = 8.6 Hz, 2 H), 7.95 (d, J = 0.70 Hz, 1 H), 7.84 (dd, J = 8.0, 1.5 Hz, 1 H), 7.70–7.74 (m, 2 H), 7.68 (t, J = 2.2 Hz, 1 H), 7.66 (d, J = 3.9 Hz, 1 H), 7.56 (d, J = 8.6 Hz, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H18IN3O3S2 587 (M), found 587. Anal. C, H, N.

2-Methoxy-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (37)

Amine 78 reacted with 2-methoxybenzenesulfonyl chloride according to general procedure B to give 37 as a yellow solid (26%); mp (MeOH/CH2Cl2) 273–276 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.49 (br s, 1 H), 8.61 (d, J = 2.0 Hz, 1 H), 8.24 (d, J = 2.4 Hz, 1 H), 7.94 (s, 1 H), 7.86 (dd, J = 7.9, 1.7 Hz, 1 H), 7.82 (dd, J = 8.0, 1.6 Hz, 1 H), 7.67–7.73 (m, 3 H), 7.56–7.62 (m, 2 H), 7.19 (d, J = 7.9 Hz, 1 H), 7.08 (dd, J = 7.6, 0.7 Hz, 1 H), 4.52 (s, 2 H), 3.88 (s, 3 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C25H20N3O4S2 491 (M − H), found 491. Anal. C, H, N.

3-Methoxy-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (38)

Amine 78 was reacted with 3-methoxybenzenesulfonyl chloride according to general procedure B to give 38 as a pale yellow solid (33%); mp (MeOH/CH2Cl2) 278–280 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.76 (br s, 1 H), 8.67 (d, J = 2.0 Hz, 1 H), 8.21 (d, J = 2.3 Hz, 1 H), 7.94 (d, J = 0.7 Hz, 1 H), 7.83 (dd, J = 8.0, 1.5 Hz, 1 H), 7.68–7.75 (m, 3 H), 7.65 (d, J = 3.9 Hz, 1 H), 7.51 (t, J = 8.0 Hz, 1 H), 7.36–7.41 (m, 1 H), 7.32 (t, J = 2.1 Hz, 1 H), 7.21 (ddd, J = 8.3, 2.6, 0.8 Hz, 1 H), 4.52 (s, 2 H), 3.79 (s, 3 H), 3.09 (s, 3 H). HRMS (ESI+) calcd for C25H22N3O4S2 492.1046 (MH+), found 492.1033. Anal. C, H, N.

4-Methoxy-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (39)

Amine 78 was reacted with 4-methoxybenzenesulfonyl chloride according to general procedure B to give 39 as a dark yellow solid (37%); mp (MeOH/CH2Cl2) 254–258 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.62 (br s, 1 H), 8.66 (d, J = 1.8 Hz, 1 H), 8.20 (d, J = 2.2 Hz, 1 H), 7.94 (s, 1 H), 7.83 (d, J = 8.3 Hz, 1 H), 7.76 (d, J = 8.9 Hz, 2 H), 7.67–7.74 (m, 3 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.10 (d, J = 8.9 Hz, 2 H), 4.52 (s, 2 H), 3.79 (s, 3 H), 3.09 (s, 3 H). HRMS (ESI+) calcd for C25H22N3O4S2 492.1046 (MH+), found 492.1033.

3,4-Dimethoxy-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide, sodium salt (40)

Amine 78 was reacted with 3,4-dimethoxybenzenesulfonyl chloride according to general procedure B to give 40 as a pale orange solid (31%). 1H NMR [400 MHz, (CD3)2SO] δ 10.57 (br s, 1 H), 8.66 (d, J = 2.0 Hz, 1 H), 8.21 (d, J = 2.4 Hz, 1 H), 7.94 (d, J = 0.7 Hz, 1 H), 7.83 (dd, J = 7.9, 1.5 Hz, 1 H), 7.69–7.75 (m, 3 H), 7.65 (d, J = 3.9 Hz, 1 H), 7.39 (dd, J = 8.5, 2.2 Hz, 1 H), 7.32 (d, J = 2.2 Hz, 1 H), 7.10 (d, J = 8.6 Hz, 1 H), 4.52 (s, 2 H), 3.79 (s, 3 H), 3.78 (s, 3 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C26H22N3O5S2 521 (M − H), found 521. In this case, the product was also converted to the sodium salt according to general procedure E to give the title compound as a yellow solid (96%); mp (EtOH) 240–244 °C. 1H NMR [400 MHz, (CD3)2SO] δ 8.00 (d, J = 2.1 Hz, 1 H), 7.89 (d, J = 0.8 Hz, 1 H), 7.86 (d, J = 2.5 Hz, 1 H), 7.79 (dd, J = 8.0, 1.6 Hz, 1 H), 7.68 (d, J = 7.9 Hz, 1 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.37–7.41 (m, 2 H), 7.28–7.32 (m, 2 H), 6.92 (d, J = 8.1 Hz, 1 H), 4.51 (s, 2 H), 3.76 (s, 3 H), 3.73 (s, 3 H), 3.08 (s, 3 H). Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-2-(trifluoromethoxy)benzenesulfonamide (41)

Amine 78 was reacted with 2-trifluoromethoxybenzenesulphonyl chloride according to general procedure B to give 41 as a pale yellow solid (42%); mp (CH2Cl2/MeOH) 251–254 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.03 (br s, 1 H), 8.67 (d, J = 1.7 Hz, 1 H), 8.23 (d, J = 2.3 Hz, 1 H), 8.09 (dd, J = 7.8, 1.6 Hz, 1 H), 7.94 (s, 1 H), 7.75–7.85 (m, 2 H), 7.68–7.74 (m, 3 H), 7.63 (d, J = 3.8 Hz, 1 H), 7.55–7.62 (m, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-3-(trifluoromethoxy)benzenesulfonamide (42)

Amine 78 was reacted with 3-trifluoromethoxybenzenesulphonyl chloride according to general procedure B to give 42 as a light brown solid (22%); mp (MeOH/CH2Cl2) 230–232 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.90 (br s, 1 H), 8.70 (d, J = 2.0 Hz, 1 H), 8.20 (d, J = 2.3 Hz, 1 H), 7.94 (s, 1 H), 7.80–7.87 (m, 2 H), 7.67–7.79 (m, 6 H), 7.65 (d, J = 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (APCI+) calcd for C25H18F3N3O4S2 546.0764 (MH+), found 546.0747.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-4-(trifluoromethoxy)benzenesulfonamide (43)

Amine 78 was reacted with 4-trifluoromethoxybenzenesulphonyl chloride according to general procedure B to give 43 as a pale yellow solid (41%); mp (CH2Cl2/MeOH) 293–296 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.88 (br s, 1 H), 8.70 (d, J = 2.0 Hz, 1 H), 8.22 (d, J = 2.3 Hz, 1 H), 7.92–7.98 (m, 3 H), 7.82 (dd, J = 8.0, 1.5 Hz, 1 H), 7.72 (m, 3 H), 7.66 (d, J = 3.9 Hz, 1 H), 7.57–7.63 (m, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-2-(trifluoromethyl)benzenesulfonamide (44)

Amine 78 was reacted with 2-(trifluoromethyl)benzenesulfonyl chloride according to general procedure B to give 44 as a pale yellow solid (67%); mp (CH2Cl2/Et2O) 271–275 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.15 (br s, 1 H), 8.68 (d, J = 1.8 Hz, 1 H), 8.25 (d, J = 2.3 Hz, 1 H), 8.21 (d, J = 7.7 Hz, 1 H), 8.03 (d, J = 7.4 Hz, 1 H), 7.84–7.96 (m, 3 H), 7.82 (dd, J = 8.0, 1.4 Hz, 1 H), 7.68–7.74 (m, 3 H), 7.63 (d, J = 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C25H18F3N3O3S2 529 (M), found 529. Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-3-(trifluoromethyl)benzenesulfonamides (45)

Amine 78 was reacted with 3-(trifluoromethyl)benzenesulfonyl chloride according to general procedure B to give 45 as a pale yellow solid (65%); mp (1,4-dioxane) 262–265 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.92 (br s, 1 H), 8.71 (d, J = 2.0 Hz, 1 H), 8.20 (d, J = 2.3 Hz, 1 H), 8.05–8.12 (m, 3 H), 7.93 (d, J = 0.7 Hz, 1 H), 7.86 (d, J = 7.9 Hz, 1 H), 7.82 (dd, J = 7.9, 1.6 Hz, 1 H), 7.68–7.74 (m, 3 H), 7.66 (d, J = 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C25H18F3N3O3S2 529 (M), found 529. Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-4-(trifluoromethyl)benzenesulfonamide (46)

Amine 78 was reacted with 4-trifluoromethylbenzenesulphonyl chloride according to general procedure B to give 46 as a pink solid (55%); mp (CH2Cl2/MeOH) 282–284 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.99 (br s, 1 H), 8.72 (d, J = 2.0 Hz, 1 H), 8.22 (d, J = 2.3 Hz, 1 H), 7.97–8.07 (m, 4 H), 7.94 (s, 1 H), 7.83 (dd, J = 8.0, 1.6 Hz, 1 H), 7.68–7.76 (m, 3 H), 7.67 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-3,5-bis(trifluoromethyl)benzenesulphonamide (47)

Amine 78 was reacted with 3,5-bis(trifluoromethyl)benzene-1-sulphonyl chloride according to general procedure B to give 47 as a beige solid (63%); mp (MeOH/CH2Cl2) 299–302 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.04 (br s, 1 H), 8.75 (d, J = 2.0 Hz, 1 H), 8.53 (s, 1 H), 8.37 (s, 2 H), 8.22 (d, J = 2.4 Hz, 1 H), 7.93 (s, 1 H), 7.82 (dd, J = 7.9, 1.4 Hz, 1 H), 7.72–7.76 (m, 2 H), 7.71 (d, J = 7.9 Hz, 1 H), 7.68 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

2-Cyano-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (48)

Amine 78 was reacted with 2-cyanobenzenesulfonyl chloride according to general procedure B to give 48 as a pale mustard-yellow solid (43%); mp (MeOH/CH2Cl2) >310 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.16 (br s, 1 H), 8.95 (d, J = 1.8 Hz, 1 H), 8.89 (d, J = 1.5 Hz, 1 H), 8.57 (t, J = 2.1 Hz, 1 H), 8.47 (d, J = 7.4 Hz, 1 H), 8.13 (d, J = 7.0 Hz, 1 H), 7.90–8.00 (m, 3 H), 7.85 (dd, J = 7.9, 1.3 Hz, 1 H), 7.79 (d, J = 3.9 Hz, 1 H), 7.76 (d, J = 3.9 Hz, 1 H), 7.72 (d, J = 8.0 Hz, 1 H), 4.53 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C25H18N4O3S2 486 (M), found 486. Anal. C, H, N. In this case the product was also converted to its sodium salt according to general procedure E to give the desired product as a pale yellow solid (100%). 1H NMR [400 MHz, (CD3)2SO] δ 8.47 (t, J = 2.5 Hz, 2 H), 8.32 (t, J = 2.2 Hz, 1 H), 7.91–7.96 (m, 2 H), 7.85 (dd, J = 8.0, 1.4 Hz, 1 H), 7.67–7.73 (m, 3 H), 7.68 (d, J = 3.9 Hz, 1 H), 7.55–7.61 (m, 2 H), 4.52 (s, 2 H), 3.09 (s, 3 H).

3-Cyano-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (49)

Amine 78 was reacted with 3-cyanobenzenesulfonyl chloride according to general procedure B to give 49 as a cream solid (39%); mp (CH2Cl2/Et2O) 282–285 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.96 (br s, 1 H), 8.71 (d, J = 2.0 Hz, 1 H), 8.30 (t, J = 1.5 Hz, 1 H), 8.22 (d, J = 2.3 Hz, 1 H), 8.15 (dt, J = 7.9, 1.2 Hz, 1 H), 8.10 (ddd, J = 8.1, 1.9, 1.1 Hz, 1 H), 7.95 (d, J = 0.8 Hz, 1 H), 7.79–7.86 (m, 2 H), 7.67–7.74 (m, 4 H), 4.51 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI+) calcd for C25H19N4O3S2 487 (MH+), found 487. Anal. C, H, N.

4-Cyano-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (50)

Amine 78 was reacted with 4-cyanobenzenesulphonyl chloride according to general procedure B to give 50 as a yellow solid (10%); mp (CH2Cl2/MeOH) 282–285 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.02 (br s, 1 H), 8.71 (d, J = 2.0 Hz, 1 H), 8.21 (d, J = 2.4 Hz, 1 H), 8.08 (d, J = 8.6 Hz, 2 H), 7.98 (d, J = 8.6 Hz, 2 H), 7.95 (s, 1 H), 7.83 (dd, J = 8.0, 1.6 Hz, 1 H), 7.74 (d, J = 4.0 Hz, 1 H), 7.72 (s, 1 H), 7.71 (d, J = 8.4 Hz, 1 H), 7.68 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

Methyl 2-(N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)sulfamoyl)benzoate (51)

Amine 78 was reacted with methyl 2-(chlorosulfonyl)benzoate according to general procedure B to give 51 as a pale orange solid (23%); mp (CH2Cl2/Et2O) 209–212 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.74 (br s, 1 H), 8.66 (d, J = 2.0 Hz, 1 H), 8.22 (d, J = 2.4 Hz, 1 H), 7.95–7.99 (m, 1 H), 7.93 (br d, J = 0.7 Hz, 1 H), 7.82 (dd, J = 8.0, 1.6 Hz, 1 H), 7.68–7.76 (m, 5 H), 7.63–7.67 (m, 2 H), 4.52 (s, 2 H), 3.85 (s, 3 H), 3.09 (s, 3 H). LRMS (APCI+) calcd for C26H22N3O5S2 520 (MH+), found 520. Anal. C, H, N.

Methyl 3-(N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)sulfamoyl)benzoate (52)

Amine 78 was reacted with methyl 3-(chlorosulfonyl)benzoate according to general procedure B giving 52 as a beige solid (24%); mp (CH2Cl2/MeOH) 250–254 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.92 (s, 1 H), 8.70 (d, J = 8.7 Hz, 1 H), 8.38 (dd, J = 1.6, 1.6 Hz, 1 H), 8.19–8.21 (m, 2 H), 8.07 (ddd, J = 7.9, 1.9, 1.1 Hz, 1 H), 7.94 (br s, 1 H), 7.77 (d, J = 7.9 Hz, 1 H), 7.70–7.75 (m, 3 H), 7.66 (d, J = 3.4 Hz, 1 H), 4.52 (s, 2 H), 3.87 (s, 3 H), 3.09 (s, 3 H). HRMS (ESI+) calcd for C26H22N3O5S2 520.0995 (MH+), found 520.1004.

Methyl 4-(N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)sulfamoyl)benzoate (53)

Amine 78 was reacted with methyl 4-(chlorosulfonyl)benzoate according to general procedure B to give 53 as a pale yellow solid (60%); mp (CH2Cl2/Et2O) 269–272 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.95 (br s, 1 H), 8.67 (d, J = 1.9 Hz, 1 H), 8.19 (d,J = 2.3 Hz, 1 H), 8.13 (d, J = 8.6 Hz, 2 H), 7.92–7.97 (m, 3 H), 7.82 (dd, J = 8.0, 1.6 Hz, 1 H), 7.68–7.73 (m, 3 H), 7.65 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.85 (s, 3 H), 3.09 (s, 3 H). LRMS (APCI+) calcd for C26H22N3O5S2 520 (MH+), found 520. Anal. C, H, N.

Ethyl 4-(N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)sulfamoyl)benzoate (54)

Amine 78 was reacted with ethyl 4-(chlorosulfonyl)benzoate according to general procedure B to give 54 as a pale yellow solid (52%); mp (CH2Cl2/Et2O) 272–275 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.93 (br s, 1 H), 8.69 (d, J = 2.0 Hz, 1 H), 8.20 (d, J = 2.4 Hz, 1 H), 8.13 (d, J = 8.6 Hz, 2 H), 7.93–7.98 (m, 3 H), 7.83 (dd, J = 8.0, 1.6 Hz, 1 H), 7.69–7.74 (m, 3 H), 7.66 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 4.31 (q, J = 7.1 Hz, 2 H), 3.09 (s, 3 H), 1.29 (t, J = 7.1 Hz, 3 H). LRMS (APCI+) calcd for C27H24N3O5S2 534 (MH+), found 534. Anal. C, H, N.

4-(N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)sulfamoyl)benzoic acid (55)

Amine 78 was reacted with 4-chlorosulphonyl benzoic acid according to general procedure B to give 55 as a pale pink solid (6%); mp (MeOH/CH2Cl2) >310 °C. 1H NMR [400 MHz, (CD3)2SO] δ 13.46 (v br s, 1 H), 10.91 (v br s, 1 H), 8.68 (d, J = 1.9 Hz, 1 H), 8.21 (d, J = 2.3 Hz, 1 H), 8.11 (d, J = 8.6 Hz, 2 H), 7.91–7.95 (m, 3 H), 7.83 (dd, J = 8.0, 1.5 Hz, 1 H), 7.69–7.73 (m, 3 H), 7.65 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C25H19N3O5S2 505 (M), found 505. Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-2-(methylsulfonyl)benzenesulfonamide (56)

Amine 78 was reacted with 2-(methanesulfonyl)benzenesulfonyl chloride according to general procedure B to give 56 as a pale orange solid (77%); mp (Et2O/CH2Cl2) 269–272 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.22 (br s, 1 H), 8.68 (d, J = 1.5 Hz, 1 H), 8.22–8.27 (m, 2 H), 8.14–8.18 (m, 1 H), 7.89–7.97 (m, 3 H), 7.82 (dd, J = 8.0, 1.5 Hz, 1 H), 7.76 (t, J = 2.2 Hz, 1 H), 7.68–7.73 (m, 2 H), 7.65 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.53 (s, 3 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C25H21N3O5S3 539 (M), found 539. Anal. C, H, N.

Sodium (5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)((4-(methylsulfonyl)phenyl)sulfonyl)amide (57)

Amine 78 was reacted with 4-(methanesulfonyl)benzenesulfonyl chloride according to general procedure B and the resulting crude product converted directly to the sodium salt according to general procedure E. The salt was recrystallised from EtOH to give 57 as a cream solid (27%); mp (EtOH) > 300 °C. 1H NMR [400 MHz, (CD3)2SO] δ 8.07 (d, J = 2.1 Hz, 1 H), 7.91–7.98 (m, 5 H), 7.90 (d, J = 0.7 Hz, 1 H), 7.81 (dd, J = 8.0, 1.5 Hz, 1 H), 7.68 (d, J = 8.2 Hz, 1 H), 7.65 (d, J = 3.8 Hz, 1 H), 7.42–7.45 (m, 2 H), 4.52 (s, 2 H), 3.19 (s, 3 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C25H21N3O5S3 539 (M − Na), found 539. HRMS (ESI+) calcd for C25H21N3NaO5S3 562.0536 (MH+), found 562.0522.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-2-nitrobenzenesulfonamide (58)

Amine 78 was reacted with 2-nitrobenzenesulfonyl chloride according to general procedure B to give 58 as a yellow solid (32%); mp (MeOH/CH2Cl2) 270–273 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.21 (bs, 1 H), 8.71 (d, J = 1.9 Hz, 1 H), 8.26 (d, J = 2.4 Hz, 1 H), 8.06–8.10 (m, 1 H), 7.98–8.01 (m, 1 H), 7.94 (d, J = 0.8 Hz, 1 H), 7.86–7.90 (m, 2 H), 7.83 (dd, J = 8.0, 1.6 Hz, 1 H), 7.72–7.75 (m, 2 H), 7.71 (d, J = 8.0 Hz, 1 H), 7.66 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H17N4O5S2 506 (M − H), found 506. Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-4-nitrobenzenesulfonamide (59)

Amine 78 was reacted with 4-nitrobenzenesulfonyl chloride according to general procedure B to give 59 as a pale yellow solid (56%); mp (MeOH/CH2Cl2) 272–275 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.09 (br s, 1 H), 8.71 (d, J = 1.9 Hz, 1 H), 8.40 (dq, J = 9.0, 5.0 Hz, 2 H), 8.22 (d, J = 2.3 Hz, 1 H), 8.07 (dq, J = 8.9, 5.0 Hz, 2 H), 7.95 (s, 1 H), 7.83 (dd, J = 7.9, 1.4 Hz, 1 H), 7.76 (t, J = 2.2 Hz, 1 H), 7.73 (d, J = 4.0 Hz, 1 H), 7.71 (d, J = 8.0 Hz, 1 H), 7.68 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C24H17N4O5S2 506 (M − H), found 506. HRMS (APCI+) calcd for C24H18N4O5S2 507.0791 (MH+), found 507.0792. In this case the product was also converted to its sodium salt according to general procedure E, giving an orange solid (89%). 1H NMR [400 MHz, (CD3)2SO] δ 8.24 (d, J = 8.8 Hz, 2 H), 8.11 (d, J = 1.6 Hz, 1 H), 7.96 (d, J = 8.8 Hz, 2 H), 7.94 (d, J = 2.4 Hz, 1 H), 7.91 (s, 1 H), 7.81 (dd, J = 8.0, 1.4 Hz, 1 H), 7.68 (d, J = 8.0 Hz, 1 H), 7.65 (d, J = 3.9 Hz, 1 H), 7.47 (t, J = 2.2 Hz, 1 H), 7.45 (d, J = 3.8 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H).

3-Chloro-2-fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (60)

Amine 78 was reacted with 3-chloro-2-fluorobenzenesulphonyl chloride according to general procedure B to give 60 as a pale yellow solid (63%); mp (CH2Cl2/MeOH) 269–272 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.30 (br s, 1 H), 8.71 (d, J = 1.9 Hz, 1 H), 8.27 (d, J = 2.3 Hz, 1 H), 7.87–7.98 (m, 3 H), 7.83 (dd, J = 7.9, 1.2 Hz, 1 H), 7.68–7.76 (m, 3 H), 7.66 (d, J = 3.9 Hz, 1 H), 7.45 (t, J = 8.0 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

4-Fluoro-2-methyl-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl) benzenesulphonamide (61)

Amine 78 was reacted with 4-fluoro-2-methylbenzene-1-sulphonyl chloride according to general procedure B to give 61 as a light-brown solid (50%); mp 280–283 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.95 (br s, 1 H), 8.65 (d, J = 2.0 Hz, 1 H), 8.23 (d, J = 2.4 Hz, 1 H), 8.05 (dd, J = 5.8, 3.1 Hz, 1 H), 7.95 (s, 1 H), 7.83 (dd, J = 7.9, 1.4 Hz, 1 H), 7.68–7.75 (m, 2 H), 7.66 (t, J = 2.2 Hz, 1 H), 7.63 (d, J = 3.9 Hz, 1 H), 7.33 (dd,J = 9.9, 2.5 Hz, 1 H), 7.27 (dt, J = 8.4, 2.6 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

4-Fluoro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-3-(trifluoromethyl)benzenesulphonamide (62)

Amine 78 was reacted with 4-fluoro-3-(trifluoromethyl)benzene-1-sulphonyl chloride according to general procedure B to give 62 as a yellow solid (40%); mp 250–252 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.93 (br s, 1 H), 8.20 (d, J = 2.4 Hz, 1 H), 8.11–8.19 (m, 2 H), 7.93 (d, J = 0.6 Hz, 1 H), 7.82 (dd, J = 8.4, 1.6 Hz, 1 H), 7.77 (d, J = 9.9 Hz, 1 H), 7.68–7.75 (m, 2 H), 7.70 (d, J = 7.9 Hz, 1 H), 7.66 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (APCI+) calcd for C25H18F4N3O3S2 548.0720 (MH+), found 548.0743.

3-Chloro-4-methyl-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (63)

Amine 78 was reacted with 3-chloro-4-methylbenzenesulphonyl chloride according to general procedure B to give 63 as an off-white solid (50%); mp (CH2Cl2/MeOH) 277–279 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.81 (br s, 1 H), 8.70 (d, J = 2.0 Hz, 1 H), 8.22 (d, J = 2.3 Hz, 1 H), 7.94 (s, 1 H), 7.80–7.86 (m, 2 H), 7.64–7.75 (m, 5 H), 7.58 (d, J = 8.2 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H), 2.36 (s, 3 H). Anal. C, H, N.

2-Chloro-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-4-(trifluoromethyl)benzenesulfonamide (64)

Amine 78 was reacted with 2-chloro-4-trifluoromethylbenzenesulphonyl chloride according to general procedure B to give 64 as a beige solid (61%); mp (CH2Cl2/MeOH) 292–295 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.39 (br s, 1 H), 8.68 (d, J = 2.0 Hz, 1 H), 8.35 (d, J = 5.0 Hz, 1 H), 8.28 (d, J = 2.4 Hz, 1 H), 8.17 (s, 1 H), 7.97 (dd, J = 8.4, 1.2 Hz, 1 H), 7.93 (s, 1 H), 7.82 (dd, J = 7.9, 1.4 Hz, 1 H), 7.67–7.75 (m, 1 H), 7.64 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

3-Bromo-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-5-(trifluoromethyl)benzenesulphonamide (65)

Amine 78 was reacted with 3-bromo-5-(trifluoromethyl)benzene-1-sulphonyl chloride according to general procedure B to give 65 as a yellow solid (26%); mp 280–283 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.01 (br s, 1 H), 8.69 (s, 1 H), 8.34 (s, 1 H), 8.24 (s, 1 H), 8.20 (d, J = 2.2 Hz, 1 H), 8.05 (s, 1 H), 7.93 (s, 1 H), 7.82 (d, J = 8.4 Hz, 1 H), 7.63–7.77 (m, 4 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)pyridine-2-sulfonamide (66)

Amine 78 with pyridine-2-sulphonyl chloride according to general procedure B to give 66 as a cream solid (20%); mp 272–275 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.01 (br s, 1 H), 8.73–8.78 (m, 1 H), 8.66 (d, J = 2.0, Hz, 1 H), 8.30 (d, J = 2.4 Hz, 1 H), 8.11 (td, J = 7.8, 1.7 Hz, 1 H), 8.60 (dt, J = 7.6, 1.0 Hz, 1 H), 7.94 (s, 1 H), 7.81–7.87 (m, 2 H), 7.73 (d, J = 3.9 Hz, 1 H), 7.71 (d, J = 8.1 Hz, 1 H), 7.66–7.70 (m, 1 H), 7.64 (d, J = 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). In this case the product was also converted to its sodium salt according to general procedure E giving a light-brown solid (89%). 1H NMR [400 MHz, (CD3)2SO] δ 8.53 (td, J = 4.7, 1.4 Hz, 1 H), 8.04 (d, J = 2.1 Hz, 1 H), 7.93 (d, J = 2.4 Hz, 1 H), 7.91 (s, 1 H), 7.83–7.87 (m, 2 H), 7.80 (dd, J = 8.0, 1.5 Hz, 1 H), 7.68 (d, J = 7.9 Hz, 1 H), 7.65 (d, J = 3.8 Hz, 1 H), 7.60 (t, J = 2.2 Hz, 1 H), 7.41 (d, J = 3.8 Hz, 1 H), 7.32–7.38 (m, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). HRMS (APCI+) calcd for C23H17N4NaO3S2 485.0713 (MH+), found 485.0710.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)pyridine-3-sulfonamide (67)

Amine 78 was reacted with pyridine-3-sulphonyl chloride according to general procedure B to give 67 as a light brown solid (45%); mp (CH2Cl2/MeOH) 283–286 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.98 (br s, 1 H), 8.97 (d, J = 2.0 Hz, 1 H), 8.83 (dd, J = 4.8, 1.4 Hz, 1 H), 8.70 (d, J = 1.9 Hz, 1 H), 8.22 (d, J = 2.2 Hz, 1 H), 8.20 (dt, J = 8.1, 1.8 Hz, 1 H), 7.94 (s, 1 H), 7.84 (d, J = 7.9 Hz, 1 H), 7.75 (t, J = 2.2 Hz, 1 H), 7.73 (d, J = 3.9 Hz, 1 H), 7.71 (d, J = 8.0 Hz, 1 H), 7.69 (d, J = 3.9 Hz, 1 H), 7.65 (dd, J = 5.2, 2.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). HRMS (APCI+) calcd for C23H18N4O3S2 463.0893 (MH+), found 463.0891.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)thiophene-2-sulfonamide (68)

Amine 78 was reacted with thiophene-2-sulphonyl chloride according to general procedure B to give 68 as a cream solid (71%); mp (CH2Cl2/MeOH) 300–304 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.91 (br s, 1 H), 8.72 (d, J = 2.0, Hz, 1 H), 8.24 (d, J = 2.3 Hz, 1 H), 7.93–7.98 (m, 2 H), 7.83 (dd, J = 8.0, 1.5 Hz, 1 H), 7.78 (t, J = 2.2 Hz, 1 H), 7.74 (d, J = 3.9 Hz, 1 H), 7.68 (d, J = 3.9 Hz, 1 H), 7.65 (dd, J = 3.8, 1.3 Hz, 2 H), 7.15 (dd, J = 4.9, 3.8 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). In this case the product was also converted to its sodium salt according to general procedure E giving a pale yellow solid (90%). 1H NMR [400 MHz, (CD3)2SO] δ 8.08 (d, J = 2.1, Hz, 1 H), 7.92 (d, J = 2.4 Hz, 1 H), 7.90 (s, 1 H), 7.80 (dd, J = 7.9, 1.5 Hz, 1 H), 7.68 (d, J = 8.0 Hz, 1 H), 7.65 (d, J = 3.8 Hz, 1 H), 7.46–7.53 (m, 2 H), 7.42 (d, J = 3.9 Hz, 1 H), 7.27 (dd, J = 3.6, 1.3 Hz, 1 H), 6.93 (dd, J = 5.0, 3.6 Hz, 1 H), 4.51 (s, 2 H), 3.08 (s, 3 H). Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)thiophene-3-sulfonamide (69)

Amine 78 was reacted with thiophene-3-sulphonyl chloride according to general procedure B to give 69 as a yellow solid (47%); mp (CH2Cl2/MeOH) > 300 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.72 (br s, 1 H), 8.68 (d, J = 1.9, Hz, 1 H), 8.31 (q, J = 1.3 Hz, 1 H), 8.24 (d, J = 2.3 Hz, 1 H), 7.95 (s, 1 H), 7.83 (dd, J = 8.0, 1.4 Hz, 1 H), 7.73–7.78 (m, 3 H), 7.70 (d, J = 8.0 Hz, 1 H), 7.66 (d, J = 3.9 Hz, 1 H), 7.32 (dd, J = 5.2, 1.4 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). Anal. C, H, N.

N-(5-(5-(2-Methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-4-(oxazol-5-yl)benzenesulfonamides (70)

Amine 78 was reacted with 4-(1,3-oxazol-5-yl)benzenesulfonyl chloride according to general procedure B to give 70 as a pale yellow solid (81%); mp (MeOH/CH2Cl2) 278–281 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.80 (br s, 1 H), 8.69 (d, J = 2.0 Hz, 1 H), 8.53 (s, 1 H), 8.23 (d, J = 2.3 Hz, 1 H), 7.86–7.96 (m, 6 H), 7.80 (dd, J = 7.9, 1.4 Hz, 1 H), 7.68–7.73 (m, 3 H), 7.66 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C27H20N4O4S2 528 (M), found 528. Anal. C, H, N.

5-(Isoxazol-5-yl)-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)thiophene-2-sulfonamide (71)

Amine 78 was reacted with 5-(5-isoxazyl)thiophene-2-sulfonyl chloride according to general procedure B to give 71 as a pale pink solid (20%); mp (MeOH/1,4-dioxane) 245–249 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.15 (br s, 1 H), 8.76 (br s, 1 H), 8.72 (d, J = 2.0 Hz, 1 H), 8.29 (d, J = 2.3 Hz, 1 H), 7.93 (d, J = 0.7 Hz, 1 H), 7.82 (dd, J = 7.9, 1.5 Hz, 1 H), 7.79 (t, J = 2.2 Hz, 1 H), 7.71–7.75 (m, 4 H), 7.70 (d, J = 3.6 Hz, 1 H), 7.11 (d, J = 1.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H). LRMS (APCI−) calcd for C25H18N4O4S3 534 (M), found 534. Anal. C, H, N.

4-(3,5-Dimethyl-1H-pyrazol-1-yl)-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)benzenesulfonamide (72)

Amine 78 was reacted with 4-(3,5-dimethyl-1H-pyrazol-1-yl)benzenesulfonyl chloride according to general procedure B to give 72 as a beige solid (14%); mp (Et2O/CH2Cl2) 265–268 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.81 (br s, 1 H), 8.70 (s, 1 H), 8.25 (d, J = 2.2 Hz, 1 H), 7.94 (br s, 1 H), 7.91 (d, J = 8.7 Hz, 2 H), 7.82 (dd, J = 8.0, 1.3 Hz, 1 H), 7.77 (d, J = 8.7 Hz, 2 H), 7.68–7.74 (m, 3 H), 7.61 (d, J = 3.9 Hz, 1 H), 6.12 (s, 1 H), 4.51 (s, 2 H), 3.09 (s, 3 H), 2.34 (s, 3 H), 2.15 (s, 3 H). LRMS (APCI−) calcd for C29H25N5O3S2 555 (M), found 555. Anal. C, H, N.

N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide (73)

Amine 78 was reacted with 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride according to general procedure B to give 73 as a yellow solid (78%). 1H NMR [400 MHz, (CD3)2SO] δ 10.64 (s, 1 H), 8.68 (d, J = 2.0 Hz, 1 H), 8.22 (d, J = 2.3 Hz, 1 H), 7.95 (br s, 1 H), 7.84 (dd, J = 8.0, 1.4 Hz, 1 H), 7.74–7.70 (m, 3 H), 7.66 (d, J = 3.9 Hz, 1 H), 7.28–7.31 (m, 2 H), 7.04 (d, J = 8.5 Hz, 1 H), 4.52 (s, 2 H), 4.28 (m, 4 H), 3.09 (s, 3 H). In this case the product was also converted to its sodium salt according to general procedure E giving a yellow solid (58%). 1H NMR [400 MHz, (CD3)2SO] δ 8.00 (d, J = 2.1 Hz, 1 H), 7.90 (br s, 1 H), 7.85 (d, J = 2.4, 1 H), 7.80 (dd, J = 8.0, 1.5 Hz, 1 H), 7.65–7.70 (m, 2 H), 7.37–7.41 (m, 2 H), 7.17–7.21 (m, 2 H), 6.82 (d, J = 8.3, 1 H), 4.51 (s, 2H), 4.21 (br s, 4H), 3.08 (s, 3H). HRMS calcd for C26H20N3NaO5S2, 541.0742 (M− + Na+); found, 541.0744.

4-Methyl-N-(5-(5-(2-methyl-1-oxoisoindolin-5-yl)thiophen-2-yl)pyridin-3-yl)thiazole-5-sulphonamide (74)

Amine 78 was reacted with 4-methylthiazole-5-sulphonyl chloride according to general procedure B, giving 74 as a brown solid (12%); mp 277–280 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.15 (br s, 1 H), 9.20 (s, 1 H), 8.78 (d, J = 2.0 Hz, 1 H), 8.25 (d, J = 2.4 Hz, 1 H), 7.95 (d, J = 0.6 Hz, 1 H), 7.83 (dd, J = 7.9, 1.5 Hz, 1 H), 7.77 (t, J = 2.2 Hz, 1 H), 7.74 (d, J = 3.9 Hz, 1 H), 7.71 (d, J = 8.0 Hz, 1 H), 6.69 (d, J = 3.9 Hz, 1 H), 4.52 (s, 2 H), 3.09 (s, 3 H), 2.52 (s, 3 H). HRMS calcd for C22H18N4O3S3, 483.06138 (M+); found, 483.06251.

Inhibition of perforin-mediated lysis of Jurkat cells

The ability of the compounds to inhibit the lysis of labelled nucleated (Jurkat T lymphoma) cells in the presence of 0.1% BSA was measured by the release of 51Cr. Jurkat target cells were labelled by incubation in medium with 100 μCi 51Cr for 1 h. The cells were then washed three times to remove unincorporated isotope and re-suspended at 1 × 105 cells per mL in RPMI buffer supplemented with 0.1% BSA. Each test compound was pre-incubated to concentrations of 20, 10, 5, 2.5 and 1.25 μM with recombinant perforin for 30 min with DMSO as a negative control. 51Cr labelled Jurkat cells were then added and cells were incubated at 37 °C for 4 h. The supernatant was collected and assessed for its radioactive content on a gamma counter (Wallac Wizard 1470 automatic gamma counter). Each data point was performed in triplicate and an IC50 was calculated from the range of concentrations described above.

KHYG1 inhibitory assay

KHYG1 cells are washed and re-suspended in RPMI/0.1% BSA at 16 × 105 cells/mL and 100 μL of the cell suspension is dispensed to each well of a 96-well V-bottom plate. Test compounds are then added (50 μL) at a final concentration of 20 μM and incubated at RT for 20 min. 51Cr-labelled K562 leukemia target cells (50 μL, 2 × 105 cells/mL) are then added to each well and incubated at 37 °C for 4 h. 51Cr release is assayed using a Skatron Harvesting Press and radioactivity estimated on a Wallac Wizard 1470 Automatic Gamma counter (Turku, Finland). The inhibitory function is then determined by identifying the number of untreated or inhibitor treated effector cells required to kill the same number of targets. The percent inhibition is calculated by the formula:where x is the point the inhibitor intersects with the DMSO on a curve. In the example shown (Fig. 3) “x” is the x-intercept corresponding to the point on the DMSO control curve that yields the same level of 51Cr release as the test compound at an E/T ratio of 16:1.

Fig. 3

Calculation of inhibitory function in the KHYG1 assay.

Toxicity to KHYG1 NK cells

The toxicity assay was carried out in exactly the same manner as the killing assay above, but instead of adding the labelled K562 target cells, 100 μL of RPMI 0.1% BSA was added. Cells were incubated for 4 h at 37 °C and then washed ×3 in RPMI + 0.1% BSA. Cells were then re-suspended in 200 μL of complete medium and incubated for 18–24 h at 37 °C. Trypan blue was added to each well. Viable (clear) cells and total (clear + blue) cells were counted, and the percentage of viable cells was calculated compared to DMSO treated cell control (% viability).