Identification of Purines and 7-Deazapurines as Potent and Selective

Sep 10, 2015 - A series of cardiac troponin I-interacting kinase (TNNI3K) inhibitors arising from 3-((9H-purin-6-yl)amino)-N-methyl-benzenesulfonamide...
1 downloads 0 Views 3MB Size
Article pubs.acs.org/jmc

Identification of Purines and 7‑Deazapurines as Potent and Selective Type I Inhibitors of Troponin I‑Interacting Kinase (TNNI3K) Brian G. Lawhorn,*,† Joanne Philp,† Yongdong Zhao,† Christopher Louer,† Marlys Hammond,† Mui Cheung,† Harvey Fries,† Alan P. Graves,‡ Lisa Shewchuk,‡ Liping Wang,‡ Joshua E. Cottom,‡ Hongwei Qi,‡ Huizhen Zhao,‡ Rachel Totoritis,‡ Guofeng Zhang,‡ Benjamin Schwartz,‡ Hu Li,‡ Sharon Sweitzer,‡ Dennis A. Holt,† Gregory J. Gatto, Jr.,† and Lara S. Kallander† †

Heart Failure Discovery Performance Unit and ‡Platform Technology and Sciences, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania 19406, United States

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

S Supporting Information *

ABSTRACT: A series of cardiac troponin I-interacting kinase (TNNI3K) inhibitors arising from 3-((9H-purin-6-yl)amino)-Nmethyl-benzenesulfonamide (1) is disclosed along with fundamental structure−function relationships that delineate the role of each element of 1 for TNNI3K recognition. An X-ray structure of 1 bound to TNNI3K confirmed its Type I binding mode and is used to rationalize the structure−activity relationship and employed to design potent, selective, and orally bioavailable TNNI3K inhibitors. Identification of the 7-deazapurine heterocycle as a superior template (vs purine) and its elaboration by introduction of C4benzenesulfonamide and C7- and C8−7-deazapurine substituents produced compounds with substantial improvements in potency (>1000-fold), general kinase selectivity (10-fold improvement), and pharmacokinetic properties (>10-fold increase in poDNAUC). Optimal members of the series have properties suitable for use in in vitro and in vivo experiments aimed at elucidating the role of TNNI3K in cardiac biology and serve as leads for developing novel heart failure medicines.



INTRODUCTION Cardiac troponin I-interacting kinase (TNNI3K or CARK) is a member of the tyrosine-like kinase (TLK) family that is selectively expressed in heart tissue, but its biological function is poorly defined.1,2 Although TNNI3K is a functional kinase capable of autophosphorylation, interacts with components of the sarcomere including cardiac troponin I, and has been linked to activation of the p38 MAP kinase pathway, no genuine substrates of TNNI3K have been identified to date.1−3 In addition, the functional role of its autophosphorylation activity and the details of how TNNI3K regulates p38 and other unidentified signaling pathways are unclear. Overexpression of Tnni3k triggers in vitro cardiac hypertrophy of neonatal rat ventricular myocytes and exacerbates disease progression in multiple in vivo settings including models of dilated cardiomyopathy, pressure overload-induced heart failure, and ischemia/reperfusion injury.3−5 In complementary studies, a Tnni3k knockout mouse exhibited reduced ischemic injury demonstrating that deletion of TNNI3K is cardioprotective.3 These studies suggest that TNNI3K inhibition could serve as a unique strategy for addressing acute ischemic injury and heart failure. Consequently, we sought to identify selective inhibitors that could be used to elucidate the cardiac biology of TNNI3K and serve as templates for the development of novel heart failure therapeutics. Herein, we provide the initial disclosure of a series of TNNI3K inhibitors arising from 3-((9H-purin-6yl)amino)-N-methyl-benzenesulfonamide (1) that are comple© XXXX American Chemical Society

mentary to the Type II compounds derived from sorafenib reported from our laboratories (Figure 1).3,6 Fundamental structure−function relationships are presented along with a cocrystal structure of 1 bound to the ATP binding site of TNNI3K, and these results are rationalized and used to design potent, selective, and orally bioavailable TNNI3K inhibitors.



RESULTS AND DISCUSSION Hit Identification. Evaluation of selected compounds from the GSK kinase inhibitor collection against the kinase panel assembled by Ambit, which includes TNNI3K, uncovered 1 (Figure 1) as a promising TNNI3K binder (Kd = 111 nM), and further evaluation of 1 in our own TNNI3K assay confirmed its affinity for TNNI3K (IC50 = 500 nM).7 Of particular interest, purine 1 exhibits excellent selectivity for TNNI3K against a broad spectrum of kinases, binding significantly to only 4% of kinases (8 of 203 in Ambit panel) at 10 μM inhibitor concentration. Compound 1 displays potent activity (IC50 = 32 nM for B-Raf V600E) against the closely related serine/ threonine-specific protein kinases B-Raf and c-Raf, which share 67% sequence identity (82% similarity) with TNNI3K among residues comprising their ATP-binding sites. Raf kinase inhibition has been linked to effects in heart failure models, and although this was not viewed as a prohibitive liability for 1, Received: June 16, 2015

A

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Figure 1. Type I and II TNNI3K inhibitors.

the development of inhibitors selective against Raf kinases would be desireable, especially in producing tools to characterize the biology of TNNI3K.8 Structure of 1 Bound to TNNI3K. A cocrystal structure of 1 bound to TNNI3K was solved to 2.7 Å resolution and revealed that 1 resides in the ATP binding site (Figure 2). In the presence of 1, the activation loop exists in the active conformation rather than the alternative “DFG-out” orientation, indicating that 1 behaves as a Type I kinase inhibitor. The purine ring of 1 contacts the kinase hinge recognition feature of TNNI3K, with N3 serving as an H-bond acceptor for the Ile542 backbone amide NH, while N9−H engages in a H-bond donor interaction with the Ile542 backbone amide CO. The benzenesulfonamide moiety projects toward the back pocket and participates in three hydrogen bonding interactions. The sulfonamide NH serves as a hydrogen bond donor to the side chain OH of Thr539, which is the gatekeeper residue of TNNI3K, while the sulfonamide oxygen atoms interact with the backbone NH of Asp606 and an active site water that bridges the Phe607 backbone NH and the carboxylate of Glu509. As a consequence of this hydrogen bond network, the sulfonamide N-Me group is directed upward where it is situated snugly into a small, lipophilic pocket formed by Ala488, Ile489, Lys490, and Ile537. In order to simultaneously accommodate the two purine hydrogen bond contacts and the three sulfonamide hydrogen bond interactions, the purine and benzene rings adopt a coplanar binding conformation (dihedral angle ∼12°). Effect of Modifications of the Purine Moiety. An initial set of derivatives of 1 were evaluated to define the role of each of its features for TNNI3K recognition. Modifications of the purine (Figure 3) included N9-methylation, which produced an inactive compound (2, IC50 > 25 000 nM), suggesting that the purine N9−H plays a key role, consistent with the cocrystal structure of 1 bound to TNNI3K which reveals that the N9−H

Figure 3. Effect of modifying the purine moiety of 1.

donates a hydrogen bond to the backbone carbonyl of Ile542. Both electron-withdrawing (e.g., Cl, 5, IC50 = 16 000 nM) and electron-donating (e.g., NH2, 4, IC50 = 8000 nM) C2substituents displayed significantly reduced TNNI3K activity in a manner that correlates with substituent size (IC50: H (500 nM) > F (3200 nM) > NH2 (8000 nM) > Cl (16 000 nM)) indicating a steric effect where this portion of the molecule contacts TNNI3K. Indeed, the structure shows C2 of 1 lying within 3.5 Å of Gln540. Translocation of N7 to N8 (6, IC50 = 50 nM) or removal of N7 (7, IC50 = 80 nM) improved TNNI3K activity, and the apparent detrimental effect of N7 was initially attributed to its desolvation upon binding to TNNI3K, where the N7 atom projects into a hydrophobic region and lacks a hydrogen bonding partner (Figure 2). The tautomeric effects unique to the purine ring system may also disrupt TNNI3K recognition. In contrast to purine 1, the pyrazolo[3,4-d]pyrimidine of 6 and pyrrolo[2,3-d]pyrimidine of 7 are predisposed for binding, as they lack the competing 7Hpurine tautomer (Figure 4) available to 1 that is unable to engage the N9−H hydrogen bond donor interaction observed as the 9H-purine tautomer of 1 binds to the TNNI3K active

Figure 2. Cocrystal structure of 1 (green) bound to TNNI3K (gray). B

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Figure 4. Structure-based rationalization of key results.

site (Figure 2).9 Thus, the 7H-purine tautomer would introduce a repulsive electrostatic interaction with the carbonyl of Ile542, and the absence of this complicating factor in 6 and 7 would enhance their interaction with TNNI3K. Further alteration of 7 by elimination of the purine N1 nitrogen to give the pyrrolo[2,3-b]pyridine 9 resulted in a substantial reduction in activity (100-fold) indicative of a pivotal function for the N1 nitrogen atom. Structural information revealed no binding contacts between N1 and TNNI3K. However, N1 does appear to allow 1 to assume a coplanar orientation that would be energetically disfavored by 9 (see Figure 4), but which is essential for TNNI3K binding by this scaffold, as illustrated by the cocrystal structure (Figure 2). Thus, N1 likely imparts a significant conformational effect on binding of 1 to TNNI3K, and the consequences that arise from this effect along with other conformational considerations will be further elaborated in a separate report. The 6-oxo-6,7dihydro-5H-pyrrolo[2,3-d]pyrimidine 10 exhibited activity similar to purine 1, and was 5-fold less active than its parent 7. Notably, 10 shares a similar electronic arrangement with 1, and also would suffer from the partial desolvation of its carbonyl upon TNNI3K binding because one of the carbonyl lone pairs is directed toward Gly544, preventing its interaction with solvent water. Effect of Benzenesulfonamide Substitutions. An assessment of substituted benzenesulfonamides demonstrated that this moiety is also sensitive to modification (Figure 5), consistent with its intimate contact within the TNNI3K active site (Figure 2). Extension of the N-Me group of 1 to the N-Et reduced affinity (11, IC50 = 2000 nM) implying a steric limitation, whereas removal of the N-Me group to give 12 (IC50 = 40 000 nM) resulted in an 80-fold reduction in activity. Furthermore, the N-Me group could not be replaced with polar atoms such as OH (13, IC50 = 20 000 nM). These results are strong evidence that the N-Me group makes hydrophobic contact within TNNI3K, consistent with the TNNI3Kcompound 1 costructure observations where this methyl group is situated comfortably in a small, lipophilic pocket formed by Ala488, Ile489, Lys490, and Ile537. The magnitude of binding energy change (2.6 kcal/mol using ΔG1 − ΔG2 = RT*ln(K1/K2) where K is approximated by IC50 values) arising from this N-Me group is beyond the level usually observed for filling a hydrophobic pocket (1.4 kcal/mol) and may reflect

Figure 5. Effect of benzenesulfonamide substitutions.

additional energetic advantages of the N-Me functionality of 1.10 Notably, 12 would presumably suffer a significant penalty for desolvation of one of its sulfonamide NH atoms which has no apparent H-bonding partner upon binding to TNNI3K, whereas the sulfonamide of 1 engages in a full complement of H-bonding interactions upon binding. Methylation (14, IC50 = 8000 nM) or removal (15, IC50 = 6300 nM) of the sulfonamide NH produced similar reductions in potency (∼15-fold) as anticipated from its role as a hydrogen bond donor (Figure 2). The 6-Me substituted benzenesulfonamide 16 is substantially less active than 1, demonstrating that this position of the benzenesulfonamide is near the wall of the binding pocket, and crystallography results confirm that C6 projects toward the catalytic side chains of Lys490 and Asp606. In contrast, 4-Me-substituted benzenesulfonamide 17 is equipotent to 1, consistent with the observation that the C4 position of the benzene points toward the opening of the pocket and is thus available for further exploration. The 7-deazapurine template 7 provided the opportunity to expand the SAR generated from the purine scaffold by examining two analogues modified at the nitrogen atom which links the two aromatic groups (Figure 5). Both the NMe variant 18 (IC50 = 5000 nM) and the O-linked 19 (IC50 = 16 000 nM) exhibited substantial reductions in TNNI3K affinity. In the case of 18, we hypothesize that this arises from disruption of the coplanar binding conformation observed in the crystal structure of 1 bound to TNNI3K (Figure 2). In a coplanar orientation, the N-Me group of 18 would interpose C

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

34%) compared to 1, which displayed no measurable oral exposure and could not be dosed intravenously due to its poor solubility in the dosing vehicle. The heightened solubility of 7 may arise from its augmented basicity (calculated BH+ pKa = 5.2) versus 1 (calculated BH+ pKa = 3.2), and this is in contrast to the potent pyrazolopyrimidine 6 (calculated BH+ pKa = 4.0), which like 1, displays poor solubility and low oral bioavailability (Cl = 72 mL/min/kg, F = 5%). Deazapurine 7 maintains the excellent broad-spectrum kinase selectivity of 1, showing >100fold selectivity against 84% (155/185) and >10-fold selectivity against 97% (180/185) of kinases tested in the Millipore KinaseProfiler panel (Millipore.com, see Supporting Information for details). Like 1, compound 7 manifests potent activity against the B-Raf oncogenic mutant (V600E), though, in the case of 7, it is nearly equipotent against TNNI3K, whereas compound 1 favors B-Raf inhibition by 15-fold, implying that 7 is a preferred scaffold for divergence into TNNI3K specific binding. An additional benefit of 7 is that it permits the appendage of substituents from both the C7 and C8 ring positions into the unfilled portion of the TNNI3K pocket. These atoms along with the benzenesulfonamide C4 site were targeted for further elaboration as the structural and biochemical results signify that such modifications would be permissible without disrupting TNNI3K recognition. Effect of C7- and C8−7-Deazapurine Substitutions. An evaluation of C7 and C8 7-deazapurine subsituents (Figure 7) confirmed that a variety of modifications are tolerated, as anticipated from the crystal structure of 1 bound to TNNI3K. The 7-Me variant (21, IC50 = 40 nM) displayed activity comparable to 7 (IC50 = 80 nM), whereas larger alkyl substitutions produced slightly weaker TNNI3K binders (22, 23, IC50 = 126 nM) than 21, suggesting that filling toward the periphery of the binding site is of little energetic consequence. The 7-Cl (25, IC50 = 32 nM) and 7-Br (26, IC50 = 32 nM) variants exhibited potencies similar to the 7-Me bearing 21, demonstrating little electronic effect on TNNI3K binding. Conversely, 25 and 26 show a marked improvement in activity against B-Raf compared to 21, and this apparent electronic effect is indicative of divergent binding interactions for B-Raf vs TNNI3K. Pertinent to this result is the observation that the

the neighboring benzene C4−H and deazapurine C7−H atoms, forcing at least one severe H/H overlapping interaction (Figure 4), and thereby cause 18 to adopt an alternate, inefficient orientation upon binding to TNNI3K.11 The O-linked 19 is also anticipated to favor an orthogonal alignment of its aryl rings, as has been observed for similar diaryl ethers, whose coplanar conformations are disfavored as a result of the increased aryl−aryl repulsion arising from the smaller linking oxygen atom.12 Thus, an orthogonal conformation of 19 allows optimal overlap between the oxygen p-orbital lone pair and the π-system of the electron-deficient pyrimidine ring while minimizing steric repulsion between the two aryl rings (Figure 4). Finally, the 5-pyridyl analogue 20 was examined and displayed activity (IC50 = 250 nM) only 3-fold less than 7, consistent with structural evidence that shows the C5 position approaches the opening of the pocket and points toward solvent space (Figure 2). Selection of a Lead Compound. On the basis of these structural and structure−function results, the 7-deazapurine 7 was selected as a suitable lead for further optimization (Figure 6). In addition to its enhanced affinity for TNNI3K, 7 exhibits

Figure 6. Comparison of hit 1 and lead 7. Kinase profiling details are available in Supporting Information.

increased aqueous solubility (50 μM) and as a result offers an improved rat pharmacokinetic profile (Cl = 68 mL/min/kg, F =

Figure 7. Effect of C7- and C8-deazapurine substituents. D

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

or pharmacokinetics, and as such, 44 as well as the C8substitutions were not further pursued. Effect of C4-Benzenesulfonamide Substitutions. An evaluation of substituted-benzenesulfonamide analogues (Figure 9) demonstrated that TNNI3K binding affinity can be

residue that lies below C7 of the deazapurine differs in TNNI3K (Leu595) and B-Raf (Phe583), and this disparity presumably gives rise to their differing responses to C7substituents (Figure 8). Importantly, C7-substitution had

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Figure 8. Crystal structure of 1 (green) bound to TNNI3K (gray) overlaid with the B-Raf structure13 (orange) (PDB code: 2FB8).

substantial effects on pharmacokinetics with the halogen bearing analogues showing up to 7-fold improvement in total oral exposure (e.g., 26, poDNAUC = 0.55 h-kg/L) compared to 7. The reduced clearance of 25 (Cl = 20 mL/min/kg) and 26 (Cl = 23 mL/min/kg) may arise from the halogen substituents blocking oxidative metabolism of the pyrrole ring, but the lower volumes of distribution of 25 (Vdss =0.45 L/kg) and 26 (Vdss = 0.63 L/kg) compared to 7 (Vdss = 1.4 L/kg) suggest that altered compound distribution may also contribute to their elevated in vivo exposure (e.g., via protective effects from increased plasma protein binding). Like the 7-Br-substituted 26, the 7-Ph analogue 27 exhibited equipotent affinity for TNNI3K (IC50 = 40 nM) but substantially improved activity against B-Raf (IC50 = 4 nM) compared to 21, perhaps reflecting the phenyl group’s greater complementarity with the aromatic Phe583 side chain of B-Raf than with the aliphatic Leu595 side chain of TNNI3K. Substitutions on the 7-Ph ring produced only slight modulations in TNNI3K and B-Raf activity, and these effects were not associated with any clear steric (cf. 27-30), electronic (cf. 27, 30, 31), or electrostatic (cf. 32-34) trends. Furthermore, the 7-Ph group displayed inferior pharmacokinetics to 26, and its high clearance (Cl = 93 mL/min/kg) could only partially be restored through substitution (30, Cl = 65 mL/min/kg). Given these limitations and their preferential binding to B-Raf over TNNI3K, 7-aryl analogues were not pursued further. Modifications of the C8 site included C8-bromination, which had little effect on TNNI3K binding (35, IC50 = 50 nM) but did improve pharmacokinetics (poDNAUC = 0.26 h-kg/L). Like the C7-phenyl compounds, the C8-phenyl variants (3641) displayed moderately enhanced (∼2−6 fold) potency against TNNI3K, and substantially improved activity (∼10−50fold) against B-Raf. The C8-Ph substituent projects into the front pocket, where residue differences between TNNI3K and B-Raf (e.g., Gly545 vs Ser535, Ser549 vs His539) may give rise to the divergent behavior of C8-substituents in binding to B-Raf and TNNI3K (Figure 8). As with the C7-Ph compound (27), the C8-Ph compound (36) displayed inferior pharmacokinetics (poDNAUC = 0.01 h-kg/L) that could be partially rescued via 4-substitution (e.g., 40, poDNAUC = 0.14 h-kg/L). The pyrimidoindole variant 44 was also evaluated, but it provided no advantage over 7, as it had little effect on binding, selectivity,

Figure 9. Effect of C4-benzenesulfonamide substituents.

modulated by C4 groups in a manner that correlates with substituent electronic properties (7, 45−47 IC50: Cl (20 nM) > H (80 nM) > Me (630 nM) > NHMe (6300 nM)), with strongly electron-donating groups proving especially detrimental to activity (e.g., 47, 80-fold reduction vs 7). Given that the C4-substituents are anticipated to project into a spacious portion of the binding pocket (Figure 2), where they are unlikely to introduce either beneficial or repulsive interactions with TNNI3K, the C4-substituents may elicit a remote electronic effect on key binding elements such as the Hbonding network formed by the sulfonamide functionality or the hydrophobic interactions of the benzene with nearby residues (e.g., Ala605). Of particular interest in light of the observed C4-substituent electronic effect, replacement of the N-methylamino substituent 47 (IC50 = 6300 nM) with the N,N-dimethylamino group (48, IC50 = 50 nM) led to a substantial (∼100-fold) improvement in binding affinity for TNNI3K, and this result was confirmed through evaluation of the related morpholine-bearing compound 49, which is also highly active (IC50 = 40 nM). This dissimilarity in TNNI3K binding behavior highlights a key stereoelectronic difference in ortho-substituted monoalkyl anilines vs ortho-substituted dialkyl anilines (Figure 10).14 Monoalkyl anilines such as 47 adopt a coplanar orientation wherein the nitrogen lone pair resides in a p-orbital parallel to the π-system of the adjacent aryl ring to maximize resonance stabilization. However, this conformation is unavailable to dialkyl aniline 48 as its additional methyl group would

Figure 10. Conformations of anilines 47 and 48. E

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

introduce a severe steric interaction with the ortho-NH group in the coplanar orientation. As a result the dimethyl aniline group of 48 must adopt a twisted conformation wherein its methyl groups move out of the plane of the neighboring NH group and its nitrogen lone pair orients perpendicular to the adjacent aryl π-system, thereby disrupting the N-aryl resonance. Thus, in the twisted conformation the strong resonance electron-donating character of the aniline nitrogen is neutralized, resulting in a significant electronic divergence between the C4-substituents of 47 and 48 that is reflected in their TNNI3K binding affinity. Dialkyl anilines 48 (Cl = 30 mL/min/kg) and 49 (Cl = 39 mL/min/kg) exhibited improved in vivo clearance compared to 7 (Cl = 68 mL/min/kg, F = 34%) demonstrating the utility of C4-substituents for modulating pharmacokinetics in the series, presumably via blocking of oxidative metabolism in the aryl ring system. The N,N-dimethylamino substituent was particularly valuable, as it also led to increased aqueous solubility (330 μM for 48 vs 50 μM for 7) and oral bioavailability (F = 100% for 48 vs 34% for 7), leading to an overall 7-fold improvement in oral exposure (poDNAUC = 0.59 h-kg/L) compared to 7. Deazapurine 48 also demonstrated good activity in a TNNI3K cellular assay (IC50 = 50 nM) and offers an enhanced selectivity profile, showing >100-fold selectivity against 95% (175/185) and >10-fold selectivity against 99% (184/185) of kinases tested in the Millipore KinaseProfiler panel (Millipore.com, see Supporting Information for details), with B-Raf and c-Raf being the only kinases significantly inhibited at 1 μM compound concentration. Given its good pharmacokinetic profile and highly specific inhibition of the structurally related TNNI3K, B-Raf, and c-Raf kinases, 48 represents an advanced lead for further development (Figure 11).

interdependency of the C4 and C7 substituents was anticipated from the structure of 1 bound to TNNI3K (Figure 2) as it suggested that the C4 and C7 substituents would project toward one another, and indeed, a subsequent crystal structure of 53 bound to TNNI3K confirmed the proximity (∼4 Å) of the C4 and C7 substituents in the TNNI3K binding site (Figure 13D). Analogues which bear a C4-morpholino group show a significant cellular activity enhancement (∼10-fold) in combination with the C7-Me (52) and C7−Br (53) modifications but to a lesser extent than the C4-NMe2 compounds, suggesting a reduced complementarity between the C4-morpholine moiety and the C7-substituents. Although multiple energetic factors (e.g., electronic, conformational, desolvation, hydrophobic effect) may be in play, a comparison of the molecular surface areas of 1, 26, 49, and 53 bound to TNNI3K (Figure 13) reveals that improved hydrophobic packing is a likely source of the synergistic effect. By contrast to the monosubstituted analogues, which are expected to make only isolated hydrophobic interactions with TNNI3K through either their C7−Br or C4-NR2 functional groups, the C7−Br and C4-NC4H8O groups of the disubstituted 53 form a large, contiguous lipophilic surface that forms multiple points of contact with Leu595 that resides at the bottom of the pocket as well as the Val477 and Ile469 side chains that lie above the inhibitor. Moreover, the C7−Br and C4-NC4H8O groups engage in an internal lipophilic interaction that may rigidify 53 into a molecular architecture that is highly complementary to the shape of the TNNI3K pocket. The C8−Br or C8−3-CF3−Ph substitutions had only a small effect on binding affinity (≤4-fold) when evaluated on either of the C4-dialkylamine templates (54−57). These results are similar to those found in the absence of a C4-substituent (Figure 7, compds 35, 40) and consistent with the structural evidence demonstrating the large distance between C4 and C8 groups upon TNNI3K binding. Overall, these results confirm a cooperative effect between C4 and C7 substituents leads to the exceptional activity of 50 and 51, which exhibit >1000-fold increase in cellular activity compared to purine 1 (cellular IC50 = 1300 nM). Chemistry. Purine 1 and analogues were assembled via substitution of chloro-heterocycles with appropriate anilines, and this operation was accomplished through one of several alternative methods (Scheme 1). In instances requiring the purine scaffold, the desired products (1−5, 11−17) were obtained by heating a mixture of the commercially available 6chloropurines and readily accessible anilines in isopropanol (Method A: 58, 59, isopropanol, 80 °C, 16 h or μwave, 150 °C, 30 min, 13−91%).15 In general, the 7-deazapurine analogues (7, 8, 18, 20, 21, 35, 43−45, 47−49, 54, 56) did not react well in refluxing isopropanol but could be accessed under AgOTfmediated coupling conditions (Method B: 59, 60, AgOTf (1 equiv), DMF, 80 °C, 16 h or AgOTf (1 equiv), μwave, 120 °C, 30 min, 3−86%).16 However, the 7-halo-7-deazapurines (24− 26, 51, 53) proved to be more active substrates and were successfully synthesized using Method A (8−53%), which also was sufficient to access some 8-aryl variants (55, 57, 13− 23%).17 Formation of pyrazolopyrimidine 6 and pyrrolopyridine 9 required prolonged reaction times in refluxing isopropanol (5 days, 8% for 6) but the reactions could be accelerated through acid catalysis (Method C: 59, 61, 1 M aq HCl (1 equiv), isopropanol, μwave, 150 °C, 3 h, 45% for 9).15 Notably, 9 was also synthesized by way of the Hartwig− Buchwald coupling method, thereby demonstrating that the 7-

Figure 11. Comparison of leads 7 and 48. Kinase profiling details are available in Supporting Information.

Combined C4-Benzenesulfonamide and C7-/C8-Deazapurine Substitutions. Given the desirable properties of the advanced lead 48, we examined the effect of C7- and C8deazapurine substitutions in combination with either the C4dimethylamino or C4-morpholino group (Figure 12). Of interest, analogues bearing the C4-NMe2 functionality in addition to either the C7-Me (50) or C7−Br group (51) showed high affinity for TNNI3K (IC50 < 10 nM), which translated into exceptional cellular potency against TNNI3K (IC50 ∼ 1 nM). The ∼50-fold improvement in cellular activity is indicative of a synergistic effect between the C4 and C7 substituents as the C7−Br and C7-Me substitutions produced only ∼2 to 3-fold improvement in TNNI3K binding in the absence of the C4 substituent (Figure 7, compds 21, 26). The F

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

Figure 12. Disubstituted 7-deazapurines. Compound IC50 values less than 10 nM could not be accurately obtained due to titration of TNNI3K in the enzyme assay.

Scheme 1

Figure 13. Comparison of 1 (A), 26 (B), 49 (C), and 53 (D) bound to TNNI3K.

azaindole does not rearrange under Method C, as might be expected based on literature reports.18 Method C also proved useful as an alternative approach to prepare some substituted 7deazapurines (46, 50, 52, 10−20%). Finally, the synthesis of Olinked 7-deazapurine 19 was accomplished through a basemediated substitution reaction (Method D: 60, 62, Cs2CO3, DMF, μwave, 180 °C, 1 h, 4%) that was not useful for 3-aminobenzenesulfonamide couplings as the sulfonamide preferentially reacted instead of the aniline nitrogen under these conditions. Postcoupling transformations of anilino-deazapurine compounds provided additional analogues of interest (Scheme 2). For instance, 10 was synthesized from 7 by way of oxidative bromination (pyridinium bromide perbromide, t-BuOH, 25 °C, 1h) followed by a zinc reduction (Zn dust, AcOH, 25 °C, 15 min, 25%, 2 steps).16 Suzuki couplings19 employing bromide 26 were only partly successful, producing some of the targeted biaryl compounds (28, 29, 31) in low yields (6−15%). Protodebromination was the predominate pathway in these reactions but could be avoided by masking the 7-deazapurine N9−H atom with an electron-withdrawing functionality. Thus, 26 was converted to the di-Boc derivative 63 (Boc2O, DMAP, MeCN, 25 °C, 1 h, 53%) and Suzuki reactions (63, ArB(OH)2, Pd(dppf)Cl2, aq Na2CO3, dioxane, 100 °C, 1 h) from this

template followed by prolonged heating of the reaction mixture to achieve Boc removal (100 °C, 16 h) afforded a wider range of products (27, 30, 32−34) in modest yields (11−54%). When using 63 as substrate, the conversions to product were high under Suzuki conditions, but often separation of the desired product from the reaction byproducts was challenging, leading to low yields. Cross coupling reactions (35, ArB(OH)2, Pd(dppf)Cl2 or Pd(PPh3)4, aq Na2CO3, dioxane, μwave, 120 G

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

Scheme 2

Scheme 4

°C, 15 min) from 8-bromo-7-deazapurine 35 were also successful using a variety of boronic acid substrates, though variable yields were observed (3−51%). 7-Alkyl-7-deazapurines 22 and 23 were prepared via reduction of their corresponding alkene functional groups, which could be readily installed onto the 7-deazapurine core (Scheme 3). In the case of 22, a cross coupling employing isopropenylboronic acid pinacol ester under the previously established conditions (63, Pd(dppf)Cl2, aq Na2CO3, dioxane, 100 °C, 15 min, 17%) produced the mono-Boc, isopropenyl derivative 64. In this instance, the long reaction times normally used to effect Boc removal produced complex mixtures. However, following hydrogenation (H2, Pd/C, MeOH, 25 °C, 16 h, 80%) of the alkene, Boc removal was easily accomplished under acidic conditions (HCl, dioxane, MeCN, 25 °C, 16 h, 97%) to give 22. The alkene precursor of 23 was installed on the 6-chloro-7-deazapurine core prior to aniline coupling (Scheme 4), and with this building block (65) in hand, 23 was assembled by way of a routine aniline substitution (AgOTf, DMF, 80 °C, 10 h) followed by hydrogenation (H2 (30 psi), Pd/C, MeOH, 25 °C, 16 h, 16%, 2 steps) (Scheme 3).

The chloro-heterocycle and aniline building blocks required to assemble each analogue were either commercially available, synthesized according to literature reports,16,20−25 or prepared by an original synthesis (Scheme 4, 5). Halogen metal exchange (n-BuLi, THF, −78 °C, 1 h) on 67 followed by alkylation with 3-bromo-2-methylpropene (THF, −78 to 0 °C, 30 min, 17%) produced 65 in a procedure analogous to that reported for the synthesis of the 6-chloro-7-Me-7-deazapurine.20 The 6-chloro8-bromo-7-deazapurine 69 was prepared via a known three-step sequence of benzenesulfonylation (NaH, PhSO2Cl, 0 to 25 °C, 4 h, 98%), bromination (LDA, BrCl2CCCl2Br, −78 °C, 2 h, 61%), and hydrolysis (KOt-Bu, THF, 25 °C, 4 h, 34%).26 The intermediate N-protected bromide 68 proved useful as a cross coupling reagent and was subjected to Suzuki conditions (3CF3−PhB(OH)2, Pd(dppf)Cl2, aq K2CO3, dioxane, 80 °C, 3 h, 47%) with subsequent sulfonamide hydrolysis (KOt-Bu, THF, 0 to 25 °C, 30 min, 99%) to give biaryl 70. Finally, the 7-CO2tBu derivative 72, which was utilized to access carboxylic acid analogue 43 was synthesized via condensation of glycine tertbutyl ester and 71 in a two-step procedure (Et3N, EtOH, 25 °C, 24 h; NaH, DMF, 0 °C, 1 h, 25%) adapted from a similar sequence developed for a related 7-deazapurine compound.27

Scheme 3

H

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

substantial reductions in activity (∼10−100-fold). These results were corroborated by an X-ray structure of 1 bound to TNNI3K, which revealed a novel coplanar binding mode consisting of bidentate hinge-recognition by the purine coupled with an elaborate hydrogen bonding network for the sulfonamide group. Notably, the sulfonamide serves as a hydrogen bond donor to the TNNI3K gatekeeper residue (Thr539) and presumably contributes to the broad spectrum kinase selectivity of the series.31 The 7-deazapurine 7 was identified as a superior lead compound vs purine 1 as it demonstrated improved potency and pharmacokinetics, and elaboration of this template by way of C4, C7, and C8substitutions produced potent (10-fold selectivity vs 97% of kinome) inhibitors, some of which (e.g., 48) display good pharmacokinetics (poDNAUC = 0.59 h-kg/L) and excellent selectivity (>100-fold selectivity vs 95% of kinome), showing appreciable activity at only the structurally related Raf kinases. Other derivatives (e.g., 51) exhibited exceptional potency improvements (>1000-fold) over 1 and displayed subnanomolar activity against TNNI3K in a cellular context (IC50 = 0.7 nM). Thus, anilino-7-deazapurine TNNI3K inhibitors suitable as in vitro and in vivo biological tools have been discovered, and these compounds along with the structure−function results established herein serve as the foundation for efforts ultimately leading to the definition of the fundamental cardiac biology of TNNI3K.3

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Scheme 5



Several benzenesulfonamide intermediates which lacked literature precedent were prepared through standard methods (Scheme 5). Methylation of 73 (H2CO, AcOH, NaBH(OAc)3, CH2Cl2, 25 °C, 15 min, 40%)28 proceeded smoothly to give NMe aniline 74 under conditions that selectively produce the monomethylated aniline vs dimethylated aniline species (4:1). 3-Hydroxy-N-Me-benzenesulfonamide 62 was synthesized from the commercially available sulfonyl chloride 75 via sequential sulfonamide formation (MeNH2, DMAP, THF, 25 °C, 30 min, 79%) and demethylation (BBr3, CH2Cl2, 0 to 25 °C, 2 h, 96%). Amino-pyridyl intermediate 79 was obtained by modification of a reported synthesis of related amino-sulfonamide pyridines.29 Thus, bromination of 76 (Br2, 130 °C, 8 h) and subsequent sulfonamide formation (CH3NH2, H2O, 0 to 25 °C, 3 h, 17%, 2 steps) afforded 78, which successfully underwent a coppercatalyzed substitution reaction (NH4OH, CuCl, sealed tube, 130 °C, 18 h, 61%) to install the aniline functionality.29 Chlorosulfonylation of 2-fluoronitrobenzene 80 (HSO3Cl, 100 °C, 18 h, 65%) to provide 81 followed by sulfonamide formation (MeNH2.HCl, Et3N, THF, −35 °C, 1 h, 90%) at low temperatures gave 82, which was utilized to access a variety of C4-substituted anilines 83.30 Addition of the appropriate amines (MeNH2, Me2NH, morpholine) to 82 (R1R2NH, Et3N, THF, 25 °C, 1 h, 90−98%)30 and subsequent reduction of the nitro group (H2, Pd/C, THF, 67−89%) delivered 3,4diaminobenzenesulfonamides 83 in good yields (Scheme 5).

EXPERIMENTAL SECTION

General Experimental. The purity of each inhibitor was determined to be >95% (except as noted) on an Agilent 1100 HPLC equipped with a Sunfire C18 5.0 μm column (3.0 mm × 50 mm) using a gradient of 10% to 100% MeCN/H2O/0.05% TFA at 1 mL/min flow rate with detection at 220 and 254 nm. Mass determinations were conducted using an Agilent 6110 Quadrupole MS with positive ESI. Preparative HPLC was conducted on a Waters 2525 system at a flow rate of 50 mL/min with 254 nm detection using either a Sunfire C18 OBD 5 μm column (30 × 150 mm) with a gradient of MeCN/H2O/0.1% TFA or an XBridge C18 OBD 5 μm column (30 × 150 mm) with a gradient of MeCN/aq NH4OH pH 10. Flash column chromatography was conducted on silica gel eluting with EtOAc-hexanes, EtOAc-petroleum ether or MeOH−CH2Cl2 mixtures. The following intermediates were synthesized following literature procedures: 4-chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidine,16 4chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine,20 4-chloro-5-fluoro7H-pyrrolo[2,3-d]pyrimidine, 21 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine,22 5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine,23 4chloro-9H-pyrimido[4,5-b]indole,24 6-bromo-4-chloro-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidine (68),26 6-bromo-4-chloro-7Hpyrrolo[2,3-d]pyrimidine (69),26 3-amino-N-hydroxy-benzenesulfonamide,24 and 3-amino-4-chloro-N-methylbenzenesulfonamide.25 Other required reagents and building blocks were either purchased and used as is or synthesized as described below.32 Method A. General Coupling Method for Anilines and ChloroPurines or Chloro-7-Deazapurines. A mixture of a 6-chloropurine or 6-chloro-7-deazapurine (i.e., 4-chloro-7H-pyrrolo[2,3-d]pyrimidine) and a 3-aminobenzenesulfonamide (1.5 equiv) in isopropanol (2 mL) was stirred at 80 °C for 16 h or subjected to microwave irradiation (150 °C) for 30 min before being cooled to room temperature. The solid was collected by filtration, washed with MeOH, and dried to afford products that were analytically pure (1−5, 11, 14, 15, 25, 51, 53) or else were subjected to preparative HPLC to give analytically pure solids (12, 13, 16, 17, 24, 26, 55, 57). Method B. General Coupling Method for Anilines and Chloro-7Deazapurines. The indicated 6-chloro-7-deazapurine, the required 3amino-benzenesulfonamide (1.3 equiv), and AgOTF (1 equiv) were dissolved in DMF (2 mL) and stirred at 80 °C overnight or were dissolved in i-PrOH (2 mL) and subjected to microwave irradiation



CONCLUSIONS A series of anilino-purines and anilino-7-deazapurines arising from 1 (Figure 1) were prepared and evaluated as inhibitors of TNNI3K. Fundamental structure−activity relationships were conducted for this initial series of Type I TNNI3K inhibitors and used to define the role of each element of 1 for TNNI3K recognition. The essential functions of the purine heterocycle, the sulfonamide moiety, and their linking N atom were established through multiple modifications that led to I

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

(120−150 °C) for 60 min before being diluted with MeOH, and filtered. Analytically pure solids were obtained after purification by reverse-phase HPLC (7, 8, 18, 20, 21, 47, 48, 49), flash chromatography (35, 43, 54, 56), precipitation (44), or SCX cation exchange (45). Method C. General Coupling Method for Anilines and ChloroHeterocycles. A mixture of the indicated chloro-heterocycle and the required 3-aminobenzenesulfonamide (1.5 equiv) in i-PrOH (2 mL) was treated with 1 M aqueous HCl (1 equiv) and subjected to microwave irradiation (150 °C) for 60 min before being filtered and subjected to reverse phase HPLC to give analytically pure products (6, 9, 46, 50, 52). N-Methyl-3-(1H-purin-6-ylamino)benzenesulfonamide (1). Compound 1 was prepared from 6-chloropurine and N-methyl 3aminobenzenesulfonamide using Method A as a yellow solid in 81% yield (80 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.34 (br. s, 1 H, aniline-NH), 8.53 (s, 1 H, purine C2-H), 8.48 (s, 1 H, purine C8H), 8.41 (s, 1 H, benzene C2-H), 8.17 (dd, J = 8.06, 1.26 Hz, 1 H, benzene C4-H), 7.57 (t, J = 7.93 Hz, 1 H, benzene C5-H), 7.48 (m, 2 H, benzene C6-H, sulfonamide-NH)), 2.48 (d, J = 4.78 Hz, 3 H, sulfonamide−CH3). MS (m/z) 305.0 (M + H+). N-Methyl-3-((9-methyl-9H-purin-6-yl)amino)benzenesulfonamide (2). Compound 2 was prepared from 6-chloro9-methyl-9H-purine and N-methyl 3-aminobenzenesulfonamide using Method A as a white solid in 50% yield (76 mg): 1H NMR (400 MHz, CD3OD) δ ppm 8.49 (m, 2 H), 8.22 (m, 1 H), 7.92 (m, 1 H), 7.82 (m, 1 H), 7.73 (m, 1 H), 3.97 (s, 3 H), 2.61 (s, 3 H). MS (m/z) 319.1 (M + H+). 3-((2-Fluoro-9H-purin-6-yl)amino)-N-methylbenzenesulfonamide (3). Compound 3 was prepared from 6-chloro-2-fluoro-9H-purine and N-methyl 3-aminobenzenesulfonamide using Method A as an off-white solid in 23% yield (27 mg, 90% purity): 1H NMR (400 MHz, DMSOd6) δ ppm 10.62 (s, 1H), 8.38 (s, 1H), 8.32 (s, 1H), 8.09 (dd, J = 1.3, 8.0 Hz, 1H), 7.60 (dd, J = 7.8, 8.0 Hz, 1H), 7.47 (m, 2H), 2.49 (d, J = 4.8 Hz, 3H). MS (m/z) 323.0 (M + H+). 3-((2-Amino-9H-purin-6-yl)amino)-N-methylbenzenesulfonamide (4). Compound 4 was prepared from 6-chloro-9H-purin-2-amine and N-methyl 3-aminobenzenesulfonamide using Method A as a white solid in 15% yield (15 mg): 1H NMR (400 MHz, CD3OD) δ ppm 8.37 (m, 1H), 8.19 (s, 1H), 8.11 (m, 1H), 7.6−7.7 (m, 2H), 2.60 (s, 3H). MS (m/z) 320.1 (M + H+). 3-((2-Chloro-9H-purin-6-yl)amino)-N-methylbenzenesulfonamide (5). Compound 5 was prepared from 2,6-dichloro-9H-purine and N-methyl 3-aminobenzenesulfonamide using Method A as a tan solid in 91% yield (342 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.89 (s, 1H), 8.59 (s, 1H), 8.40 (m, 1H), 8.08 (m, 1H), 7.61 (dd, J = 8.0, 8.1 Hz, 1H), 7.49 (m, 2H), 2.51 (s, 3H). MS (m/z) 338.9 (M + H+). 3-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (6). Compound 6 was prepared from 4-chloro-1Hpyrazolo[3,4-d]pyrimidine and N-methyl 3-aminobenzenesulfonamide using Method C as a white solid in 8% yield (112 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 13.75 (s, 1H), 10.33 (s, 1H), 8.47 (s, 1H), 8.36 (s, 1H), 8.32 (s, 1H), 8.29 (d, J = 8.3 Hz, 1H), 7.63 (dd, J = 7.8 Hz, 8.0 Hz, 1H), 7.50 (m, 2H), 2.48 (d, J = 5.0 Hz, 3H). MS (m/z) 305.0 (M + H+). 3-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzenesulfonamide (7). Compound 7 was prepared from 4-chloro-7Hpyrrolo[2,3-d]pyrimidine and N-methyl 3-aminobenzenesulfonamide using Method B as a white solid in 80% yield (330 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.23 (s, 1H, pyrrolopyrimidine-NH), 10.21 (s, 1H, aniline-NH), 8.40 (s, 1H, pyrrolopyrimidine C2-H), 8.23 (s, 1H, benzene C2-H), 8.19 (d, J = 8.0 Hz, 1H, benzene C4-H), 7.64 (dd, J = 7.9, 8.0 Hz, 1H, benzene C5-H), 7.52 (d, J = 7.9 Hz, 1H, benzene C6-H), 7.50 (m, 1H, sulfonamide-NH), 7.38 (dd, J = 2.5, 3.0 Hz, 1H, pyrrolopyrimidine-C6-H), 6.84 (dd, J = 1.5, 3.0 Hz, 1H, pyrrolopyrimidine-C5-H), 2.48 (d, J = 4.8 Hz, 3H, sulfonamide− CH3). MS (m/z) 304.0 (M + H+). N-Methyl-3-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzenesulfonamide (8). Compound 8 was prepared from 4-chloro7-methyl-7H-pyrrolo[2,3-d]pyrimidine and N-methyl 3-aminobenzenesulfonamide using Method B as a white solid in 86% yield (111 mg,

92% purity): 1H NMR (400 MHz, CD3OD) δ ppm 8.35 (s, 1 H), 8.18 (s, 1 H), 7.91 (d, J = 7.5 Hz, 1 H), 7.79 (d, J = 8.1 Hz, 1 H), 7.72 (dd, J = 7.5, 8.1 Hz, 1 H), 7.39 (d, J = 3.51 Hz, 1 H), 6.77 (d, J = 3.51 Hz, 1 H), 3.91 (s, 3 H), 2.62 (s, 3 H). MS (m/z) 318.0 (M + H+). 3-((1H-Pyrrolo[2,3-b]pyridin-4-yl)amino)-N-methylbenzenesulfonamide (9). Compound 9 was prepared from 4-chloro-1H-pyrrolo[2,3-b]pyridine and N-methyl 3-aminobenzenesulfonamide using Method C as a white solid in 19% yield (13 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.39 (br. s., 1 H), 10.28 (s, 1 H), 8.11 (d, J = 7.03 Hz, 1 H), 7.76 (s, 1 H), 7.65−7.75 (m, 3 H), 7.61 (q, J = 5.02 Hz, 1 H), 7.43−7.47 (m, 1 H), 6.81−6.87 (m, 2 H), 2.48 (d, J = 5.02 Hz, 3 H). MS (m/z) 303.0 (M + H+). N-Methyl-3-((6-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzenesulfonamide (10). A solution of 7 (100 mg, 0.330 mmol) in tert-butanol (20 mL) was treated with pyridine hydrobromide perbromide (211 mg, 0.659 mmol), and the mixture was stirred at 25 °C for 1 h before being poured into water and extracted with EtOAc. The organic extracts were washed (H2O, saturated aqueous NaCl), dried (Na2SO4), and concentrated to give a yellowbrown solid. The residue was dissolved in AcOH (15 mL), treated with zinc dust (300 mg, 4.59 mmol), and the mixture was stirred for 15 min before being filtered, concentrated, and subjected to reverse phase HPLC (10−50% MeCN/water/0.1%TFA) to give 10 (12 mg, 11%) as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 11.13 (s, 1 H), 9.24 (s, 1 H), 8.36 (s, 1 H), 8.12 (t, J = 1.88 Hz, 1 H), 8.07 (dd, J = 8.03, 1.25 Hz, 1 H), 7.54 (t, J = 8.03 Hz, 1 H), 7.44 (q, J = 5.02 Hz, 1 H), 7.38 (d, J = 8.28 Hz, 1 H), 2.55 (s, 2 H), 2.45 (d, J = 5.02 Hz, 3 H). MS (m/z) 320.0 (M + H+). 3-((9H-Purin-6-yl)amino)-N-ethylbenzenesulfonamide (11). Compound 11 was prepared from 6-chloropurine and 3-amino-Nethylbenzenesulfonamide using Method A as a pale yellow solid in 89% yield (94 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.10 (br. s, 1H), 10.02 (br. s, 1H), 8.37 (d, J = 2.01 Hz, 1H), 8.27 (br. s, 1H), 8.20 (d, J = 6.78 Hz, 1H), 7.54−7.67 (m, 2H), 7.48 (d, J = 7.28 Hz, 1H), 7.29−7.40 (m, 1H), 6.83 (dd, J = 1.88, 3.39 Hz, 1H), 2.74−2.93 (m, 2H), 1.01 (t, J = 7.28 Hz, 3H). MS (m/z) 318.1 (M + H+). 3-((9H-Purin-6-yl)amino)benzenesulfonamide (12). Compound 12 was prepared from 6-chloropurine and 3-aminobenzenesulfonamide using Method A as an off-white solid in 44% yield (42 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 13.25 (br. s., 1 H, purine N9-H), 10.13 (br. s., 1 H, aniline-NH), 8.61 (br. s., 1 H, purine C2-H), 8.43 (s, 1 H, purine C8-H), 8.34 (s, 1 H, benzene-C2-H), 8.09 (dt, J = 7.74, 1.79 Hz, 1 H, benzene C4-H), 7.44−7.56 (m, 2 H, benzene C5-H, C6H), 7.36 (s, 2 H, SO2NH2). MS (m/z) 291.1 (M + H+). 3-((9H-Purin-6-yl)amino)-N-hydroxybenzenesulfonamide (13). Compound 13 was prepared from 6-chloropurine and 3-amino-Nhydroxybenzenesulfonamide24 using Method A as a white solid in 24% yield (35 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.22 (s, 1 H, aniline-NH), 9.60 (s, 2 H, SO2NHOH), 8.61 (br. s., 1 H, purine C2H), 8.44 (s, 1 H, purine C8-H), 8.36 (s, 1 H, benzene C2-H), 8.22 (d, J = 8.0 Hz, 1 H, benzene C4-H), 7.58 (dd, J = 7.8, 8.0 Hz, 1 H, benzene C5-H), 7.50 (d, J = 7.8 Hz, 1 H, benzene C6-H). MS (m/z) 307.0 (M+H+). The structure of 13 was further confirmed by its successful conversion to 3-((9H-purin-6-yl)amino)benzenesulfinic acid. Thus, 13 (3 mg) was dissolved in 0.5 mL of 1N NaOH and heated at 80 °C for 5 min to give a yellow solution which consisted of 3-((9H-purin-6-yl)amino)benzenesulfinic acid (100%) based on LCMS analysis: MS (m/z) 276.0 (M + H+). 3-((9H-Purin-6-yl)amino)-N,N-dimethylbenzenesulfonamide (14). Compound 14 was prepared from 6-chloropurine and 3-amino-N,Ndimethylbenzenesulfonamide using Method A as a white solid in 77% yield (160 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.06 (br. s., 1 H), 8.63 (br. s., 2 H), 8.45 (t, J = 1.76 Hz, 1 H), 8.26 (dd, J = 8.16, 1.13 Hz, 1 H), 7.66 (t, J = 7.91 Hz, 1 H), 7.46 (d, J = 7.28 Hz, 1 H), 2.67 (s, 6 H). MS (m/z) 319.1 (M + H+). N-(3-(Ethylsulfonyl)phenyl)-9H-purin-6-amine (15). Compound 15 was prepared from 6-chloropurine and 3-(ethylsulfonyl)aniline using Method A as a pale yellow solid in 64% yield (66 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.06 (br. s., 1 H), 8.65 (d, J = 2.52 Hz, 2 H), 8.59 (t, J = 1.76 Hz, 1 H), 8.23−8.28 (m, 1 H), 7.68 (dd, J = 7.8, J

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

A mixture of 63 (500 mg, 0.86 mmol), Pd(dppf)Cl2−CH2Cl2 (250 mg, 0.34 mmol), and isopropenylboronic acid pinacol ester (430 mg, 2.6 mmol) in 1,4-dioxane (12 mL) was treated with 0.4 M aq Na2CO3 (8.6 mL, 3.4 mmol), purged with nitrogen, and stirred at 100 °C for 15 min before being treated with water and extracted with EtOAc. The organic extract was washed with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (50% EtOAc/ hexanes) to give 64 (65 mg, 17%) as a yellow solid following trituration with MeOH: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.08 (br. s, 1H), 8.45 (s, 1H), 8.42 (t, J = 1.88 Hz, 1H), 8.37 (s, 1H), 8.01 (dd, J = 1.38, 8.16 Hz, 1H), 7.56−7.62 (m, 1H), 7.45−7.52 (m, 2H), 5.25 (s, 1H), 5.06 (s, 1H), 3.31 (s, 3H), 2.21 (s, 3H), 1.25 (s, 9H); MS (m/z) 443.9 (M + H+). N-Methyl-3-{[5-(1-methylethyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}benzenesulfonamide hydrochloride (22). A solution of 64 (65 mg, 0.15 mmol) in MeOH (25 mL) was treated with 10% Pd/C (60 mg, 0.056 mmol) and stirred at 25 °C under a balloon of H2 overnight. After 16 h, the mixture was filtered, and the filtrate was concentrated to give tert-butyl (3-((5-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)sulfonyl(methyl)carbamate (52 mg, 80%) as a gray solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 11.67 (br. s, 1H), 8.45 (br. s, 1H), 8.32−8.36 (m, 1H), 8.27 (s, 1H), 8.08 (dd, J = 1.63, 7.65 Hz, 1H), 7.58 (t, J = 8.03 Hz, 1H), 7.46−7.52 (m, 1H), 7.08 (s, 1H), 3.31 (s, 3H), 1.27 (s, 3H), 1.26 (s, 3H), 1.24 (s, 9H); MS (m/z) 446 (M + H+). tert-Butyl (3-((5-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)sulfonyl(methyl)carbamate (52 mg, 0.117 mmol) in CH3CN (5 mL) at 25 °C was treated with HCl (0.5 mL of a 4 M solution in 1,4-dioxane, 2.00 mmol) and stirred overnight before being concentrated to give an oil. The oil was dissolved in MeOH and filtered, and the filtrate was concentrated to give 22 (48 mg, 97%) as a pale yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.26 (br. s, 1H), 9.18 (br. s, 1H), 8.31 (s, 1H), 8.05 (s, 1H), 7.91 (d, J = 7.78 Hz, 1H), 7.49−7.71 (m, 3H), 7.24 (br. s, 1H), 3.56−3.69 (m, 1H), 2.48 (s, 3H), 1.28 (d, J = 6.53 Hz, 6H). MS (m/z) 346.0 (M + H+).4-Chloro5-(2-methyl-2-propen-1-yl)-1H-pyrrolo[2,3-d]pyrimidine (65) N-Methyl-3-{[5-(2-methylpropyl)-1H-pyrrolo[2,3-d]pyrimidin-4yl]amino}benzenesulfonamide Trifluoroacetate (23) A mixture of 5bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (67)21 (500 mg, 2.2 mmol) in THF (15 mL) at −78 °C was treated dropwise with n-BuLi (4.3 mL of 2.5 M in hexanes, 10.75 mmol). After 1 h, 3-bromo-2methyl-1-propene (2.89 g, 21.5 mmol) was added, and the mixture was stirred and allowed to warm to room temperature. After 30 min, the reaction was quenched with the addition of water (1 mL), and the mixture was concentrated and subjected to reverse phase HPLC (MeCN/H2O/0.1% TFA) to give 65 (100 mg, 22%) as a brown solid: 1 H NMR (400 MHz, DMSO-d6) δ ppm 12.33 (br. s, 1 H), 8.49 (s, 1 H), 7.43 (s, 1 H), 4.75 (br. s, 1 H), 4.44 (s, 1 H), 3.47 (s, 2 H), 1.73 (s, 3 H). MS (m/z) 208.0 (M + H+).N-Methyl-3-{[5-(2-methyl-2propen-1-yl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}benzenesulfonamide (66) A mixture of 65 (100 mg, 0.483 mmol) and 3-amino-Nmethylbenzenesulfonamide (135 mg, 0.726 mmol) in DMF (5 mL) was treated with AgOTf (124 mg, 0.483 mmol) and heated at 80 °C for 10 h before being cooled, diluted with water, and extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts were dried (Na2SO4) and concentrated to give 66 (150 mg, 87%) as a black oil: MS (m/z) 358 (M + H+). N-Methyl-3-{[5-(2-methylpropyl)-1H-pyrrolo[2,3-d]pyrimidin-4yl]amino}benzenesulfonamide Trifluoroacetate (23). A solution of 66 (150 mg, 0.42 mmol) in MeOH (20 mL) was treated with 10% Pd/C (100 mg) and stirred a 25 °C under 30 psi of H2 for 16 h before being filtered, concentrated, and subjected to reverse phase HPLC (MeCN/H2O/0.1% TFA) to give 23 (24 mg, 16%) as a brown solid: 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.83 (br. s, 1 H), 8.59 (br. s, 1 H), 8.26 (s, 1 H), 8.15 (s, 1 H), 7.91 (d, J = 9.30, 1 H), 7.57 (t, J = 7.91 Hz, 1 H), 7.45 (m, 2 H), 7.10 (s, 1 H), 2.82 (d, J = 7.03 Hz, 2 H), 2.47 (d, J = 5.02 Hz, 3 H), 1.80−1.87 (m, 1 H). MS (m/z) 360.2 (M + H+).

8.0 Hz, 1 H), 7.61 (d, J = 7.81 Hz, 1 H), 3.30 (q, J = 7.30 Hz, 2 H), 1.15 (t, J = 7.43 Hz, 3 H). MS (m/z) 304.0 (M + H+). 5-((9H-Purin-6-yl)amino)-N,2-dimethylbenzenesulfonamide (16). Compound 16 was prepared from 6-chloropurine and 5-amino-N,2dimethylbenzenesulfonamide using Method A as a yellow solid in 14% yield (48 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.17 (s, 1 H), 8.40−8.47 (m, 2 H), 8.37 (s, 1 H), 8.08 (dd, J = 8.28, 2.26 Hz, 1 H), 7.49 (q, J = 4.77 Hz, 1 H), 7.37 (d, J = 8.53 Hz, 1 H), 2.53 (s, 3 H), 2.51 (s, 3H). MS (m/z) 319.0 (M + H+). 3-((9H-Purin-6-yl)amino)-N,4-dimethylbenzenesulfonamide (17). Compound 17 was prepared from 6-chlorpurine and 3-amino-N,4dimethylbenzenesulfonamide using Method A as a yellow solid in 13% yield (43 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 9.82 (br. s., 1 H), 8.39 (s, 1 H), 8.35 (s, 1 H), 7.91 (d, J = 1.51 Hz, 1 H), 7.58 (dd, J = 1.8, 8.0 Hz, 1 H), 7.53 (d, J = 8.0 Hz, 1 H), 7.43 (q, J = 4.94 Hz, 1 H), 2.46 (d, J = 4.77 Hz, 3 H), 2.33 (s, 3 H). MS (m/z) 319.0 (M + H+). N-Methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzenesulfonamide (18). Compound 18 was prepared from 4chloro-7H-pyrrolo[2,3-d]pyrimidine and N-methyl-3-(methylamino)benzenesulfonamide (74) using Method B as a white solid in 18% yield (26 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.09 (br. s, 1 H), 8.40 (s, 1 H), 7.83−7.72 (m, 4 H), 7.58 (q, J = 4.94 Hz, 1 H), 2.43 (d, J = 5.02 Hz, 3 H). MS (m/z) 318.0 (M + H+). Method D. Synthesis of 3-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)oxy)N-methylbenzenesulfonamide (19). A mixture of 4-chloro-7Hpyrrolo[2,3-d]pyrimidine (41.0 mg, 0.267 mmol), 3-hydroxy-Nmethylbenzenesulfonamide (62) (50 mg, 0.267 mmol), and Cs2CO3 (174 mg, 0.534 mmol) in DMF (2 mL) was subjected to microwave irradiation (180 °C) for 60 min. The solid was removed by filtration, and the filtrate was diluted with MeOH and subjected to reverse phase HPLC (20−60% MeCN/water/0.1%TFA) to give 19 as an off-white solid (4 mg, 4%): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.32 (br. s., 1 H), 8.34 (s, 1 H), 7.68−7.74 (m, 2 H), 7.58−7.64 (m, 2 H), 7.52−7.58 (m, 2 H), 2.46 (d, J = 5.02 Hz, 3 H). MS (m/z) 305.0 (M + H+). 5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylpyridine-3sulfonamide (20). Compound 20 was prepared from 4-chloro-7Hpyrrolo[2,3-d]pyrimidine and 5-amino-N-methylpyridine-3-sulfonamide (79) using Method B as an orange solid in 6% yield (6 mg): 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.96 (br. s, 1H), 9.87 (s, 1H), 9.35 (d, J = 2.26 Hz, 1H), 8.86 (t, J = 2.26 Hz, 1H), 8.54 (d, J = 2.01 Hz, 1H), 8.39 (s, 1H), 7.74 (q, J = 4.94 Hz, 1H), 7.30−7.44 (m, 1H), 6.83 (dd, J = 1.76, 3.26 Hz, 1H), 2.50−2.55 (m, 3H). MS (m/z) 304.9 (M + H+). N-Methyl-3-[(5-methyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]benzenesulfonamide (21). Compound 21 was prepared from 4chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine20 and 3-amino-N-methylbenzenesulfonamide using Method B as a white solid in 3% yield (25 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.52 (br. s, 1H), 8.39 (s, 1H), 8.21 (s, 1H), 8.19 (t, J = 1.82 Hz, 1H), 7.98 (dd, J = 1.24, 8.13 Hz, 1H), 7.51 (t, J = 7.94 Hz, 1H), 7.35−7.45 (m, 2H), 6.98−7.06 (m, 1H), 2.46−2.54 (presumed 3H, obscured by DMSO peak), 2.44 (d, J = 5.02 Hz, 3H). MS (m/z) 318.1 (M + H+).1,1-Dimethylethyl 5bromo-4-[(3-{[{[(1,1-dimethylethyl)oxy]carbonyl}(methyl)amino]sulfonyl}phenyl)amino]-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (63) N-Methyl-3-{[5-(1-methylethyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}benzenesulfonamide hydrochloride (22) A mixture of 26 (10.0 g, 23.88 mmol) in CH3CN (200 mL) at 25 °C was treated with Boc2O (16.64 mL, 71.7 mmol) and DMAP (5.84 g, 47.8 mmol) and stirred for 15 min before being concentrated. The residue was dissolved in EtOAc and washed with water and brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (20% EtOAc/ hexanes) to afford a yellow solid, which was triturated with EtOAc to give 63 (7.5 g, 54%) as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.23 (s, 1H), 8.94 (s, 1H), 8.75−8.79 (m, 1H), 8.45−8.51 (m, 1H), 8.34 (s, 1H), 7.99−8.10 (m, 2H), 3.74 (s, 3H), 2.04 (s, 9H), 1.69 (s, 9H).1,1-Dimethylethyl methyl[(3-{[5-(1-methylethenyl)-1Hpyrrolo[2,3-d]pyrimidin-4-yl]amino}phenyl)sulfonyl]carbamate (64) K

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

N-Methyl-3-({5-[4-(methyloxy)phenyl]-1H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)benzenesulfonamide Trifluoroacetate (31). Compound 31 was prepared from 26 and 4-methoxyphenylboronic acid using Method E as a white solid in 11% (28 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.18 (br. s, 1H), 8.39 (s, 1H), 8.12 (s, 1H), 7.97 (br. s, 1H), 7.70−7.77 (m, 1H), 7.49−7.58 (m, 3H), 7.46 (q, J = 4.94 Hz, 1H), 7.35−7.43 (m, 2H), 7.07 (d, J = 8.53 Hz, 2H), 3.82 (s, 3H), 2.45 (d, J = 5.02 Hz, 3H). MS (m/z) 410.0 (M + H+). N-Methyl-3-{[5-(4-pyridinyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}benzenesulfonamide (32). Compound 32 was prepared from 63 and 4-pyridylboronic acid using Method E as a pale orange solid in 26% yield (26 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.42 (br. s, 1H), 8.54−8.61 (m, 3H), 8.41 (s, 1H), 8.07 (s, 1H), 7.86 (d, J = 8.28 Hz, 1H), 7.76 (s, 1H), 7.56 (d, J = 6.02 Hz, 2H), 7.51 (t, J = 7.91 Hz, 1H), 7.43 (q, J = 4.77 Hz, 1H), 7.37 (d, J = 7.78 Hz, 1H), 2.46 (d, J = 5.02 Hz, 3H). MS (m/z) 380.1 (M + H+). 3-({5-[4-(Aminomethyl)phenyl]-1H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)-N-methylbenzenesulfonamide (33). Compound 33 was prepared from 63 and 4-aminomethylphenylboronic acid using Method E as a tan solid in 54% yield (57 mg): 1H NMR (400 MHz, CD3OD) δ ppm 8.40−8.43 (m, 1H), 8.15−8.19 (m, 1H), 7.69− 7.75 (m, 1H), 7.52−7.63 (m, 4H), 7.46−7.50 (m, 2H), 7.30−7.33 (m, 1H), 3.95 (s, 2H), 2.55−2.59 (m, 3H). MS (m/z) 409.1 (M + H+). 4-[4-({3-[(Methylamino)sulfonyl] phenyl}amino)-1H-pyrrolo[2,3d]pyrimidin-5-yl]benzoic acid (34). Compound 34 was prepared from 63 and (4-methoxycarbonyl)phenylboronic acid using Method E as a brown solid in 41% yield (45 mg, 93% purity): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.89 (br. s, 1H), 12.28−12.37 (m, 1H), 8.40 (s, 1H), 8.31 (s, 1H), 8.14 (s, 1H), 7.97−8.04 (m, J = 8.28 Hz, 2H), 7.78 (dd, J = 1.38, 7.91 Hz, 1H), 7.67−7.72 (m, J = 8.53 Hz, 2H), 7.64 (d, J = 2.26 Hz, 1H), 7.50 (t, J = 7.91 Hz, 1H), 7.43 (q, J = 4.85 Hz, 1H), 7.36 (d, J = 8.03 Hz, 1H), 2.46 (d, J = 5.02 Hz, 3H). MS (m/z) 424.0 (M + H+). 3-[(6-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methylbenzenesulfonamide (35). Compound 35 was prepared from 6926 and 3-amino-N-methylbenzenesulfonamide using Method B as an offwhite solid in 53% yield (413 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.68 (br. s, 1H), 9.66 (s, 1H), 8.24−8.36 (m, 3H), 7.57 (t, J = 8.06 Hz, 1H), 7.45 (q, J = 4.95 Hz, 1H), 7.40 (d, J = 7.81 Hz, 1H), 6.92 (s, 1H), 2.46 (d, J = 5.04 Hz, 3H). MS (m/z) 382.0 (M + H+). N-Methyl-3-[(6-phenyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]benzenesulfonamide (36). Compound 36 was prepared from 35 and phenylboronic acid using Method E as a yellow solid in 38% yield (19 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.39 (s, 1H), 9.75 (s, 1H), 8.32−8.39 (m, 3H), 7.86 (d, J = 7.81 Hz, 2H), 7.54−7.61 (m, 1H), 7.32−7.54 (m, 5H), 7.25 (d, J = 1.76 Hz, 1H), 2.47−2.49 (m, 3H). MS (m/z) 380.0 (M + H+). N-Methyl-3-{[6-(2-methylphenyl)-1H-pyrrolo[2,3-d]pyrimidin-4yl]amino}benzenesulfonamide (37). Compound 37 was prepared from 35 and 2-methylphenylboronic acid using Method E as a tan solid in 7% yield (8 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.12 (br. s, 1H), 9.70 (s, 1H), 8.33−8.39 (m, 3H), 7.54−7.62 (m, 2H), 7.28−7.47 (m, 5H), 7.00 (s, 1H), 2.50 (2.47−2.54 presumed 3H, obscured by DMSO peak), 2.47 (d, J = 4.78 Hz, 3H). MS (m/z) 394.1 (M + H+). N-Methyl-3-{[6-(3-methylphenyl)-1H-pyrrolo[2,3-d]pyrimidin-4yl]amino}benzenesulfonamide (38). Compound 38 was prepared from 35 and 3-methylphenylboronic acid using Method E as a brown solid in 51% yield (40 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.35 (br. s, 1H), 9.74 (s, 1H), 8.27−8.44 (m, 3H), 7.62−7.75 (m, 1H), 7.58 (t, J = 7.81 Hz, 1H), 7.43−7.54 (m, 1H), 7.34−7.43 (m, 2H), 7.23−7.32 (m, 1H), 7.17 (d, J = 7.55 Hz, 1H), 7.03 (br. s, 1H), 2.48 (d, J = 4.78 Hz, 3H), 2.39 (s, 3H). MS (m/z) 394.1 (M + H+). N-Methyl-3-{[6-(4-methylphenyl)-1H-pyrrolo[2,3-d]pyrimidin-4yl]amino}benzenesulfonamide (39). Compound 39 was prepared from 35 and 4-methylphenylboronic acid using Method E as a tan solid in 17% yield (9 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.32 (br. s, 1H), 9.71 (s, 1H), 8.29−8.41 (m, 3H), 7.74 (d, J = 8.06 Hz, 2H), 7.58 (t, J = 8.06 Hz, 1H), 7.45 (q, J = 4.78 Hz, 1H), 7.39 (d, J = 7.81 Hz, 1H), 7.30 (d, J = 8.06 Hz, 2H), 7.18 (d, J = 2.01 Hz, 1H), 2.47 (d, J = 4.78 Hz, 3H), 2.35 (s, 3H). MS (m/z) 394.1 (M + H+).

3-[(5-Fluoro-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methylbenzenesulfonamide Trifluoroacetate (24). Compound 24 was prepared from 4-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine21 and 3-amino-N-methylbenzenesulfonamide using Method A as an white solid in 53% yield (68 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.81 (br. s, 1H), 9.25 (br. s, 1H), 8.30 (s, 1H), 8.26 (t, J = 1.76 Hz, 1H), 8.03−8.13 (m, 1H), 7.51−7.64 (m, 1H), 7.37−7.51 (m, 2H), 7.30 (t, J = 2.51 Hz, 1H), 2.47 (d, J = 4.77 Hz, 3H). MS (m/z) 322.0 (M + H+). 3-[(5-Chloro-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methylbenzenesulfonamide Hydrochloride (25). Compound 25 was prepared from 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine22 and 3amino-N-methylbenzenesulfonamide using Method A as an tan solid in 36% yield (72 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.25 (br. s, 1H), 8.62 (s, 1H), 8.33 (s, 1H), 8.27 (t, J = 1.76 Hz, 1H), 8.00− 8.06 (m, 1H), 7.55−7.61 (m, 1H), 7.54 (d, J = 2.76 Hz, 1H), 7.43− 7.49 (m, 2H), 2.47 (d, J = 5.02 Hz, 3H). MS (m/z) 322.0 (M + H+). 3-[(5-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methylbenzenesulfonamide Trifluoroacetate (26). Compound 26 was prepared from 5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine22 and 3-amino-N-methylbenzenesulfonamide using Method A as yellow solid in 17% yield (157 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.37 (br. s, 1H), 8.55 (s, 1H), 8.35 (s, 1H), 8.27 (t, J = 1.76 Hz, 1H), 7.98− 8.04 (m, 1H), 7.55−7.61 (m, 2H), 7.43−7.50 (m, 2H), 2.47 (d, J = 5.02 Hz, 3H). MS (m/z) 382.0 (M + H+). Method E. General Suzuki Coupling Method for Aryl Boronic Acids and 7- or 8-Bromo-7-Deazapurines.19 A mixture of the appropriate bromide (26, 35, or 63), aryl boronic acid (3 equiv), PdCl2(dppf)−CH2Cl2 (0.4 equiv) or Pd(PPh3)4 (0.2 equiv), and 0.4 M aqueous Na2CO3 (4 equiv) in 1,4-dioxane (5 mL) was heated at 100 °C for 16 h or subjected to microwave irradiation (160 °C) for 15 min before being diluted with water and extracted with EtOAc. The organic extract was washed (saturated aqueous NaCl), dried (Na2SO4), and concentrated to give pure products (34), or else the solids were subjected to flash chromatography (27, 32, 33, 36, 37, 39), preparative HPLC (28, 29, 30, 31, 40, 41) or SCX cation exchange (MeOH wash, NH3/MeOH elution, 38, 42) to give analytically pure solids. N-Methyl-3-[(5-phenyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]benzenesulfonamide Trifluoroacetate (27). Compound 27 was prepared from 63 and phenylboronic acid using Method E as a white solid in 45% yield (13 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.19 (br. s, 1H), 8.40 (s, 1H), 8.13 (t, J = 1.89 Hz, 1H), 7.95 (s, 1H), 7.72 (dd, J = 1.26, 8.06 Hz, 1H), 7.60 (d, J = 7.05 Hz, 2H), 7.46− 7.54 (m, 3H), 7.44 (br. s, 1H), 7.34−7.41 (m, 3H), 2.45 (s, 3H). MS (m/z) 380.0 (M + H+). 3-{[5-(2-Chlorophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}N-methylbenzenesulfonamide Trifluoroacetate (28). Compound 28 was prepared from 26 and 2-chlorophenylboronic acid using Method E as a white solid in 15% yield (19 mg): 1H NMR (400 MHz, DMSOd6) δ ppm 12.30 (br. s, 1H), 8.40 (s, 1H), 8.03 (s, 1H), 7.69 (br. s, 1H), 7.57−7.65 (m, 2H), 7.52−7.57 (m, 1H), 7.42−7.52 (m, 5H), 7.36 (d, J = 7.53 Hz, 1H), 2.44 (d, J = 5.02 Hz, 3H). MS (m/z) 414.0 (M + H+). 3-{[5-(3-Chlorophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}N-methylbenzenesulfonamide Trifluoroacetate (29). Compound 29 was prepared from 26 and 3-chlorophenylboronic acid using Method E as a white solid in 6% yield (8 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.33 (br. s, 1H), 8.41 (s, 1H), 8.39 (br. s, 1H), 8.07−8.12 (m, 1H), 7.77−7.84 (m, 1H), 7.61−7.66 (m, 2H), 7.45−7.55 (m, 3H), 7.41−7.45 (m, 1H), 7.35−7.41 (m, 2H), 2.46 (d, J = 5.02 Hz, 3H). MS (m/z) 414.0 (M + H+). 3-{[5-(4-Chlorophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}N-methylbenzenesulfonamide Trifluoroacetate (30). Compound 30 was prepared from 63 and 4-chlorophenylboronic acid using Method E as a white solid in 11% yield (15 mg): 1H NMR (400 MHz, DMSOd6) δ ppm 12.27 (br. s, 1H), 8.39 (s, 1H), 8.29 (br. s, 1H), 8.08−8.15 (m, 1H), 7.74−7.82 (m, 1H), 7.48−7.61 (m, 6H), 7.44 (q, J = 4.85 Hz, 1H), 7.38 (d, J = 7.28 Hz, 1H), 2.46 (d, J = 4.77 Hz, 3H). MS (m/ z) 414.0 (M + H+). L

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

δ ppm 11.76 (br. s, 1H, pyrrolopyrimidine NH), 8.78 (s, 1H, aniline NH), 8.20 (s, 1H, pyrrolopyrimidine C2-H), 8.11 (d, J = 2.26 Hz, 1H, benzene C2-H)), 7.49 (dd, J = 2.26, 8.53 Hz, 1H, benzene C6-H), 7.29 (q, J = 4.85 Hz, 1H, SO2NHMe), 7.18−7.23 (m, 2H, benzene C5-H, pyrrolopyrimidine C6-H), 6.55 (dd, J = 1.63, 3.14 Hz, 1H, pyrrolopyrimidine C5-H), 2.76 (s, 6H, N(CH3)2), 2.43 (d, J = 5.02 Hz, 3H, SO2NHCH3). 13C NMR (500 MHz, DMSO-d6) δ ppm 154.86 (pyrrolopyrimidine C4), 151.92 (pyrrolopyrimidine C7a), 151.86 (pyrrolopyrimidine C2), 151.04 (benzenesulfonamide C4), 131.71 (benzene C3), 131.64 (benzene C1), 125.72 (benzene C2), 124.32 (benzene C6), 123.17 (pyrrolopyrimidine C6), 118.97 (benzene C5), 104.27 (pyrrolopyrimidine C4a), 99.40 (pyrrolopyrimidine C5), 43.31 (N(CH3)2), 29.59 (SO2NHCH3). For compound 48, the 1H NMR and 13C NMR resonances were assigned on the basis of gCOSY45, gHMQC, gHMBC, and 13C GASPE spectra, and all 2D correlations are in agreement with the assigned structure. MS (m/z) 347.0 (M + H+). N-Methyl-4-(4-morpholinyl)-3-(1H-pyrrolo[2,3-d]pyrimidin-4ylamino)benzenesulfonamide (49). Compound 49 was prepared from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-4-(morpholino)-N-methylbenzenesulfonamide (83c) using Method B as tan solid in 37% yield (47 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.86 (br. s, 1H), 8.67 (s, 1H), 8.52 (d, J = 2.01 Hz, 1H), 8.29 (s, 1H), 7.50 (dd, J = 2.13, 8.41 Hz, 1H), 7.39 (q, J = 4.94 Hz, 1H), 7.33 (d, J = 8.53 Hz, 1H), 7.25−7.31 (m, 1H), 6.54−6.61 (m, 1H), 3.65−3.76 (m, 4H), 2.91−2.98 (m, 4H), 2.45 (d, J = 5.02 Hz, 3H). MS (m/z) 389.1 (M + H+). 4-(Dimethylamino)-N-methyl-3-[(5-methyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]benzenesulfonamide Trifluoroacetate (50). Compound 50 was prepared from 4-chloro-5-methyl-7H-pyrrolo[2,3d]pyrimidine20 and 3-amino-4-(dimethylamino)-N-methylbenzenesulfonamide (83b) using Method C as an orange solid in 20% yield (28 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.04 (br. s, 1 H), 9.17 (br. s, 1H), 8.60 (br. s, 1H), 8.29 (s, 1 H), 7.54 (m, 1 H), 7.38−7.46 (m, 2 H), 7.21 (br. s, 1 H), 2.76 (s, 6H), 2.53 (s, 3 H), 2.46 (d, J = 5.02 Hz, 3 H). MS (m/z) 361.1 (M + H+). 3-[(5-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-4-(dimethylamino)-N-methylbenzenesulfonamide Hydrochloride (51). Compound 51 was prepared from 5-bromo-4-chloro-7H-pyrrolo[2,3d]pyrimidine22 and 3-amino-4-(dimethylamino)-N-methylbenzenesulfonamide (83b) using Method A as a gray solid in 8% yield (25 mg): 1 H NMR (400 MHz, DMSO-d6) δ ppm 12.38 (s, 1H), 9.31 (s, 1H), 9.26 (d, J = 2.01 Hz, 1H), 8.43 (s, 1H), 7.61 (s, 1H), 7.38−7.51 (m, 3H), 2.74 (s, 6H), 2.47 (d, J = 5.02 Hz, 3H). MS (m/z) 424.9 (M + H+). N-Methyl-3-[(5-methyl-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]4-(4-morpholinyl)benzenesulfonamide (52). Compound 52 was prepared from 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine20 and 3-amino-4-(morpholino)-N-methylbenzenesulfonamide (83c) using Method C as gray solid in 10% yield (12 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.65 (br. s, 1 H), 9.26 (d, J = 2.01 Hz, 1 H), 8.75 (s, 1 H), 8.34 (s, 1 H), 7.57 (d, J = 8.28 Hz, 1 H), 7.43 (m, 2 H), 7.12 (s, 1 H), 3.77−3.84 (m, 4 H), 2.90−2.97 (m, 4 H), 2.66 (s, 3 H), 2.47 (s, 3 H). MS (m/z) 403.1 (M + H+). 3-[(5-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methyl4-(4-morpholinyl)benzenesulfonamide Hydrochloride (53). Compound 53 was prepared from 5-bromo-4-chloro-7H-pyrrolo[2,3d]pyrimidine22 and 3-amino-4-(morpholino)-N-methylbenzenesulfonamide (83c) using Method A as a pale gray solid in 32% yield (35 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.41 (br. s, 1H), 9.13 (d, J = 2.01 Hz, 1H), 9.03 (s, 1H), 8.41 (s, 1H), 7.64 (d, J = 2.51 Hz, 1H), 7.39−7.55 (m, 3H), 3.84−3.91 (m, 4H), 2.91−2.98 (m, 4H), 2.47 (d, J = 5.02 Hz, 3H). MS (m/z) 466.9 (M + H+). 3-[(6-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-4-(dimethylamino)-N-methylbenzenesulfonamide (54). Compound 54 was prepared from 68 and 3-amino-4-(dimethylamino)-N-methylbenzenesulfonamide (83b) using Method B as a white solid in 36% yield (162 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.56 (br. s, 1H), 8.89 (s, 1H), 8.17 (s, 1H), 8.00 (d, J = 2.01 Hz, 1H), 7.46−7.54 (m, 1H),

N-Methyl-3-({6-[3-(trifluoromethyl)phenyl]-1H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)benzenesulfonamide (40). Compound 40 was prepared from 35 and 3-trifluoromethylphenylboronic acid using Method E as a white solid in 38% yield (45 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 8.46 (br. s, 3 H), 8.13−8.18(m, 3 H), 7.82 (d, J = 7.78 Hz, 2 H), 7.62−7.69 (m, 2 H), 7.48−7.55 (m, 2 H), 2.55 (d, 3 H (obscured by solvent)). MS (m/z) 448.1 (M + H+). 3-{[6-(4-Hydroxyphenyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}N-methylbenzenesulfonamide Trifluoroacetate (41). Compound 41 was prepared from 35 and 4-hydroxphenylboronic acid using Method E as a white solid in 3% yield (4 mg): 1H NMR (400 MHz, MeOD) δ ppm 8.31 (s, 1 H), 8.12 (s, 1 H), 7.84−7.92 (m, 2 H), 7.74−7.80 (m, 1 H), 7.63−7.70 (m, 2 H), 6.88−6.95 (m, 3 H), 2.62 (s, 3 H). MS (m/ z) 396.1 (M + H+). N-Methyl-3-{[6-(4-pyridinyl)-1H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}benzenesulfonamide (42). Compound 42 was prepared from 35 and 4-pyridylboronic acid using Method E as a brown solid in 50% yield (37 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.35 (br. s, 1H), 9.74 (s, 1H), 8.30−8.40 (m, 3H), 7.34−7.72 (m, 5H), 7.23−7.32 (m, 1H), 7.17 (d, J = 7.55 Hz, 1H), 7.03 (br. s, 1H), 2.48 (d, J = 4.78 Hz, 3H). MS (m/z) 381.0 (M + H+). 4-({3-[(Methylamino)sulfonyl] phenyl}amino)-1H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (43). Compound was prepared from 72 and 3-amino-N-methylbenzenesulfonamide using Method B as a pale purple 43 solid in 57% yield (38 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.59 (br. s, 1H), 9.99 (br. s, 1H), 8.43 (s, 1H), 8.33−8.39 (m, 1H), 8.30 (d, J = 7.78 Hz, 1H), 7.55−7.67 (m, 2H), 7.41−7.55 (m, 2H), 2.47 (d, J = 4.77 Hz, 3H). MS (m/z) 347.9 (M + H+). N-Methyl-3-(1H-pyrimido[4,5-b]indol-4-ylamino)benzenesulfonamide (44). Compound 44 was prepared from 4chloro-9H-pyrimido[4,5-b]indole23 and 3-amino-N-methylbenzenesulfonamide using Method B as a pale lavender solid in 90% yield (156 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.27 (s, 1H), 9.27 (br. s., 1H), 8.50 (s, 1H), 8.46 (d, J = 7.81 Hz, 1H), 8.23 (t, J = 1.76 Hz, 1H), 8.07 (dd, J = 1.01, 8.06 Hz, 1H), 7.61 (t, J = 7.93 Hz), 7.53−7.58 (m, 1H), 7.44−7.53 (m, 3H), 7.29−7.36 (m, 1H), 2.48 (d, J = 4.78 Hz, 3H). MS (m/z) 354.1 (M + H+). N,4-Dimethyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)benzenesulfonamide (45). Compound 45 was synthesized from 4chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-N,4-dimethylbenzenesulfonamide using Method B as a pale green solid in 20% yield (75 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.75 (br. s, 1H), 9.10 (s, 1H), 8.14 (s, 1H), 7.84 (d, J = 1.51 Hz, 1H), 7.48−7.57 (m, 2H), 7.42 (q, J = 4.85 Hz, 1H), 7.18−7.24 (m, 1H), 6.49 (dd, J = 1.76, 3.26 Hz, 1H), 2.45 (d, J = 5.02 Hz, 3H), 2.31 (s, 3H). MS (m/z) 318.0 (M + H+). 4-Chloro-N-methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)benzenesulfonamide Trifluoroacetate (46). Compound 46 was prepared from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-4chloro-N-methylbenzenesulfonamide25 using Method C as a tan solid in 12% yield (24 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.09 (br. s., 1 H), 9.76 (br. s., 1 H), 8.25 (s, 1 H), 8.13 (d, J = 2.26 Hz, 1 H), 7.85 (d, J = 8.53 Hz, 1 H), 7.55−7.71 (m, 2 H), 7.32−7.37 (m, 1 H), 6.67 (br. s., 1 H), 2.49 (d, J = 5.02 Hz, 3 H). MS (m/z) 338.0 (M + H+). 3-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methyl-4(methylamino)benzenesulfonamide (47). Compound 47 was prepared from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-Nmethyl-4-(methylamino)benzenesulfonamide (83a) using Method B as a tan solid in 15% yield (16 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.64 (br. s., 1 H), 8.72 (s, 1 H), 8.10 (s, 1 H), 7.52 (dd, J = 8.66, 2.13 Hz, 1 H), 7.48−7.50 (m, 1 H), 7.12−7.15 (m, 1 H), 7.05 (q, J = 5.27 Hz, 1 H), 6.73 (d, J = 8.53 Hz, 1 H), 6.31 (br. s., 1 H), 5.91−6.02 (m, 1 H), 2.77 (d, J = 4.77 Hz, 3 H), 2.37 (d, J = 5.27 Hz, 3 H). MS (m/z) 333.0 (M + H+). 4-(Dimethylamino)-N-methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4ylamino)benzenesulfonamide (48). Compound 48 was prepared from 4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-4-(dimethylamino)-N-methylbenzenesulfonamide (83b) using Method B as an off-white solid in 22% yield (51 mg): 1H NMR (400 MHz, DMSO-d6) M

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

7.28 (d, J = 5.04 Hz, 1H), 7.19 (d, J = 8.56 Hz, 1H), 6.67 (s, 1H), 2.75 (s, 6H), 2.42 (d, J = 5.04 Hz, 3H). MS (m/z) 425.0 (M + H+). 4-(Dimethylamino)-N-methyl-3-({6-[3-(trifluoromethyl)phenyl]1H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)benzenesulfonamide Trifluoroacetate (55). Compound 55 was prepared from 70 and 3amino-4-(dimethylamino)-N-methylbenzenesulfonamide (83b) using Method A as a white solid in 13% yield (18 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.52 (br. s, 1 H), 8.87 (s, 1 H), 8.28 (s, 1 H), 8.23 (m, 1 H), 8.12 (d, J = 7.53 Hz, 1 H), 7.67−7.74 (m, 2 H), 7.50 (dd, J = 8.53, 2.26 Hz, 1 H), 7.31 (q, J = 4.94 Hz, 1 H), 7.22−7.29 (m, 2 H), 2.79 (s, 6 H), 2.44 (d, J = 5.02 Hz, 3 H). MS (m/z) 491.1 (M + H+). 3-[(6-Bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-N-methyl4-(4-morpholinyl)benzenesulfonamide (56). Compound 56 was prepared from 68 and 3-amino-4-(morpholino)-N-methylbenzenesulfonamide (83c) using Method B as a pale yellow powder in 65% yield (68 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.64 (br. s, 1 H), 8.73 (s, 1 H), 8.34 (d, J = 2.01 Hz, 1 H), 8.23 (s, 1 H), 7.51 (dd, J = 8.31, 2.01 Hz, 1 H), 7.37 (q, J = 5.04 Hz, 1 H), 7.30 (d, J = 8.56 Hz, 1 H), 6.69 (s, 1 H), 3.64−3.72 (m, 4 H), 2.91−2.98 (m, 4 H), 2.44 (d, J = 5.04 Hz, 3 H). MS (m/z) 467.0 (M + H+). N-Methyl-4-(4-morpholinyl)-3-({6-[3-(trifluoromethyl)phenyl]-1Hpyrrolo[2,3-d]pyrimidin-4-yl}amino)benzenesulfonamide Trifluoroacetate (57). Compound 57 was prepared from 70 and 3-amino4-(morpholino)-N-methylbenzenesulfonamide (83c) using Method A as a white solid in 23% yield (35 mg, 93% purity): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.88 (br. s, 1 H), 8.36 (s, 1 H), 8.24 (br. s, 1 H), 8.17 (m, 2 H), 7.71−7.78 (m, 2 H), 7.59−7.66 (m, 2 H), 7.45 (m, 1 H), 7.38 (m, 1 H), 7.26 (br. s, 1 H), 2.96−3.04 (m, 4 H), 2.46 (d, J = 5.02 Hz, 3 H). MS (m/z) 533.2 (M + H+). 4-Chloro-6-[3-(trifluoromethyl)phenyl]-1H-pyrrolo[2,3-d]pyrimidine (70). A mixture of 6825 (1.97 g, 4.34 mmol), 3(trifluoromethyl)phenylboronic acid (0.823 g, 4.34 mmol) and K2CO3 (0.959 g, 6.94 mmol) in 1,4-dioxane (36 mL) and water (12 mL) was purged with nitrogen and treated with PdCl2(dppf)−CH2Cl2 (0.5 g, 0.612 mmol). The mixture was stirred at 82 °C for 3 h before being cooled and partitioned between 200 mL EtOAc and 30 mL brine. The organic layer was washed with brine, dried over MgSO4, concentrated, and subjected to flash column chromatography (0−35% EtOAc-hexanes) to give 4-chloro-7-(phenylsulfonyl)-6-[3(trifluoromethyl)phenyl]-7H-pyrrolo[2,3-d]pyrimidine (0.893 g, 47.0%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.90 (s, 1 H), 7.89−7.96 (m, 5 H), 7.78 (t, J = 7.53 Hz, 2 H), 7.61− 7.69 (m, 2 H), 7.16 (s, 1 H). MS (m/z) 438.0 (M + H+). A solution of 4-chloro-7-(phenylsulfonyl)-6-[3-(trifluoromethyl)phenyl]-7H-pyrrolo[2,3-d]pyrimidine (500 mg, 1.14 mmol) in THF (20 mL) was treated with KOt-Bu (641 mg, 5.71 mmol) at 0 °C before being warmed to 25 °C. After 30 min, the mixture was diluted with 50 mL of sat. aqueous Na2CO3 and extracted with EtOAc (100 mL × 2). The combined organic extracts were washed with brine, dried over MgSO4, and concentrated to give 70 (345 mg, 100%) as a light brown solid: MS (m/z) 298.0 (M + H+). 1,1-Dimethylethyl 4-chloro-1H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (72). To a suspension of 4,6-dichloro-pyrimidine-5carbaldehyde (71) (2 g, 11 mmol) in EtOH (50 mL) at 25 °C was added glycine tert-butyl ester (1.894 g, 11 mmol) followed by Et3N (3.9 mL, 28 mmol). The reaction mixture was stirred for 24 h before being concentrated, and the residue was taken up in water and extracted with CH2Cl2. The organic layer extract was concentrated and subjected to flash chromatography (0−90% EtOAc-hexanes) to give 1,1-dimethylethyl 4-chloro-5-hydroxy-5,6-dihydro-1H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (1.38 g, 44.9%) as a mix of two isomers, a light yellow solid: MS (m/z) 272.1 (M + H+). To a solution of 1,1-dimethylethyl 4-chloro-5-hydroxy-5,6-dihydro1H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (1.38 g, 5.06 mmol) in DMF (20 mL) at 0 °C was added NaH (0.186 g of a 60% dispersion in mineral oil, 4.64 mmol). The resulting mixture was stirred for 1 h before being quenched slowly with the addition of 2 mL of water and partitioned between 30 mL sat. aqueous NH4Cl solution and 250 mL EtOAc. The organic layer was washed with sat. aqueous NH4Cl (2 × 20 mL) then brine (30 mL), dried over MgSO4, and concentrated.

The resulting yellowish solid was suspended in 10 mL of hexanes and filtered to give 72 (718 mg, 56%) as an off-white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 13.33 (br. s, 1H), 8.74 (s, 1H), 7.14 (s, 1H), 1.58 (s, 9H). MS (m/z) 254.1 (M + H+). N-Methyl-3-(methylamino)benzenesulfonamide (74). A mixture of N-methyl-3-aminobenzenesulfonamide (1 g, 5.37 mmol) and formaldehyde (0.440 mL, 5.91 mmol) in CH2Cl2 at 25 °C was treated with acetic acid (0.338 mL, 5.91 mmol) and stirred for 5 min before sodium triacetoxyborohydride (1.707 g, 8.05 mmol) was added. The mixture was stirred for 10 min before being treated with saturated aqueous NaHCO3 and extracted with CH2Cl2. The combined organic extracts were washed with brine, dried (Na2SO4), concentrated, and subjected to flash chromatography (35−40% EtOAc-hexanes) to give 74 (438 mg, 40%) as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.30−7.26 (m, 2H), 6.91−6.89 (m, 2H), 6.75 (d, J = 7.3 Hz, 1H), 6.18 (q, J = 5.0 Hz, 1H), 2.70 (d, J = 5 0.0 Hz, 3H), 2.40 (d, J = 3.8 Hz, 3H). MS (m/z) 201.0 (M + H+). 3-Hydroxy-N-methylbenzenesulfonamide (62). A mixture of 3(methyloxy)benzenesulfonyl chloride (250 mg, 1.21 mmol) and DMAP (29.6 mg, 0.24 mmol) in a solution of methylamine (2 M in THF, 4.8 mL, 9.6 mmol) was stirred at 25 °C for 30 min before being filtered and concentrated to give N-methyl-3-(methyloxy)benzenesulfonamide (267 mg, 85% purity, 93% yield) as a yellow oil: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.50−7.57 (m, 1 H), 7.47 (br. s., 1 H), 7.35 (d, J = 7.78 Hz, 1 H), 7.28 (t, J = 2.01 Hz, 1 H), 7.23 (dd, J = 8.28, 2.26 Hz, 1 H), 3.83 (s, 3 H), 2.41 (s, 3 H). MS (m/z) 202.0 (M + H+). A mixture of N-methyl-3-(methyloxy)benzenesulfonamide (267 mg, 1.13 mmol) in CH2Cl2 (2.8 mL) at 0 °C was treated with boron tribromide (2.26 mL of 1 M in CH2Cl2, 2.26 mmol) and allowed to warm to 25 °C. After 2 h the reaction was carefully quenched by the cautious addition of dry methanol (∼1 mL) and then concentrated and the residue partitioned between DCM and water. The organic layer was then collected via hydrophobic frit and concentrated to give 62 (56 mg, 27%) as a brown oil. The aqueous layer was passed through an OASIS cartridge (Waters) eluting with MeOH to give additional 62 (146 mg, 69%) as a yellow oil: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.89−10.19 (m, 1 H), 7.33−7.42 (m, 2 H), 7.12− 7.21 (m, 2 H), 6.97−7.03 (m, 1 H), 2.39 (d, J = 4.77 Hz, 3 H). MS (m/z) 188.0 (M + H+). 5-Bromo-3-pyridinesulfonyl Chloride (77). A mixture of 3pyridinesulfonyl chloride hydrochloride (8.9 g, 44 mmol) and bromine (14 g, 88 mmol) was heated to 130 °C for 8 h. The resulting mixture of 77 was cooled and used immediately in the subsequent step. 5-Bromo-N-methyl-3-pyridinesulfonamide (78). A solution of CH3NH2 (50 mL of a 23−30% in H2O) at 0 °C was treated with 77 (44 mmol), warmed to 25 °C, and stirred for 3 h before being extracted with EtOAc. The organic extract was concentrated washed with 10:1 hot petroleum ether-EtOAc to give 78 (2.4 g, 18% yield, 2 steps) as a brown solid that was used immediately in the subsequent step. 5-Amino-N-methyl-3-pyridinesulfonamide (79). A mixture of 78 (2.4 g, 9.6 mmol), CuCl (0.100 g, 1.01 mmol), and concentrated aqueous NH4OH (5 mL) was heated to 130 °C for 18 h in a sealed tube. The reaction mixture was then treated with sodium sulfide and extracted with EtOAc. The combined organic extracts were concentrated and the residue was washed with 20:5:3 hot petroleum ether-EtOAc-MeOH to give 79 (1.1 g, 61%) as a brown solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.11 (d, J = 2.51 Hz, 1H), 8.04 (d, J = 1.76 Hz, 1H), 7.47 (br. s, 1H), 7.24 (t, J = 2.13 Hz, 1H), 5.83 (br. s, 2H), 2.44 (s, 3H); MS (m/z) 188.1 (M + H+). 4-Fluoro-3-nitrobenzenesulfonyl Chloride (81). 1-Fluoro-2-nitrobenzene (80) (50.0 g, 0.354 mol) was added to chlorosulfonic acid (91 g, 0.778 mol) at 65 °C. The resulting mixture was then heated to 100 °C for 18 h. The mixture was cooled to 25 °C, poured over ice and extracted with CH2Cl2. The combined organic layers were then washed with sat. aqueous NaHCO3, then brine, dried over MgSO4, filtered, and concentrated to give 81 (55.3 g, 65%) as a brown oil: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.24 (dd, J = 7.40, 2.13 Hz, 1 H), N

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

7.98 (ddd, J = 8.60, 4.45, 2.26 Hz, 1 H), 7.55 (dd, J = 11.29, 8.53 Hz, 1 H). 4-Fluoro-N-methyl-3-nitrobenzenesulfonamide (82). A solution of 81 (43 g, 179.5 mmol) in THF (500 mL) was treated with Et3N (150 mL, 1.08 mol) and then cooled to −35 °C. Then a solution of CH3NH2−HCl (14.5 g, 215.4 mmol) in water was added dropwise and the mixture was stirred for 1 h before being warmed to 25 °C, diluted with water and extracted with EtOAc. The organic extract was washed with saturated aqueous NaHCO3, then brine, dried over MgSO4, filtered, concentrated, and subjected to flash chromatography (20% EtOAc-petroleum ether) to give 82 (38 g, 90%) as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.47 (dd, J = 7.03, 2.51 Hz, 1 H), 8.17 (ddd, J = 8.72, 3.95, 2.38 Hz, 1 H), 7.81−7.89 (m, 2 H), 2.47 (d, J = 5.02 Hz, 3 H). MS (m/z) 235.0 (M + H+). 3-Amino-4-(dimethylamino)-N-methylbenzenesulfonamide (83b). A mixture of 82 (1.8 g, 7.7 mmol) and Me2NH-HCl (0.63 g, 7.7 mmol) in THF (100 mL) at −40 °C was treated with Et3N (1.8 mL, 12.8 mmol). The mixture was warmed to room temperature and stirred for 30 min before being concentrated and subjected to flash chromatography (20% EtOAc-petroleum ether) to give 4-(dimethylamino)-N-methyl-3-nitrobenzenesulfonamide (1.8 g, 90%) as a yellow solid: MS (m/z) 260.0 (M+H+). A solution of 4-(dimethylamino)-N-methyl-3-nitrobenzenesulfonamide (1.8 g, 6.9 mmol) in THF (100 mL) was treated with 10% Pd/C (0.7 g) and stirred at 50 °C under an atmosphere of hydrogen for 16 h before being filtered and concentrated to give 83b (1.5 g, 94%) as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.03−7.10 (m, 2H), 7.00 (d, J = 8.28 Hz, 1H), 6.93 (dd, J = 2.13, 8.16 Hz, 1H), 5.13 (s, 2H), 2.62 (s, 6H), 2.38 (d, J = 5.02 Hz, 3H). MS (m/z) 230.2 (M +H+). 3-Amino-N-methyl-4-(methylamino)benzenesulfonamide (83a). Compound 83a was prepared from 82 and MeNH2−HCl by a procedure analogous to that for 83b to give 83a (1.3 g, 66%) as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 6.95 (dd, J = 8.16, 2.13 Hz, 1 H), 6.91 (d, J = 2.01 Hz, 1 H), 6.82 (q, J = 5.10 Hz, 1 H), 6.43 (d, J = 8.28 Hz, 1 H), 5.32 (q, J = 4.52 Hz, 1 H), 4.86 (s, 2 H), 2.77 (d, J = 5.02 Hz, 3 H), 2.32 (d, J = 5.02 Hz, 3 H). MS (m/z) 216.0 (M+H+). 3-Amino-N-methyl-4-(4-morpholinyl)benzenesulfonamide (83c). Compound 83c was prepared from 82 and morpholine by a procedure analogous to that for 83b to give 83c (1.98 g, 85%) as a white solid: 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.07−7.17 (m, 2H), 7.01 (d, J = 8.28 Hz, 1H), 6.94 (dd, J = 1.88, 8.16 Hz, 1H), 5.20 (s, 2H), 3.72− 3.81 (m, 4H), 2.80−2.89 (m, 4H), 2.38 (d, J = 4.77 Hz, 3H). MS (m/ z) 272.2 (M+H+). TNNI3K Assay Reagents.32 His6-MBP-TEV-Full length human TNNI3K (hTNNI3K) was expressed in a Baculovirus system and purified from amylase affinity chromatography followed by Superdex200. The fluorescent ligand 5-({[2-({[3-({4-[(5-hydroxy-2methylphenyl)amino]-2-pyrimidinyl}amino)phenyl]carbonyl}amino)ethyl]amino}carbonyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (84) was prepared as described.33 Buffer components, including MgCl2, Bis-Tris, DTT and CHAPS were purchased from SigmaAldrich. TNNI3K Enzyme Assay.32 A fluorescence polarization assay was used to determine the concentration-dependent binding of compounds to the ATP binding pocket of hTNNI3K. The binding of 84 to the hTNNI3K ATP binding pocket results in an increase in fluorescence polarization. Addition of compound displaces the fluorescent probe, which leads to a decrease in the polarization signal. Ten milliliters of a 5 nM solution of 84 (Solution 1) was prepared by mixing 5 μL of 1 M DTT, 80 μL of 10% (w/v) CHAPS and 5 μL of a stock solution of 84 (10 μM in DMSO) into 9910 μL of buffer (20 mM Tris, 15 mM MgCl2, pH 7.5). Solution 2 was formed by mixing 53.8 μL of 2.6 μM hTNNI3K with a 6946.2 μL aliquot of Solution 1 to make up a 7 mL mixture of hTNNI3K and 84 (Solution 2). Fifty (50) nL of inhibitors in DMSO (or DMSO controls) were stamped into a 384well low volume Greiner black plate, followed by addition of 5 μL of Solution 1 to column 18 and 5 μL Solution 2 to columns 1−17 and 19−24 of the plate. The plate was then spun at 500 rpm for 30 s and

incubated at room temperature for 60 min. Fluorescence polarization was measured on an Analyst plate reader (Ex/Em: 485/530 nm, Dichroic: 505). For dose-response experiments, normalized data were fit by ABASE/XC50 to the following equation:

pIC50 = (log((b − y)/(y − a)))/d − log(x) where x is the compound concentration, y is the % activity at specified compound concentration, a is the minimum % activity, b is the maximum % activity, and d is the Hill slope. The pIC50 values were averaged to determine a mean value, for a minimum of 2 experiments, but typically for ≥4 experiments. On average, pIC50 values fell within 0.3 units for individual experiments with a given compound. Compound IC50 values less than 10 nM could not be accurately obtained due to titration of TNNI3K in the assay. TNNI3K Cellular Assay. The ability of compounds to inhibit TNNI3K autophosphorylation was measured in HEKMSRII cells overexpressing human myc-TNNI3K. After cell lysis, myc-TNNI3K is captured using an antimyc tag antibody, and phosphorylation is detected using europium-tagged antiphosphotyrosine antibody (EuPY20, PerkinElmer Catalog No: AD0038) and measured by timeresolved fluorescence of europium on an EnVision plate reader (PerkinElmer). This technique is known as dissociation-induced lanthanide fluorescence intensity assay (DELFIA). HEKMSRII cells transduced with human myc-TNNI3K-expressing Bacmam were cultured in DMEM/F12 with 0.1% FBS and 1% penicillinstreptomycin at 37 °C in a 5% CO2 incubator overnight. These myc-TNNI3K overexpressing cells were treated with test compounds and incubated for 30 min. Pervanadate solution (0.8 mM vanadate, 0.01% H2O2, DMEM/F12 with 0.1% FBS and 1% penicillinstreptomycin) was then added to cells. After 20 min of incubation at room temperature, media was aspirated, and cells were lysed with Cell Extraction Buffer (Invitrogen cat. No FNN0011) containing Complete Protease Inhibitor Cocktail (Roche cat. no. 11 697 498 001). Fifteen microliters of cell lysate was transferred to a black Maxisorp plate (Nunc cat no. 460518) coated with anti-Myc Tag (clone 4A6, Millipore, cat. no. 05-724), which had been blocked and washed with Superblock-TBS and TBS-T, respectively. After the plates were incubated for 2 h at room temperature, they were washed with TBS-T and treated with Superblock T20 (TBS) containing 100 μg/ mL Eu-PY20 antibody, and the plates were incubated for 1 h at room temperature. The cells were then washed extensively with TBS-T, DELFIA enhancement solution (PerkinElmer) was added and the cells incubated for 20 min at room temperature, and fluorescence was quantified on an EnVision plate reader using an Europium-615 nm filter. For dose response experiments, normalized data were fit by ABASE/XC50 to the following equation:

pIC50 = (log((b − y)/(y − a)))/d − log(x) where x is the compound concentration, y is the % activity at specified compound concentration, a is the minimum % activity, b is the maximum % activity, and d is the Hill slope. The pIC50 values were averaged to determine a mean value, for a minimum of 2 experiments. On average, pIC50 values fell within 0.3 units for individual experiments with a given compound. B-Raf Enzyme Assay.32,34 Selected compounds were tested for BRaf protein serine kinase inhibitory activity in a B-Raf Accelerated MEK ATPase assay (BRAMA). Baculovirus-expressed His6-tagged BRAFV600E full-length (amino acids 2−766) was used in the BRAMA assay. The BRAMA assay is a high-sensitivity assay which measures an intrinsic MEK-mediated ATP hydrolysis uncoupled from downstream ERK phosphorylation by coupling the formation of ADP to NADH oxidation through the enzymes pyruvate kinase and lactate dehydrogenase. When ADP production is initiated by addition of catalytic amounts of an activated Raf enzyme and nonphosphorylated MEK, one observes robust ADP production concomitant with Rafmediated phosphorylation of MEK. The assay was conducted as disclosed34 but with the following changes: (1) the assay was performed with a final MEK concentration of 150 nM, and (2) the assay was read at a single end point instead of a kinetic read. O

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

Notes

Acceleration of MEK ATPase activity was determined from the data and plotted as a function of inhibitor concentration to afford concentration response curves, from which the pIC50 values were generated following a standard pIC50 fitting protocol. For key analogues, the concentration−response experiments were repeated and, on average, the pIC50 values fell within 0.2 units from individual experiments with a given compound. Pharmacokinetics. Rat pharmacokinetics were determined in male Sprague−Dawley rats in a noncrossover fashion. Two rats (fed) received the designated compound as a 30 min intravenous infusion (1 mg/kg target dose; 4 mL/kg dose volume) formulated as a solution in 20% aqueous cavitron with 3−5% DMSO at pH 4−4.5. Two additional rats (fed) received the designated compound as a gastric bolus (2 mg/kg target dose, 16 mL/kg dose volume) formulated as a solution in 6% cavitron with 3−5% DMSO at pH 4. Blood samples were collected from the femoral artery at various times following dosing and centrifuged to obtain plasma. Plasma concentrations of the test compound were quantified by LC/MS/MS (LOQ = 1 ng/mL). Noncompartmental methods were used for analysis of concentration versus time data. Cl = iv plasma clearance; F = oral bioavailability; poDNAUC = dose-normalized area under the oral concentration− time curve. All studies were conducted after review by the GSK Institutional Animal Care and Use Committee and in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. Protein X-ray Crystal Structures. TNNI3K protein (residues 402− 730 for complex 1 and residues 421−730 for complex 53) was complexed with 5-fold molar excess of inhibitor for 1 h on ice. The complex was concentrated to 8−10 mg/mL in 25 mM HEPES, 400 mM NaCl pH 8, 5 mM DTT, and 1 mM CHAPS. Crystals were grown by sitting-drop vapor diffusion at 4 °C. Drops (600 nL) were set with a Mosquito instrument (TTP LabTech; Melbourne, United Kingdom) in MRC 2 Well Crystallization Plates (Swissci; Zug, Switzerland) by combining 300 nL TNNI3K−inhibitor complex and 300 nL of well solution. For complex 1, the well solution contained 0.1 M HEPES 7.0 and 0.3 M sodium potassium tartrate. For complex 53, the well solution contained 0.1 M HEPES pH 7.0, 0.1 M ammonium sulfate, and 8% PEG3350. TNNI3K−inhibitor complexed crystals were harvested and cryoprotected with 25% glycerol (complex 1) or 35% ethylene glycol (complex 53) prior to data collection. The initial structure of TNNI3K was solved by molecular replacement using MOLREP35 in the CCP4 program suite and Tak1 as a search model (RCSB 2EVA). These crystals belonged to the P21 space group with 4 mol/asu. Data for the 1 and 53 complexes were collected on a Saturn 944+/FRE+ Super Bright and reduced and scaled with HKL2000.36 The structure was built using COOT37 and refined with REFMAC.38 Statistics for the data can be found in Supplementary Table 1. Atomic coordinates and structure factors have been deposited into the Protein Data Bank (PDB: 4YFI and PDB: 4YFF). Images of the X-ray crystal structures (Figures 2, 8, 13) were generated using the PyMol Molecular Graphics System, Version 1.7.2.1 Schrödinger, LLC.



The authors declare the following competing financial interest(s): Authors affilliated with GlaxoSmithKline have received compensation in the form of salary and stock.



ACKNOWLEDGMENTS We gratefully acknowledge Nathan Gaul, Peter Caprioli, Evanson Agyeman, Michael Shaber, Brian Dombroski, and Laurie Carson for conducting the TNNI3K and B-Raf assays and Will Burkhart for supplying recombinant TNNI3K protein for crystallization studies.



ABBREVIATIONS USED TNNI3K, troponin I-interacting kinase; CARK, cardiac ankyrin repeat kinase; DNAUC, dose-normalized area under the curve; TLK, tyrosine-like kinase; GSK, GlaxoSmithKline; kcal, kilocalories; Cl, iv clearance; Vdss, volume of distribution; F, oral bioavailability; μwave, microwave; i-Bu, isobutyl; dppf, 1,1′Bis(diphenylphosphino)ferrocene; SCX, strong cation exch an ge; Bis-Tris, Bis(2-hy droxy et hyl)-am ino -t ris(hydroxymethyl)-methane; CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; DELFIA, dissociationinduced lanthanide fluorescence intensity assay; DMEM/F12, Dulbecco’s modified eagle medium−nutrient mixture F12; FBS, fetal bovine serum; TBS, tris buffered saline; BRAMA, B-Raf Accelerated MEK ATPase Assay; MEK, mitogen-activated protein kinase kinase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00931. Table of statistics for X-ray crystal structures and kinase selectivity results for 1, 7, and 48 (PDF) Molecular Formula Strings (CSV) Accession Codes

4YFI (TNNI3K-1), 4YFF (TNNI3K-53).



REFERENCES

(1) Zhao, Y.; Meng, X. M.; Wei, Y. J.; Zhao, X. W.; Liu, D. Q.; Cao, H. Q.; Liew, C. C.; Ding, J. F. Cloning and characterization of a novel cardiac-specific kinase that interacts specifically with cardiac troponin I. J. Mol. Med. (Berl) 2003, 81, 297−304. (2) Lal, H.; Ahmad, F.; Parikh, S.; Force, T. Troponin I-interacting protein kinase: a novel cardiac-specific kinase, emerging as a molecular target for the treatment of cardiac disease. Circ. J. 2014, 78, 1514− 1519. (3) Vagnozzi, R. J.; Gatto, G. J., Jr.; Kallander, L. S.; Hoffman, N. E.; Mallilankaraman, K.; Ballard, V. L. T.; Lawhorn, B. G.; Stoy, P.; Philp, J.; Graves, A. P.; Naito, Y.; Lepore, J. J.; Gao, E.; Madesh, M.; Force, T. Inhibition of the cardiomyocyte-specific TNNI3K limits oxidative stress, injury, and adverse remodeling in the ischemic heart. Sci. Transl. Med. 2013, 5, 207ra141. (4) Wang, L.; Wang, H.; Ye, J.; Xu, R. X.; Song, L.; Shi, N.; Zhang, Y. W.; Chen, X.; Meng, X. M. Adenovirus-mediated overexpression of cardiac troponin I-interacting kinase promotes cardiomyocyte hypertrophy. Clin. Exp. Pharmacol. Physiol. 2011, 38, 278−284. (5) Wheeler, F. C.; Tang, H.; Marks, O. A.; Hadnott, T. N.; Chu, P. L.; Mao, L.; Rockman, H. A.; Marchuk, D. A. Tnni3k modifies disease progression in murine models of cardiomyopathy. PLoS Genet. 2009, 5, e1000647. (6) Stoy, P. unpublished results. (7) (a) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. A quantitiative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127−131. (b) Fabian, M. A.; Biggs, W. H., III; Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 2005, 23, 329−335.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (610) 270-5347. P

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

(8) (a) Harris, I. S.; Zhang, S.; Treskov, I.; Kovacs, A.; Weinheimer, C.; Muslin, A. J. Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload. Circulation 2004, 110, 718−723. (b) Muslin, A. J. Role of Raf proteins in cardiac hypertrophy and cardiomyocyte survival. Trends Cardiovasc. Med. 2005, 15, 225−229. (9) Gonnella, N. C.; Roberts, J. D. Studies of the tautomerism of purine and the protonation of purine and its 7- and 9-methyl derivatives by nitrogen-15 nuclear magnetic resonance spectroscopy. J. Am. Chem. Soc. 1982, 104, 3162−3164. (10) Leung, C. S.; Leung, S. S. F.; Tirado-Rives, J.; Jorgensen, W. L. Methyl effects on protein-ligand binding. J. Med. Chem. 2012, 55, 4489−4500. (11) Schmid, S.; Rottgen, M.; Thewalt, U.; Austel, V. Synthesis and conformational properties of 2,6-bis-anilino-3-nitropyridines. Org. Biomol. Chem. 2005, 3, 3408−3421. (12) Uno, B.; Kawakita, T.; Kano, K.; Ezumi, K.; Kubota, T. Spectroscopic analysis and geometry assignment of the minimum energy conformations of 2-phenoxypyridines and diphenyl ethers. Bull. Chem. Soc. Jpn. 1992, 65, 2697−2703. (13) King, A. J.; Patrick, D. R.; Batorsky, R. S.; Ho, M. L.; Do, H. T.; Zhang, S. Y.; Kumar, R.; Rusnak, D. W.; Takle, A. K.; Wilson, D. M.; Hugger, E.; Wang, L.; Karreth, F.; Lougheed, J. C.; Lee, J.; Chau, D.; Stout, T. J.; May, E. W.; Rominger, C. M.; Schaber, M. D.; Luo, L.; Lakdawala, A. S.; Adams, J. L.; Contractor, R. G.; Smalley, K. S. M.; Herlyn, M.; Morrissey, M. M.; Tuveson, D. A.; Huang, P. S. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res. 2006, 66, 11100−11105. (14) Ahlbrecht, H.; Duber, E. O.; Epsztajn, J.; Marcinkowski, R. M. K. Delocalisation, conformation and basicity of anilines. Tetrahedron 1984, 40, 1157−1165. (15) (a) Zhang, C.; Shokat, K. M. Enhanced selectivity for inhibition of analog sensitive kinases through scaffold optimization. Tetrahedron 2007, 63, 5832−5838. (b) Tasler, S.; Muller, O.; Wieber, T.; Herz, T.; Krauss, R.; Totzke, F.; Kubbutat, M. H. G.; Schachtele, C. Nsubstituted 2′-(aminoaryl)benzothiazoles as kinase inhibitors: Hit identification and scaffold hopping. Bioorg. Med. Chem. Lett. 2009, 19, 1349−1356. (c) Liu, X.-P.; Narla, R. K.; Uckun, F. M. Organic phenyl arsonic acid compounds with potent antileukemic activity. Bioorg. Med. Chem. Lett. 2003, 13, 581−583. (d) Bridges, A. J.; Denny, W. A.; Fry, D.; Kraker, A.; Meyer, R. F.; Rewcastle, G. W.; Thompson, A. M. Bicyclic compounds capable of inhibiting tyrosine kinases of the epidermal growth factor receptor family. U.S. Patent 5,654,307, 1997. (e) Gangjee, A.; Namjoshi, O. A.; Ihnat, M. A.; Buchanan, A. The contribution of a 2-amino group on receptor tyrosine kinase inhibition and antiangiogenic activity in 4-anilinosubstituted pyrrolo[2,3-d]pyrimidines. Bioorg. Med. Chem. Lett. 2010, 20, 3177−3181. (f) Deng, X.; Okram, B.; Ding, Q.; Zhang, J.; Choi, Y.; Adrian, F. J.; Wojciechowski, A.; Zhang, G.; Che, J.; Bursulaya, B.; Cowan-Jacob, S. W.; Rummel, G.; Sim, R.; Gray, N. S. Expanding the diversity of allosteric Bcr-Abl inhibitors. J. Med. Chem. 2010, 53, 6934−6946. (16) Sun, L.; Cui, J.; Liang, C.; Zhou, Y.; Nematalla, A.; Wang, X.; Chen, H.; Tang, C.; Wei, J. Rational design of 4,5-disubstituted-5,7dihydro-pyrrolo[2,3-d]pyrimidine-6-ones as a novel class of inhibitors of epidermal growth factor receptor (EGF-R) and Her2(p185erbB) tyrosine kinases. Bioorg. Med. Chem. Lett. 2002, 12, 2153−2157. (17) Voronkov, M. V.; Gu, K.; Baugh, S. D. P.; Becker, M. R. A modular approach to 4,5-diaminopyrrolo[2,3-d]pyrimidines and 2,4,5triaminopyrrolo[2,3-d]pyrimidines. Tetrahedron Lett. 2006, 47, 4149− 4151. (18) (a) Guillard, J.; Decrop, M.; Gallay, N.; Espanel, C.; Boissier, E.; Herault, O.; Viaud-Massuard, M.-C. Synthesis and biological evaluation of 7-azaindole derivatives, synthetic cytokinin analogues. Bioorg. Med. Chem. Lett. 2007, 17, 1934−1937. (b) Girgis, N. S.; Larson, S. B.; Robins, R. K.; Cottam, H. B. The synthesis of 5azaindoles by substitution-rearrangement of 7-azaindoles upon treatment with certain primary amines. J. Heterocycl. Chem. 1989, 26, 317− 325.

(19) (a) Axten, J. M.; Romeril, S. P.; Shu, A.; Ralph, J.; Medina, J. R.; Feng, Y.; Li, W. H. H.; Grant, S. W.; Heerding, D. A.; Minthorn, E.; Mencken, T.; Gaul, N.; Goetz, A.; Stanley, T.; Hassell, A. M.; Gampe, R. T.; Atkins, C.; Kumar, R. Discovery of GSK2656157: An optimized PERK inhibitor selected for preclinical development. ACS Med. Chem. Lett. 2013, 4, 964−968. (b) Devine, S. M.; Lim, S. S.; Chandrashekaran, I. R.; Macraild, C. A.; Drew, D. R.; Debono, C. O.; Lam, R.; Anders, R. F.; Beeson, J. G.; Scanlon, M. J.; Scammells, P. J.; Norton, R. S. A critical evaluation of pyrrolo[2,3-d]pyrimidine-4amines as Plasmodium falciparum apical membrane antigen 1 (AMA1) inhibitors. MedChemComm 2014, 5, 1500−1506. (c) Reader, J. C.; Matthews, R. P.; Klair, S.; Cheung, K.-M. J.; Scanlon, J.; Proisy, N.; Addison, G.; Ellard, J.; Piton, N.; Taylor, S.; Cherry, M.; Fisher, M.; Boxall, K.; Burns, S.; Walton, M. I.; Westwood, I. M.; Hayes, A.; Eve, P.; Valenti, M.; De Haven Brandon, A.; Box, G.; Van Montfort, R. L. M.; Williams, D. H.; Aherne, G. W.; Raynaud, F. I.; Eccles, S. A.; Garrett, M. D.; Collins, I. Structure-guided evolution of potent and selective CHK1 inhibitors through scaffold morphing. J. Med. Chem. 2011, 54, 8328−8342. (d) Khalaf, A. I.; Huggan, J. K.; Suckling, C. J.; Gibson, C. L.; Stewart, K.; Giordani, F.; Barrett, M. P.; Wong, P. E.; Barrack, K. L.; Hunter, W. N. Structure-based design and synthesis of antiparasitic pyrrolopyrimidines targeting pteridine reductase 1. J. Med. Chem. 2014, 57, 6479. (20) Pudlo, J. S.; Nassiri, M. R.; Kern, E. R.; Wotring, L. L.; Drach, J. C.; Townsend, L. B. Synthesis, antiproliferative, and antiviral activity of certain 4-substituted and 4,5-disubstituted 7-[(1,3-dihydroxy-2propoxy)methyl]pyrrolo[2,3-d]pyrimidines. J. Med. Chem. 1990, 33, 1984−1992. (21) Wang, X.; Seth, P. P.; Ranken, R.; Swayze, E. E.; Migawa, M. T. Synthesis and biological activity of 5-fluorotubercidin. Nucleosides, Nucleotides Nucleic Acids 2004, 23, 161−170. (22) Pudlo, J. S.; Saxena, N. K.; Nassiri, M. R.; Turk, S. R.; Drach, J. C.; Townsend, L. B. Synthesis and antiviral activity of certain 4- and 4,5-disubstituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidines. J. Med. Chem. 1988, 31, 2086−2092. (23) (a) Showalter, H. D. H.; Bridges, A. J.; Zhou, H.; Sercel, A. D.; McMichael, A.; Fry, D. W. Tyrosine kinase inhibitors. 16. 6,5,6tricyclic benzothieno[3,2-d]pyrimidines and pyrimido[5,4-b]- and [4,5-b]indoles as potent inhibitors of the epidermal growth factor receptor tyrosine kinase. J. Med. Chem. 1999, 42, 5464−5474. (b) Reader, J. C.; Matthews, T. P.; Klair, S.; Cheung, K.-M. J.; Scanlon, J.; Proisy, N.; Addison, G.; Ellard, J.; Piton, N.; Taylor, S.; Cherry, M.; Fisher, M.; Boxall, K.; Burns, S.; Walton, M. I.; Westwood, I. M.; Hayes, A.; Eve, P.; Valenti, M.; Brandon, A. d. H.; Box, G.; van Montfort, R. L. M.; Williams, D. H.; Aherne, G. W.; Raynaud, F. I.; Eccles, S. A.; Garrett, M. D.; Collins, I. Structure guided evolution of potent and selective CHK1 inhibitors through scaffold morphing. J. Med. Chem. 2011, 54, 8328−8342. (24) Mincione, F.; Menabuoni, L.; Briganti, F.; Mincione, G.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors: inhibition of isozymes I, II, and IV with N-hydroxysulfonamides − a novel class of intraocular pressure lowering agents. J. Enzyme Inhib. 1998, 13, 267−284. (25) Petrow, V.; Stephenson, O.; Wild, A. M. Studies in the field of diuretic agents part V. A new route to disulphamyl derivatives of benzene. J. Pharm. Pharmacol. 1960, 12, 705−719. (26) Mayasundari, A.; Fujii, N. Efficient formation of 4,6disubstituted pyrollo[2,3-d]pyrimidines: a novel route to TWS119, a glycogen synthase kinase-3β inhibitor. Tetrahedron Lett. 2010, 51, 3597−3598. (27) Clark, M. P.; George, K. M.; Bookland, R. G.; Chen, J.; Laughlin, S. K.; Thakur, K. D.; Lee, W.; Davis, J. R.; Cabrera, E. J.; Brugel, T. A.; VanRens, J. C.; Laufersweiler, M. J.; Maier, J. A.; Sabat, M. P.; Golebiowski, A.; Easwaran, V.; Webster, M. E.; De, B.; Zhang, G. Development of new pyrrolopyrimidine-based inhibitors of Janus kinase 3 (JAK3). Bioorg. Med. Chem. Lett. 2007, 17, 1250. (28) (a) Abdel-Magid, A. F.; Carson, K. C.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. Reductive amination of aldehydes and ketones with sodium triacetoxyborohoydride. Studies on direct and indirect Q

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX

Downloaded by RUTGERS UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 10, 2015 | doi: 10.1021/acs.jmedchem.5b00931

Journal of Medicinal Chemistry

Article

reductive amination procedures. J. Org. Chem. 1996, 61, 3849−3862. (b) Sabatucci, J. P.; Ashwell, M. A.; Trybulski, E.; O’Donnell, M.-M.; Moore, W. J.; Harnish, D. C.; Chadwick, C. C. Substituted 4hydroxyphenyl sulfonamides as pathway-selective estrogen receptor ligands. Bioorg. Med. Chem. Lett. 2006, 16, 854−858. (29) Morisawa, Y.; Kataoka, M.; Nagahori, H.; Sakamoto, T.; Kitano, N.; Kusano, K.; Sato, K. Studies on anticoccidial agents. 13. Synthesis and anticoccidial activity of nitropyridine-2- and −3-sulfonamides and derivatives. J. Med. Chem. 1980, 23, 1376−1380. (30) Wendt, M. D.; Shen, W.; Kunzer, A.; McClellan, W. J.; Bruncko, M.; Oost, T. K.; Ding, H.; Joseph, M. K.; Zhang, H.; Nimmer, P. N.; Ng, S.-C.; Shoemaker, A. R.; Petros, A. M.; Oleksijew, A.; Marsh, K.; Bauch, J.; Oltersdorf, T.; Belli, B. A.; Martineau, D.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo. J. Med. Chem. 2006, 49, 1165−1181. (31) For an example of using the H-bond donating ability of a gatekeeper Thr to introduce kinase selectivity, see: Martin, M. W.; Newcomb, J.; Nunes, J. J.; Boucher, C.; Chai, L.; Epstein, L. F.; Faust, T.; Flores, S.; Gallant, P.; Gore, A.; Gu, Y.; Hsieh, F.; Huang, X.; Kim, J. L.; Middleton, S.; Morgenstern, K.; Oliveira-dos-Santos, A.; Patel, V. F.; Powers, D.; Rose, P.; Tudor, Y.; Turci, S. M.; Welcher, A. A.; Zack, D.; Zhao, H.; Zhu, L.; Zhu, X.; Ghiron, C.; Ermann, M.; Johnston, D.; Saluste, C.-G. P. Structure-based design of novel 2-amino-6-phenylpyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-ones as potent and orally active inhibitors of lymphocyte specific kinase (Lck): Synthesis, SAR, and in vivo anti-inflammatory activity. J. Med. Chem. 2008, 51, 1637−1648. (32) Hammond, M.; Kallander, L. S.; Lawhorn, B. G.; Philp, J.; Sarpong, M. A.; Seefeld, M. A. Azolopyrimidine compounds as kinase inhibitors and their preparation and use in the treatment of disesases and methods. PCT Int. Appl. WO2011149827, 2011. (33) Casillas, L. N.; Chakravorty, S. J.; Charnley, A. K.; Eidam, P.; Haile, P. A.; Hughes, T. V.; Jeong, J. U.; Kang, J.; Lakdawala, S. A.; Leister, L. K.; Marquis, R. W.; Miller, N. A.; Price, D. J.; Sehon, C. L.; Wang, G. Z.; Zhang, D. Preparation of indazolyl-substituted pyrimidinediamine derivatives as RIP2 kinase inhibitors. PCT Int. Appl. WO2011120025, 2011. (34) May, E. W.; Rominger, C. M.; Schaber, M. D. Assay for B-Raf activity based on intrinsic MEK ATPase activity. PCT Int. Appl. US 20060211073, 2006. (35) Vagin, A.; Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 1997, 30, 1022−1025. (36) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. In Methods in Enzymology; Carter, C. W., Jr., Sweet, R. M., Eds.; Academic Press: New York, 1997; pp 307−326. (37) Emsley, P.; Cowtan, K. Coot: Model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132. (38) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240−255.

R

DOI: 10.1021/acs.jmedchem.5b00931 J. Med. Chem. XXXX, XXX, XXX−XXX