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Pyrazolylamine derivatives reveal the conformational switching between type-I and type-II binding modes of anaplastic lymphoma kinase (ALK) Chih-Hsiang Tu, Wen-Hsing Lin, Yi-Hui Peng, Tsu Hsu, Jian-Sung Wu, Chun-Yu Chang, Cheng-Tai Lu, Ping-Chiang Lyu, Chuan Shih, Weir-Torn Jiaang, and Suying Wu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00106 • Publication Date (Web): 31 Mar 2016 Downloaded from http://pubs.acs.org on March 31, 2016

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Journal of Medicinal Chemistry

Pyrazolylamine derivatives reveal the conformational switching between type-I and type-II binding modes of anaplastic lymphoma kinase (ALK) Chih-Hsiang Tu, †∥ Wen-Hsing Lin, †§ Yi-Hui Peng, †§ Tsu Hsu, † Jian-Sung Wu,† Chun-Yu Chang,† Cheng-Tai Lu,† Ping-Chiang Lyu, ∥ Chuan Shih,† Weir-Torn Jiaang, †* Su-Ying Wu†*

†

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC

∥ Institute

of Bioinformatics and Structural Biology, National Tsing Hua University, 101, Sect.2, Guangfu Road, Hsinchu 300, Taiwan, ROC

§ W.-H. L. and Y.-H. P. contributed equally

KEYWORDS: anaplastic lymphoma kinase (ALK), type-II kinase inhibitors, DFG (Asp-PheGly) motif, juxtamembrane (JM) domain.

ACS Paragon Plus Environment


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ABSTRCT Most Anaplastic lymphoma kinase (ALK) inhibitors adopt a type-I binding mode, but only limited type-II ALK structural studies are available. Herein, we present the structure of ALK in complex with N1-(3-4-[([5-(tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]-3-methylphenyl-1H5-pyrazolyl)-4-[(4-methylpiperazino)methyl]benzamide (5a), a novel ALK inhibitor adopting a type-II binding mode. It revealed binding of 5a resulted in the conformational change and reposition of the activation loop, αC-helix, and juxtamembrane domain, which are all important domains for the autoinhibition mechanism and downstream signal pathway regulation of ALK. A structure-activity relationship study revealed that modifications to the structure of 5a led to significant differences in the ALK potency and altered the protein structure of ALK. To the best of our knowledge, this is the first structural biology study to directly observe how changes in the structure of a small molecule can regulate the switch between the type I and type II binding modes, and induce dramatic conformational changes.

Introduction Anaplastic lymphoma kinase (ALK) was first discovered as the Nucleophosmin (NPM)-ALK fusion protein in the anaplastic large cell lymphoma (ALCL) of non-Hodgkin’s lymphoma (NHL) patients in 1994.1 It is classified as a receptor tyrosine kinase (RTK), and consists of an extracellular ligand binding domain, a transmembrane domain, a juxtamembrane domain, an intracellular kinase domain, and a C-terminal tail.2, 3 Midkine (MK) and pleiotrophin (PTN), two neurite growth-promoting factors (NEGF), have been reported as the activating ligands for mammalian ALK.2, 3 Since the first discovery of NPM-ALK in NHL patients, various ALKfused partners have been identified, including cysteinyl-tRNA synthetase (CARS)-ALK in


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inflammatory myofibroblastic tumors (IMT); clathrin (CLTC)-ALK in diffuse large B-cell lymphoma (DLBCL); and echinoderm microtubule associated protein like 4 (EML4)-ALK in non-small-cell lung carcinoma (NSCLC).2 Among these, EML4-ALK, identified in about 3 to 7% of NSCLS patients,4 is one of the most extensively studied ALK fusions. The N-terminal portion of EML4 facilitates ALK oligomerization, kinase activation, and subsequently initiates the signal transduction network. Studies have shown that mutations, gene amplification and chromosomal translocation of ALK are associated with various cancers.2 Therefore, ALK has been proposed as a drug target in the treatment of tumours; and the inhibition of ALK by small molecules constitutes a promising approach due to the low ALK mRNA and protein expression levels in normal human tissue.2,

5

Currently, several ALK inhibitors, including crizotinib,6

ceritinib,7 and alectinib5 are FDA-approved for the treatment of ALK positive NSCLC patients; and several second-generation inhibitors such as N2-[2-Methoxy-4-[4-(4-methyl-1-piperazinyl)1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfonyl]phenyl]-1,3,5-triazine-2,4-diamine (ASP3016),8

5-Chloro-N2-{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N4-[2-

(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine

(AP26113),9

and

(10R)-7-Amino-12-

fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922)10 are in clinical trials. Unfortunately, some of these are associated with adverse effects. For example, nausea, vomiting, diarrhea, visual disorders and peripheral edema were observed in patients after receiving crizotinib treatment.11,

12

Ceritinib produced adverse effects; nausea, diarrhea, vomiting and

fatigue were observed in clinical trials.13 The reported side effects of alectinib were dysgeusia, anemia, elevated aminotransferase levels, rash and constipation.14 Hence, further development of ALK inhibitors for the treatment of cancer is needed.



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Type-I kinase inhibitors are ATP competitors and target the DFG-in kinase conformation whereas type-II kinase inhibitors recognize the DFG-out conformation.15 In addition, type-II kinase inhibitors not only occupy the ATP binding site but also exploit the less conserved allosteric pocket by flipping the DFG motif, which might improve kinase selectivity and reduce the potential for resistance.16, 17 Moreover, type-II kinase inhibitors generally display a lower dissociation rate constant (koff) with prolonged residence time, which would extend the efficacy both in vitro and in vivo.18, 19 However, most type-II inhibitors tend to have high molecular weight and lipophilicity in order to exploit the additional hydrophobic pocket. In consequence, the majority of type-II inhibitors have the potential problems of limited solubility and worse pharmacokinetic (PK) properties.15 These require further efforts in the late stage of drug development to improve the druggability of type-II inhibitors. Nevertheless, the use of type II kinase inhibitors, such as imatinib,20 has been viewed as beneficial approach for the treatment of cancers. Unfortunately, due to the limitations of the traditional enzymatic assay, few type-II ALK kinase inhibitors have been identified.21,

22

In

addition, limited DFG-out ALK/inhibitor structures available for structure-guided in drug design may impede the effort on the design and optimization of type-II ALK inhibitors. In this study, a screening of in-house kinases led to the identification of 5a, a 3-phenyl-1H-5pyrazolylamine-based compound bearing a urea substituent, as an ALK inhibitor with an IC50 value of 177 nM. Herein, we describe structural biology studies of 5a with ALK, revealing it to adopt a type-II binding mode and concurrently rearrange the conformations of the activation loop, αC-helix, and juxtamembrane domain. Upon binding to ALK, compound 5a induces movement of the αC-helix which allosterically regulates the autoinhibition state of the juxtamembrane domain from anti-parallel β-sheets to a less compact, short helix conformation. A subsequent


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structure-activity relationship (SAR) study revealed that modifications to the structure of 5a led to a significant difference in the ALK inhibitor potency. Moreover, comparison of the co-crystal structure of ALK in complex with 5a with that of its analogue, 4, demonstrated that modification of inhibitor structure could switch it between a type I and type II binding mode, and induce dramatic conformational changes.

Results Identification of pyrazolylamine derivatives as ALK inhibitors. A novel class of 3-phenyl1H-5-pyrazolylamine derivatives23 has been utilized as a versatile template for the development of kinase inhibitors. Further screening of this series of compounds against in-house kinases led to the identification of urea-substituted series of compounds as ALK inhibitors. As shown in Table 1, 5a was the most potent ALK inhibitor, with an IC50 value of 177 nM. The general synthetic route to 3-phenyl-1H-5-pyrazolylamine benzamides, 5a and its analogs (Table 1), is shown in Scheme 1. Structure-activity relationship (SAR) studies showed that modifications to the ureasubstituted tail moiety (R1 group in Table 1) could significantly affect ALK potency. Removing the urea-substituted moiety of 5a to give 4 resulted in the complete loss of activity (IC50 > 10 uM), suggesting the urea-substituted moiety to be very important for the inhibition of ALK. Replacement of the isoxazole ring of 5a with a phenyl ring to give 5b decreased the activity to IC50 = 6.2 uM. Adding a methyl group next to the urea moiety to give 5c also reduced the activity (IC50 = 4.5 uM). The effect of alkyl substituents on the terminal isoxazole ring on ALK inhibition was also evaluated. In comparison with the bulky t-butyl group of 5a, compounds bearing the less sterically hindered isopropyl group (5d), ethyl group (5e) and methyl group (5f) were



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synthesized and tested, and the substitution found to result in a 2-fold, 4-fold and 32-fold decrease in potency, respectively. Removal of the methyl substituent (R2 group in Table 1) on the phenyl ring connected to the pyrazolylamine group to give 5g and 5h resulted in decreased ALK inhibition compared with their methyl analogues, 5a and 5d. The effect of adding a water-solubilizing group on ALK inhibition was investigated. Previous studies have demonstrated the importance of a water-solubilizing substituent on the benzamide moiety for optimal activity toward the FLT3 kinase.23-25 In this study, addition of various watersolubilizing benzamide substituents to the urea substituted moiety had dramatic effects on potency. With conservation of the 5-tert-butyl-isoxazole urea moiety, inhibitors bearing a pyrrolidine group tethered by either a two-carbon (5i) or three-carbon ether (5j) were found to be 3-fold and 7-fold less potent than a N-methylpiperazine group linked by a one-carbon tether (5a). A sharp drop in potency was observed when the pyrrolidine group (5i) was replaced by a morpholine group (5k), or an N-methylpiperazine group was directly attached to benzamide (5l). Both compounds 5k and 5l showed no inhibition (IC50 > 10 µM).

Crystal structure of 5a. To further elucidate the binding mode and get structural insights into the detailed interactions between compound 5a and ALK, a structural biology study of 5a in complex with the ALK kinase domain (amino acids 1084-1410) was performed. Compound 5a was soaked into the ALK native crystal and the structure of 5a bound to ALK determined to a resolution of 1.92 Å. Data collection and refinement statistics are summarized in Table 2. All the protein residues were well defined in the electron density map except for some flexible loops, including residues 1084-1092, 1124-1127, 1137-1143, 1216-1219, 1275-1287 and 1405-1410.


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The structural moieties of ALK in complex with 5a were: the small N-terminal lobe (N-lobe); the large C-terminal lobe (C-lobe); and the interconnection hinge region. The N-lobe is comprised of five antiparallel β-sheets and one dominant helix (αC-helix). The C-lobe is comprised predominantly of eight α-helices and an activation-loop (A-loop). The electron density map clearly shows that 5a is located in the ATP binding site between the N-lobe and Clobe. Unlike most known ALK inhibitors (which present as type-I inhibitors), 5a adopts the typeII inhibitor binding mode and exploits the additional hydrophobic pocket, creating by the rearrangement of the activation loop of ALK from the DFG-in to the DFG-out conformation. Indeed, 5a bears several structural features that bind to ALK: the hinge scaffold (pyrazolylamine), the water-solubilizing head group (N-methylpiperazine), the middle linker (tolyl), hydrogen-bonding moiety (urea) and hydrophobic tail group (t-butyl-isoxazole) (Figure 1A). The pyrazolylamine scaffold of 5a forms hydrogen bonds with Glu1197 and Met1199 in the hinge region (Figure 1B), stabilizing the binding orientation and coordinating 5-t-butyl-isoxazole group deeply into the hydrophobic pocket. The tolyl group at the 3-position next to the pyrazolylamine scaffold makes hydrophobic interactions with the surrounding residues, including Val1130, Leu1196, Leu1256, and Phe1271. Moreover, the rearrangement of the sidechain of Phe1271 of DFG motif provides the edge-to-face hydrophobic interactions with the tolyl group. The methyl substituent in the tolyl group has interactions with Phe1271 and also contributes to the inhibitory activity to ALK. Removal of this methyl substitute results in 5-fold decreased activity of 5g as compared with 5a. The N-methylpiperazine head group is located at the acidic zone, formed by the side chains of Asp1203, Ser1206 and Glu1210 and the backbone carbonyl oxygens of Ala1200 and Gly1201 (Figure 1B). The methylpiperazine group is a watersolubilizing group, often used in kinase inhibitor design to improve the physicochemical



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properties of compounds.26 However, the methylpiperazine group not only improved the physicochemical properties of 5a, but also significantly influences potency against ALK, as revealed in the aforementioned SAR study. The two tertiary amines of the N-methylpiperazine of 5a serve as bases to form the long-range, charge-charge interactions with the carboxylic acid group of Glu1210 and the backbone carbonyl oxygens of Ala1200 and Gly1201. With an increase (5k) or decrease (5l) in chain length, the inhibitory activities decrease. One reason for the dramatic loss of activity of 5k could be that the water-solubilizing group is away from the acidic region, which leads to the loss of charge-charge interactions with the surrounding residues. Another possibility is that the basicity of the water-solubilizing group might also contribute to the binding with the protein. The pKa of the nitrogen atom in the morpholine-4-ylmethoxy group of 5k is 4.09, while pKa of two nitrogen atoms in N-methylpiperazine of 5a are 7.84 and 8.07 as calculated by MarvinSketch program implemented in ChemAxon.27 The nitrogen atom in the morpholine-4-yl-methoxy group with the lower pKa value and less basicity might have weaker charge interactions with the acidic residues of ALK, resulting in a loss in activities. Moreover, the reason for the decreased activity of 5l could be that the shorter chain length restricted the orientation of N-methylpiperazine to form optimized interactions with the surrounding acidic residues. The urea moiety and the t-butyl isoxazole group of 5a are located at the additional hydrophobic pocket, creating by the shift of the DFG motif from the DFG-in to the DFG-out conformation. The urea moiety forms one hydrogen bond with the backbone of Asp1270 in the DFG motif, and two hydrogen bonds with the sidechain of Glu1167 in αC-helix. The t-butyl isoxazole group extends into the allosteric site surrounded by the HRD motif and the αC-helix, where it makes extensive hydrophobic interactions with Ile1171, Phe1174, Ile1179, Phe1245,


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His1247 and Ile1268. That these interactions between the t-butyl moiety and these hydrophobic residues contribute to the activity of 5a is evidenced by compounds 5d, 5e and 5f. When the tbutyl moiety is replaced with the smaller alkyl substituents, such as iso-propyl (5d), ethyl (5e), or methyl (5f), the activity decreases accordingly, suggesting that the hydrophobicity of the alkyl substituents correlates with the activity of compounds. Indeed, superimposition of the crystal structure of ALK/5a and of ALK/5d shows that the iso-propyl group of 5d loss of the hydrophobic interactions (Phe1174, Ile1179 and Phe1245) compared with the t-butyl group of 5a (Figure 2).

5a induces a dramatic conformational change of ALK. Superimposition of the ALK/5a complex structure with the apo ALK structure reveals that ALK undergoes significant conformational changes upon 5a binding. The root-mean-square deviation (RMSD) of these two aligned structures is 2.11 Å for 282 Cα atoms. Binding of 5a, a type-II kinase inhibitor, not only induces the conformational flipping of DFG motif but also results in the reconstitution of the αChelix, activation loop (A-loop) and juxtamembrane (JM) domain. (i) Rearrangement of Activation-loop (A-loop) and αC-helix. To elucidate the conformational change induced by 5a, the apo ALK crystal structure was solved to a resolution of 1.50 Å and compared with ALK/5a complex structure (Figure 3). There are various differences between these two structures. Firstly, in the apo ALK structure, the non-conserved region of the A-loop following the DFG motif adopted a short helix (α-AL, residue 1272-1280) and positioned orthogonally underneath the αC-helix to form the salt bridge and hydrogen bond interactions with αC-helix (i.e. Gln1159 in αC-helix/Arg1279 in α-AL; Asp1163 in αC-helix/Arg1275 and Arg1279

in

α-AL;

Glu1167

in

αC-helix/


Arg1275

in

α-AL).


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Tyr1278, located in the terminal of α-AL, is an autophosphorylation site in the A-loop and plays an important role in the regulation of ALK activation.28 Through edge-to-face, π-π interactions with Tyr1096 and the hydrogen bonding with backbone nitrogen atom of Ser1097 in JM domain, Tyr1278 serves as the latch which clamps the orientation of αC-helix and stabilizes the JM domain in an anti-parallel β-turn conformation (Figure 3A). In comparison, binding of 5a unfolded the α-AL to a non-structural loop and moved α-AL away from the αC-helix (Figure 3B), resulting in the loss of the salt bridge and hydrogen bond interactions with αC-helix. The loss of the interactions between α-AL and αC-helix moves the αC-helix 2 Å away from C-lobe to an open state (Figure 3C). Consequently, the interactions of Tyr1278 in α-AL with Tyr1096 and S1097 in the JM domain were concurrently lost. Secondly, 5a flipped the DFG motif from an in to an out conformation. This conformational change altered the internal architecture of the ALK kinase domain. In the apo ALK structure, the neighbouring residues (His1247 in HRD motif, Phe1271 in DFG motif, Ile1171 in the αC-helix and Cys1182 in the β4-strand) constitute the functional hydrophobic network, which is designated as regulatory spine (R-spine) (Figure 4A). The backbone nitrogen of His1247 in Rspine hydrogen bonded to the sidechain of Asp1311 in the helix-F, served as the base for the Rspine. Studies have shown that assembling and disassembling of the R-spine regulates the activity of protein kinase.29-31 Upon the binding of 5a to ALK, the R-spine is disrupted by the movement of Phe1271 in the DFG-motif and re-accommodation by the t-butyl-isoxazole group of 5a. In addition, the catalytic spine (C-spine) also showed differences between the apo ALK and ALK/5a complex structures. It also regulates activity of protein kinase; however, assembling or disassembling of the C-spine is dependent on the incorporation of ATP or substrate. In the apo ALK structure, without the ATP or substrate, the C-spine is incomplete; while in the ALK/5a


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complex structure, the pyrazole group of 5a interconnects the N-lobe and C-lobe through neighbouring hydrophobic residues and completes the C-spine (Figure 4B). (ii) Conformational change of juxtamembrane (JM) domain. Most of the published ALK structures revealed that the juxtamembrane (JM) domain (residues 1087-1115), flanked by the αC-helix, contain an N-terminal helix segment (residues 1086-1092) and an antiparallel β-turn motif (residues 1096-1103) (Figure 3A and Figure 3C). The β-turn motif in the JM domain is a unique feature for ALK, compared with other kinase structures. The hydrophobic residues from the JM domain, including Ile1088, Met1089, Pro1094, Tyr1096, Phe1098, Ile1105 and Leu1108, facilitate the close packing of the JM domain on the interface of αC-helix. In addition, the interactions between the JM domain (Tyr1096 and Ser1097) and the α-AL (Tyr1278) also assist the stabilization of the ALK kinase domain in the native conformation (Figure 3A). Interestingly, upon the binding of 5a, the JM domain transformed from the anti-parallel β-turn conformation to the helix conformation (residue 1096-1102), with concomitant disorder of the N-terminal helix segment of the JM domain (Figure 3B and Figure 3C). This conformational change in the JM domain results in significant loss of the contacted interface area with αC-helix, and disrupts the interactions with α-AL. Superimposition of the apo ALK structure with the ALK/5a complex structure revealed that the opened state αC-helix induced by the binding of 5a would clash with the JM domain (Figure 3D). Ile1088 and Met1089 in the N-terminal JM domain experience a steric clash with the Glu1158 and Glu1161 in the N-terminal αC-helix; while Tyr1096 and Phe1098 in the β-turn motif of the JM domain clash with Ile1170 and Phe1174 in the C-terminal part of αC-helix, respectively (Figure 3D). To avoid this, the JM domain undergoes a conformational change, from β-turn motif to the helix conformation and also



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disorders the N-terminal helix segment to a more flexible conformation to accommodate the opened state αC-helix. As a result of the conformational change of the JM domain in the ALK/5a structure, two potential autophosphorylation tyrosine residues, Tyr1092 and Tyr109632, 33 were repositioned. In the apo-ALK structure, Tyr1092 in the N-terminal helix segment was exposed to solvent region while Tyr1096 was deeply immersed in a hydrophobic pocket composed of Phe1098, Leu1169, Ile1170, Lys1173, Phe1174, Phe1245 and Tyr1278 (Figure 3A). However, in the ALK/5a structure, Tyr1092 was structurally disordered and Tyr1096 was exposed to solvent, where it forms hydrogen bonds with Asp1163 in the αC-helix and Asn1093 in the JM domain (Figure 3B). In the previous studies, Tyr1096 has been identified as the phosphoprotein binding domain (PBD) for insulin receptor substrate1 (IRS1) and Suc1-associated neurotrophic factor-induced tyrosinephosphorylated target (SNT).32 IRS1 binds to pTyr1096 and activates the downstream of cell division of the Ras/Erk1/2 signal pathway. It is suggested that binding of 5a to ALK results in reconstitution of the JM-domain and alters the interaction network of Tyr1096 with its surroundings, which may affect the autoinhibition state of ALK and the downstream signalling pathway inhibition. Tyr1096 is not only a key autophosphorylation site, but might also serve as a structural switcher to regulate the JM domain autoinhibition and kinase catalytic activity. The conformational change of the JM domain induced by the type-II kinase inhibitors kinase has also been observed for other tyrosine kinases, including FLT3/ 1-(5-tert-butyl-1,2-oxazol-3yl)-3-[4-[6-(2-morpholin-4-ylethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl]urea (AC220) (PDB code: 4XUF),34 c-KIT/imatinib (PDB code: 1T46)35 and VEGFR/sorafenib (PDB code: 4ASD).36 In addition, reposition of those auto-phosphorylation tyrosine residues has always accompanied rearrangement of the JM domain. For example, in the c-KIT structure, Tyr568 and


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Tyr570 are key auto-phosphorylation tyrosine residues that regulate the autoinhibition state of cKIT. The binding of imatinib to c-KIT led to the rearrangement of the JM domain and the repositioning of Tyr568 and Tyr570. However, most of those type-II inhibitors would directly clash with the JM domain; hence, the conformational change of JM domain is required to avoid this. In contrast, 5a is far away and has no directly steric clash with the JM domain as revealed in the ALK/5a complex structure. 5a induced an allosteric effect through the conformational change of αC-helix moving toward the JM domain, and consequently led to the rearrangement of JM domain to be compatible with the opened state of αC-helix. In conclusion, 5a induced conformation changes in the α-AL, αC-helix and JM domain, and thus disrupted the extensive interaction networks in the ALK structure. Consequently, the interactions between Tyr1278 in α-AL and Tyr1096 and S1097 in the JM domain were lost. These results suggest that 5a altered the interaction network formed by αC-helix, α-AL and JM domain, which regulates the autoinhibition and activation of ALK.

The impact of substituted-urea tail moiety on 5a binding. As revealed in the SAR study, 4 did not impart any ALK inhibitory activity (IC50 >10 µM), suggesting that the urea-substituted tail moiety was the dominant feature of 5a and contributed to the kinase inhibition activities. In order to evaluate the effect of the urea-substituted tail group on the binding of ALK at the molecular level, 4, a non-urea aryl analogue of 5a, was soaked into ALK crystal and the structure of the complex resolved to a resolution of 1.87 Å. The electron density map clearly shows that 4 presented as a type-I inhibitor, bound in the DFG-in conformation. The structure of ALK/4 is significantly different from the ALK/5a structure. Superimposition of these two structures gave a RMSD of 2.147 Å for 280 Cα atoms. Firstly, in the ALK/5a



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structure, the DFG motif adopts the out conformation to allocate the urea-substituted tail and makes the face-to-edge π-π interactions with the pyrazolylamine scaffold of 5a; while in ALK/4 structure, in the absence of the urea-substituted tail, the DFG motif adopts the in conformation, and has no direct interactions with the pyrazolylamine scaffold of 4 (Figure 5A). Secondly, in the ALK/5a structure, the αC-helix exhibits the open state conformation and the Glu1167 in the αC-helix forms hydrogen bonding interactions with the urea moiety (Figure 1). The JM domain was fully unfolded due to the allosteric effect derived from movement of αChelix. However, in the ALK/4 structure, the αC-helix adopts the close state conformation and the Glu1167 interacts with the amino-substituent on the 4-position of the tolyl group, through a water-mediated hydrogen bond (Figure 5B). Without the ligand-induced allosteric effect, the αChelix and JM domain of ALK/4 structure would adopt a similar conformation to the apo ALK structure (Figure 3A) and other published ALK complex structures with type I inhibitors. As revealed in the structural biology studies of 5a and 4, 5a is a type-II inhibitor bound to the DFG-out conformation, while 4 is a type-I inhibitor bound to the DFG-in conformation. Many reports have shown that the binding kinetics of type-II inhibitors and type-I inhibitors are different.18 Therefore, the binding kinetics of 5a and 4 were further evaluated by surface plasmon resonance (SPR). Binding of 4 showed a fragment-like squared-shaped sensorgram, indicating very fast on and fast off rates (Supporting information, Figure S1). In contrast, the association rate (kon) and dissociation rate (koff) for 5a were 5.17x104 1/Ms and 2.4x10-3 1/s, respectively, suggesting that 5a bound to ALK with slow association and dissociation rates. It has been reported that these slow binding kinetics correlate with protein conformation change induced by the ligand binding.37, 38 The urea-substituted tail moiety endows 5a with a characteristic slow offrate, consistent with the requirement of DFG motif conformational change.


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In addition to the ALK/5a structure, the structure of ALK/4 also compared with apo ALK structure. The most significant difference between ALK/4 and apo ALK structures is the conformation change of the phosphate-binding loop (P-loop) upon the binding of 4. In the ALK/4 structure, the P-loop adopts a bent conformation and orients the conserved aromatic residue Phe1127 into the catalytic site, directly stacked onto the tolyl group of 4 by edge-to-face π-π interaction, whereas the P-loop adopted an extended conformation in most of the ALK structures (Figure 5C). In many kinase/inhibitor complex structures, bending of the P-loop always creates the face-to-face or edge-to-face aromatic interaction between the conserved aromatic residue and inhibitors such as Aurora-A/N-[4-[4-(4-methylpiperazin-1-yl)-6-[(5methyl-1H-pyrazol-3-yl)amino]pyrimidin-2-yl]sulfanylphenyl]cyclopropanecarboxamide

(VX-

680) (PDB code: 3E5A),39 c-Abl/imatinib (PDB code: 1IEP),40 and p38α/1-[5-tert-butyl-2-(4methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea

(BIRB796)

(PDB code: 1KV2)18 complex structures.

Comparing 5a with others ALK kinase inhibitors. To elucidate the similarities and differences of structure conformation and protein-ligand interactions, crystal structures of 5a and different ALK kinase inhibitor (Figure 6A) complex structures were superimposed. Superimposition of 5a and crizotinib (PDB code: 2XP2)6 revealed that the structural conformations are quite different (Figure 6 B and C). The structure of ALK/crizotinib resembles apo ALK and the binding of crizotinib to ALK does not induce dramatic conformational changes. The conformation of ALK/crizotinib is the DFG-in conformation and the αC-helix adopts the close state accompanying with the JM domain in the anti-parallel β-sheets conformation. In



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contrast, ALK/5a was found to present the DFG-out conformation, where the αC-helix adopts the open state accompanying with the JM domain in the helix conformation. Both the pyrazolyl-5-amine of 5a and pyridine-2-amine of crizotinib (Figure 6A, highlighted in cyan) formed hydrogen bonds to the hinge residues Glu1197 and Met1199. However, the most significant differences between 5a and crizotinib are that the t-butyl-isoxazole urea moiety (Figure 6A, highlighted in purple) of 5a extended to the allosteric pocket, whereas the 2,6dichloro-3-fluoro-phenyl group (Figure 6A, highlighted in red) of crizotinib was oriented to small hydrophobic cavity underneath the P-loop instead. Conformational changes in ALK induced by 5a, accompanied by additional interactions with the allosteric pocket, led to the slowbinding kinetics of 5a as compared with crizotinib. The binding association and dissociation rate constants of 5a (kon: 5.17x104 1/Ms; koff: 2.4 x10-3 1/s) were about 226-fold and 15-fold slower than crizotinib (kon: 1.17x107 1/Ms; koff: 3.55x10-2 1/s). The slower association rate of 5a may due to the higher energy barrier for inducing ALK conformational change. Slow dissociation-rate has shown to be correlated with the potency and prolonged drug effects both in vitro and in vivo.18, 37, 38, 41 Next, the structure of 5a was further compared with 6, a type-II ALK inhibitor (PDB code: 4FNY).22 Superimposing the crystal structure of 5a with 6 show differences between these two structures. Firstly, the JM domain is visible in ALK/5a structure but absent in ALK/6 structure (Figure 6D). Binding of 5a reorients the αC-helix to an open state and allosteric effects regulate the refolding of the JM domain to a short helix segment. However, in the ALK/6 structure, the refolding of the JM domain is not observed due to the disordered in this region. Secondly, in the ALK/5a structure, α-AL was unfolded to a non-structural loop and moved away from the αChelix while α-AL was completely invisible in the ALK/6 structure.


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Moreover, by superimposing the structure complex of 6 onto 5a, it revealed that pyrazolyl-5amine (Figure 6A, highlighted in cyan) of 5a formed three hydrogen bonds with hinge residues Glu1197 and Met1199, while the nitrogen atom of the dimethoxyquinoline group (highlighted in cyan) in 6 made one hydrogen bond with Met1199. Moreover, the phenyl group together with the N-methylpiperidine group of 5a extend to the solvent exposed region of ALK to form hydrophobic interactions with the surrounding residues and made charge-charge interactions with the acidic zone. In contrast, in ALK/6 complex structure, the solvent exposed region is unoccupied and two methoxy substitutions at quinoline group of 6 were much less exposed to the bulk solvent and had no interactions with the acidic zone.

Discussion and Conclusions. In this study, 5a, a 3-phenyl-1H-5-pyrazolylamine-based compound bearing a urea substituent, was identified by in-house kinase screening as a novel ALK inhibitor. Further structural biology studies of 5a with ALK revealed that 5a adopts a typeII binding mode. The urea-substituted moiety of 5a flips the DFG-motif from an in-to-out inactive conformation and coordinates the orientation of 5a through the hydrogen bonds with Glu1167 in the αC-helix and Asp1270 in DFG motif. In addition, the t-butyl group of 5a allows for a hydrophobic interaction with the allosteric pocket created by the conformational change of DFG motif. Interestingly, binding of 5a induces a dramatic conformational change of ALK by spatial rearrangement of A-loop, αC-helix, and JM domain. 5a unfolded the A-loop to a nonstructural loop and moved the α-AL away from the αC-helix, resulting in the loss of the salt bridge and hydrogen bond interactions with αC-helix. The loss of the interactions between α-AL and the αC-helix reorients the αC-helix 2 Å away from the C-lobe to an opened state. Furthermore, the binding of 5a results in the transformation of the JM domain from the



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antiparallel β-turn conformation to the helix conformation and the disorder of N-terminal helix segment of JM domain. The conformational change of JM domain significantly reduces the contacted interface area with αC-helix and disrupts the interactions with α-AL. As a consequence of this conformational change and spatial rearrangement, the binding of 5a to the ALK kinase domain alters the interaction network of Tyr1096, an important autophosphorylation residue located in juxtamembrane domain, and may affect the autoinhibition mechanism of ALK and downstream signal pathway regulation. Further comparison of the co-crystal structures of ALK/5a with ALK/4, revealed that the substituted-urea tail moiety controls the conversion between the DFG-in and DFG-out conformation and re-allocate the A-loop, αC-helix, and JM domain. Further studies of 5a and 4 showed these two compounds have different molecular properties, such as the polarizability, lipophilicity and molecular surface area. The polarizability of compound 5a and 4 are 62.85 Å3 and 46.99 Å3, respectively, indicating they have different electronic properties. The logP values of compound 5a and 4 are 4.54 and 2.61, respectively, suggesting 5a exhibited higher lipophilicity. Moreover, the molecular surface area of compound 5a is 863.6 Å2 and that of compound 4 is 615.3 Å2, suggesting they have different impacts on steric effects with the protein. The differences in physicochemical and structural properties of inhibitors would also contribute to the conversion between the type-I and type-II binding modes and the conformational change of ALK. Furthermore, the ligand efficiency (LE) of 5a and analogues were calculated (Table 1). Based on the structural observation from ALK/5a, it is suggested that replacing the N-methylpiperazine group of 5a with a guanidine group may potentially provide electrostatic interactions and hydrogen bond interactions with the acid region in ALK to improve the potency (Figure S3 in the


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supporting information). In addition, the replacement of N-methylpiperazine group of 5a with a guanidine group would reduce the molecular weight (Figure S3 in the supporting information), which would potentially improve the ligand efficiency of 5a. To the best of our knowledge, this is the first structural biology study to directly observe how changes in a small molecule can regulate the switch between the type I and type II binding modes, and induce dramatic conformational changes in protein structures. Our results are expected to be useful for activation/inhibition mechanism studies of ALK, and should give insights into the development of the novel type-II inhibitors for ALK or the other kinases in the future.

Experimental Section ALK Expression, Purification and Crystallization. Human ALK kinase domain (residues 1084-1410) was expressed using baculovirus system (Sf21 cells). The residue Cys1097 was mutated to serine to facilitate protein expression and crystallization as reported previously.22 Cell suspension was centrifuged at 4500 r.p.m. for 15 minutes and the cell pellet was suspended in Tris buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM imidazole and 1mM TCEP) and lysed by sonication. The supernatant was filtered with 0.22 filters and loaded onto HiTrap HP Ni-affinity column (GE Life Sciences). ALK was eluted with 75 mM imidazole in wash buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and buffer exchanged to Factor Xa cleavage buffer (50 mM Tris-HCl, 100 mM NaCl. 5 mM CaCl2, pH 8.0) by Hiprep 26/10 desalting column (GE Life Sciences). His tag was then removed by Factor Xa (Novagen) cleavage at 4°C. ALK was finally exchanged to the protein buffer (25 mM Tris-HCl, 125 mM NaCl, 14 mM β-ME, pH 8.5) and concentrated to 12-15 mg/ml.



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ALK was crystallized by sitting drop method. Equal volumes of reservoir solution (18% PEG2000 MME, 0.1 M Tris-HCl pH 8.6) and ALK were mixed and grown at 4°C. ALK crystals were soaked with 0.5 mM tested compounds for more than 6 hours. The ALK crystal was then quickly immersed in the reservoir solution with additional 20% ethylene glycol and then cooled in the liquid nitrogen. Diffraction data were collected at beamline BL15A1 (NSRRC, Taiwan), BL12B2 and 44XU (Spring-8, Japan). Data were processed by HKL2000 program.42 The phase of initial ALK structure was resolved by molecular replacement using PHENIX.43 Structure refinement and model building were performed using PHENIX and COOT-0.8.1.44 Figures were prepared with PyMOL v1.7.2 (Schrodinger, LLC).

Kinase Inhibition Assay. In vitro kinase assays were carried out in 96-well plate with tested compounds in the reaction buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, 4 mM MnCl2, 0.5 mM Na3VO4, 2 mM DTT, 0.02% TritonX-100, and 0.01% BSA). Total of 50 µl of reaction mixture contained the testing compounds, l µM ATP (Sigma), 2 µM polyGlu4:Tyr peptide (Sigma), and 100 ng ALK protein. The reaction was performed at 30°C for an hour. The kinase assay was run with phosphorylated ALK. Equal volume of kinase-Glo plus reagent (Promega) was added after one hour of incubation. The reaction mixture was transferred to a black plate and recorded the luminescence by Wallac Vector 1420 multilabel counter (PerkinElmer,Shelton, CT, USA). Surface Plasmon Resonance (SPR). The ligand was immobilized on the CM5 chip (GE Life Sciences) using amine-coupling method. The immobilization procedure was based on the default setting on the Biacore-T200 control software. PBS buffer (pH 7.5, contained 5% fresh DMSO) was used as the running buffer. 5a (50 mM) in 100% DMSO was dissolved in 1x running buffer to give the analyte solution concentration 1.25 to 0.046 µM. Crizotinib (1mM) was used to


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prepare analyte solution 40 to 2.5 nM. Three cycles of start-up and one cycle of solvent correction were performed before sample-cycle run. Solvent correction (8 concentrations, ranged from 4.5% to 5.5% DMSO in 1xPBS pH 8.5) was used to adjust response error due to the content mismatch between running buffer and sample. The LMW single-cycle kinetics analysis consisted of 120 seconds of association (30 µl/min), 300 seconds of dissociation (30 µl/min), 30 seconds of carry-over control (40 µl/min) and finally 60 seconds of surface stabilization (30 µl/min). Except five concentrations of sample injection, two double reference cycle-run were repeated. Biacore-T200 evaluation software version2.0 was used for the affinity and kinetic analysis. Molecular properties calculation. The polarizability and molecular surface area

of

compound 5a and 4 were calculated by MarvinSketch program implemented in ChemAxon.27 The logP value of compound 5a and 4 were calculated by Chemdraw Ultra 7.0.

Chemistry methods. All commercial chemicals and solvents are reagent grade and were used without further treatment unless otherwise noted. 1H NMR spectra were obtained with a Varian Mercury-300 or a Varian Mercury-400 spectrometer. Chemical shifts were recorded in parts per million (ppm, δ) and were reported relative to the solvent peak or TMS. LC/MS data were measured on an Agilent MSD-1100 ESI-MS/MS System. High-resolution mass spectra (HRMS) were measured with a Thermo Finnigan (TSQ Quantum) electrospray ionization (ESI) mass spectrometer. Flash column chromatography was done using silica gel (Merck Kieselgel 60, No. 9385, 230-400 mesh ASTM). Reactions were monitored by TLC using Merck 60 F254 silica gel glass backed plates (5 × 10 cm); zones were detected visually under ultraviolet irradiation (254 nm) or by spraying with phosphomolybdic acid reagent (Aldrich) followed by heating at 80 °C.



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All starting materials and amines were commercially available unless otherwise indicated. The purity of compounds was determined by a Hitachi 2000 series HPLC system. Compound purities were determined by reverse phase C18 column (Agilent ZORBAX Eclipse XDB-C18 5 µm, 4.6 mm × 150 mm) under the following gradient elution condition: Mobile phase A-acetonitrile (10% to 90%, 0 to 45 min) and mobile phase B-10 mM NH4OAc aqueous solution containing 0.1% formic acid (90% to 10%, 0 to 45 min). The flow-rate was 0.5 ml/min and the injection volume was 20 µl. The system operated at 25 °C. Peaks were detected at 254 nm.

N-[3-(4-Amino-3-methylphenyl)-1H-pyrazol-5-yl]-4-[(4-methylpiperazin-1yl)methyl]benzamide (4). Mp 229–230 °C. 1H NMR (300 MHz, DMSO-d6): 11.12 (s, 1H), 8.11 (d, J = 8.1 Hz, 2H), 8.00 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.1 Hz, 2H), 7.77 (s, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.03 (s, 1H), 4.51 (bs, 2H), 3.78-3.30 (m, 6H), 3.15 (s, 3H), 2.90-2.70 (m, 4H), 2.43 (s, 3H); MS (ES+) m/z calcd. for C23H28N6O: 404.23; found: 405.2 (M+H+). HPLC: tR = 14.09 min, 95.0%. N1-(3-4-[([5-(Tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]-3-methylphenyl-1H-5pyrazolyl)-4-[(4-methylpiperazino)methyl]benzamide (5a). Mp 263–264 °C. 1H NMR (300 MHz, DMSO-d6): 10.78 (s, 1H), 9.95 (s, 1H), 8.35 (s, 1H), 7.99-7.97 (m, 3H), 7.73 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.11 (s, 1H), 6.48 (s, 1H), 3.52 (s, 2H), 3.35 (s, 4H), 2.37 (s, 4H), 2.30 (s, 3H), 2.15 (s, 3H), 1.30 (s, 9H); MS (ES+) m/z calcd. for C31H38N8O3: 570.31; found: 571.4 (M+H+); HRMS (ESI) calcd. for C31H39N8O3: 571.3145; found: 571.3136 (M+H+). HPLC: tR = 22.66 min, 99.1%. N-[3-(4-{[(3-Fluorophenyl)carbamoyl]amino}phenyl)-1H-pyrazol-5-yl]-4-[(4methylpiperazin-1-yl)methyl]benzamide (5b). Mp 225–227 °C. 1H NMR (400 MHz, DMSO-


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d6): 12.80 (s, 1H), 10.78 (s, 1H), 9.07 (s, 1H), 8.99 (s, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H), 7.49 (s, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.31 (q, J = 7.8 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.97 (s, 1H), 6.79 (td, J = 8.4, 2.4 Hz , 1H), 3.67 (s, 2H), 2.62-2.00 (m, 8H), 2.16 (s, 3H); MS (ES+) m/z calcd. for C29H30FN7O2: 527.24; found: 528.2 (M+H+). HPLC: tR = 21.21 min, 98.7%. N1-[3-(4-[([5-(Tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]methylphenyl)-1H-5pyrazolyl]-4-[(4-methylpiperazino)methyl]benzamide (5c). Mp 226–227 oC. 1H NMR (300 MHz, DMSO-d6): 10.82 (s, 1H), 10.71 (s, 1H), 9.50 (s, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.36 (t, J = 8.4 Hz, 2H), 7.02 (t, J = 5.7 Hz, 2H), 6.38 (s, 1H), 4.35 (d, J = 5.7 Hz, 2H), 3.52 (s, 2H), 3.25 (s, 4H), 2.37 (s, 4H), 2.15 (s, 3H), 1.27 (s, 9H); MS (ES+) m/z calcd. for C31H38N8O3: 570.31; found: 571.4 (M+H+); HRMS (ESI) calcd. for C31H39N8O3: 571.3140; found: 571.3147 (M+H+). HPLC: tR = 20.98 min, 98.1%. 4-[(4-Methylpiperazin-1-yl)methyl]-N-{3-[3-methyl-4-({[5-(propan-2-yl)-1,2-oxazol-3yl]carbamoyl}amino)phenyl]-1H-pyrazol-5-yl}benzamide (5d). Mp 235–236 °C. 1H NMR (400 MHz, DMSO-d6): 12.80 (s, 1H), 10.78 (s, 1H), 9.93 (s, 1H), 8.35 (s, 1H), 8.05-7.95 (m, 3H), 7.62 (s, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.00 (s, 1H), 6.49 (s, 1H), 3.52 (s, 2H), 3.02 (sep, J = 7.2 Hz, 1H), 2.60-2.00 (m, 8H), 2.37 (s, 3H), 1.25 (d, J = 7.2 Hz, 6H); MS (ES+) m/z calcd. for C30H36N8O3: 556.29; found: 557.3 (M+H+). HRMS (ESI) calcd. for C30H37N8O3: 557.2989; found: 557.2994 (M+H+). HPLC: tR = 21.79 min, 99.9%. N1-3-[4-([(5-Ethyl-3-isoxazolyl)amino]carbonylamino)-3-methylphenyl]-1H-5-pyrazolyl4-[(4-methylpiperazino)methyl]benzamide (5e). Mp 225–227 °C.

1

H NMR (400 MHz,

DMSO-d6): 12.80 (s, 1H), 10.77 (s, 1H), 9.91 (s, 1H), 8.36 (s, 1H), 7.99-7.97 (m, 3H), 7.62-7.56 (m, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.00 (s, 1H), 6.52 (s, 1H), 3.50 (s, 2H), 3.17 (d, J = 4.8 Hz,



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3H), 2.67 (q, J = 7.6 Hz, 2H), 2.37 (br s, 8H), 2.15 (s, 3H), 1.22 (t, J = 7.6 Hz, 3H); MS (ES+) m/z calcd. for C29H34N8O3: 542.28; found: 543.3 (M+H+). HRMS (ESI) calcd. for C29H35N8O3: 543.2832; found: 543.2839 (M+H+). HPLC: tR = 20.16 min, 95.1%. N1-3-[3-Methyl-4-([(5-methyl-3-isoxazolyl)amino]carbonylamino)phenyl]-1H-5pyrazolyl-4-[(4-methylpiperazino)methyl]benzamide (5f). Mp 155–157 °C.1H NMR (300 MHz, DMSO-d6): 10.78 (s, 1H), 9.89 (s, 1H), 8.36 (s, 1H), 7.99-7.97 (m, 3H), 7.62 (s, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.00 (s, 1H), 6.52 (s, 1H), 3.52 (s, 2H), 3.36 (s, 4H), 2.37 (s, 7H), 2.30 (s, 3H), 2.15 (s, 3H); MS (ES+) m/z calcd. for C28H32N8O3: 528.26; found: 529.3 (M+H+). HRMS (ESI) calcd. for C28H32N8O3: 528.2597; found: 528.2598 (M+). HPLC: tR = 18.55 min, 99.5%. N1-(3-4-[([5-(Tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]phenyl-1H-5-pyrazolyl)-4[(4-methylpiperazino)methyl]benzamide (5g). Mp 247–248 °C. 1H NMR (300 MHz, DMSOd6): 10.79 (s, 1H), 9.73 (s, 1H), 9.23 (s, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 6.97 (s, 1H), 6.52 (s, 1H), 3.51 (d, J = 5.1 Hz, 2H), 3.28 (t, J = 4.8 Hz, 4H), 2.37 (t, J = 4.8 Hz, 4H), 2.16 (s, 3H), 1.30 (s, 9H); MS (ES+) m/z calcd. for C30H36N8O3: 556.29; found: 557.4 (M+H+); HRMS (ESI) calcd. for C30H37N8O3: 557.2993; found: 557.2988 (M+H+). HPLC: tR = 22.29 min, 93.3%. 4-[(4-Methylpiperazin-1-yl)methyl]-N-{3-[4-({[5-(propan-2-yl)-1,2-oxazol-3yl]carbamoyl}amino)phenyl]-1H-pyrazol-5-yl}benzamide (5h). Mp 229–230 °C. 1H NMR (400 MHz, DMSO-d6): 12.82 (s, 1H), 10.78 (s, 1H), 9.60 (s, 1H), 9.09 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 7.6 Hz, 2H), 6.98 (s, 1H), 6.54 (s, 1H), 3.52 (s, 2H), 3.03 (sep, J = 6.8 Hz, 1H), 2.60-2.02 (m, 8H), 2.16 (s, 3H), 1.25 (d, J


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= 6.8 Hz, 6H); MS (ES+) m/z calcd. for C29H34N8O3: 542.28; found: 543.3 (M+H+). HPLC: tR = 21.28 min, 97.2%. N1-(3-4-[([5-(Tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]phenyl-1H-5-pyrazolyl)-4-(2tetrahydro-1H-1-pyrrolylethoxy)benzamide (5i). Mp 256–257 oC. 1H NMR (400 MHz, DMSO-d6): 12.79 (s, 1H), 10.68 (s, 1H), 9.59 (s, 1H), 8.99 (s, 1H), 8.02 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.96 (bs, 1H), 6.52 (s, 1H), 4.16 (t, J = 5.8 Hz, 2H), 2.83 (t, J = 5.8 Hz, 2H), 2.62-2.50 (m, 4H), 1.80-1.64 (m, 4H), 1.32 (s, 9H); MS (ES+) m/z calcd. for C30H35N7O4: 557.64; found: 558.6(M+H+); HRMS (ESI) calcd. for C30H36N7O4: 558.2823; found: 558.2841 (M+H+). HPLC: tR = 42.79 min, 95.6%. N-[3-4-{[(5-(Tert-butyl)-1,2-oxazol-3-yl)carbamoyl]amino}phenyl]-1H-pyrazol-5-yl]-4-(3(pyrrolidin-1-yl)propoxy]benzamide (5j). Mp 172–174 °C. 1H NMR (300 MHz, DMSO-d6): 10.65 (s, 1H), 9.77 (s, 1H), 9.61 (s, 1H), 7.98 (d, J = 9.3 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.86 (bs, 1H), 6.48 (s, 1H), 4.08 (t, J = 6.2 Hz, 2H), 3.10-2.60 (m, 6H), 2.03 (t, J = 7.1 Hz, 2H), 1.85-1.60 (m, 4H), 1.25 (s, 9H); MS (ES+) m/z calcd. for C31H37N7O4: 571.29; found: 572.1(M+H+). HPLC: tR = 24.24 min, 95.6%. N1-(3-4-[([5-(Tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]-3-methylphenyl-1H-5pyrazolyl)-4-(2-morpholinoethoxy)benzamide (5k). Mp 258–260 oC. 1H NMR (300 MHz, DMSO-d6): 12.77 (s, 1H), 10.69 (s, 1H), 9.98 (s, 1H), 8.38 (s, 1H), 7.99 (t, J = 9.6 Hz, 3H), 7.61-7.55 (m, 2H), 7.04 (d, J = 9.0 Hz, 2H), 6.91 (s, 1H), 6.48 (s, 1H), 4.16 (t, J = 5.7 Hz, 2H), 3.58 (t, J = 4.5 Hz, 4H), 2.71 (t, J = 5.7 Hz, 2H), 2.50-2.46 (m, 4H), 2.30 (s 3H), 1.30 (s, 9H); MS (ES+) m/z calcd. for C31H37N7O5: 587.29; found: 588.4 (M+H+); HRMS (ESI) calcd. for C31H38N7O5: 588.2934; found: 588.2926 (M+H+). HPLC: tR = 23.60 min, 98.0%.



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Page 26 of 48

N1-(3-4-[([5-(Tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]phenyl-1H-5-pyrazolyl)-4-(4methylpiperazino)benzamide (5l). Mp 332–334 oC. 1H NMR (400 MHz, DMSO-d6): 12.74 (s, 1H), 10.51 (s, 1H), 9.58 (s, 1H), 8.99 (s, 1H), 7.92 (d, J = 9.2 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 6.99-6.92 (m, 3H), 6.53 (s, 1H), 3.29-3.27 (m, 4H), 2.47-2.43 (m, 4H), 2.23 (s, 3H), 1.27 (s, 9H); MS(ES+) m/z calcd. for C29H34N8O3: 542.28; found: 543.3 (M+H+); HRMS (ESI) calcd. for C29H34N8O3: 543.2827; found: 543.2832 (M+H+). HPLC: tR = 22.70 min, 99.9%.


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ASSOCIATED CONTENT Supporting Information Binding kinetics analysis of compound 5a, 4 and crizotinib. The density maps of 5a, 4 and 5d in the active site of ALK. The docking position of the designed ALK inhibitor in the binding site of ALK. This material is available free of charge via the Internet at http://pubs.acs.org. PDB ID Codes: 5IUG (ALK/5a), 5IUH(ALK/5d), 5IUI(ALK/4) Authors will release the atomic coordinates with experimental data upon article publication.

AUTHOR INFORMATION Corresponding Author * Phone: +886-37-246-166 extension 35713. Fax: + 886-37-586-456. Email: [email protected] for SYW. Phone: +886-37-246-166 extension 35712. Fax: + 886-37-586-456. Email: [email protected] for WTJ Funding Sources This work was supported by National Health Research Institute, Taiwan, ROC and National Science Council (NSC 104-2325-B-400 -003). ABBREVIATIONS ALK, anaplastic lymphoma kinase; NHL, non-Hodgkin’s lymphoma; CARS, cysteinyl-tRNA synthetase ; IMT, inflammatory myofibroblastic tumors; DLBCL, diffuse large B-cell lymphoma; EML4, echinoderm microtubule associated protein like 4; NSCLC, non-small-cell lung carcinoma; DFG, Asp-Phe-Gly; HRD, His-Arg-Asp; JM domain, juxtamembrane domain;



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A-loop, activation loop; P-loop, phosphate-binding loop; R-spine, regulatory spine; C-spine, catalytic spine.


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etheno)pyrazolo[4,3-h][2,5,11]-

benzoxadiazacyclotetradecine-3-carbonitrile

a

(PF-06463922),

macrocyclic

inhibitor

of

anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure and broad-spectrum potency against ALK-resistant mutations. J. Med. Chem. 2014, 57,


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Page 37 of 48

Table 1. ALK inhibition of 5a and analogous H N

N

H N 3

R

O

1

R

2

R

R1

ID

R2

R3

ALK inhibitiona IC50 (nM)

Ligand Efficiency (LE)b

> 10000

-

177

0.225

6203

0.182

4500

0.178

402

0.218

805

0.213

5642

0.188

860

0.207

855

0.212

559

0.213

N N

CH3

H 2N

4

HN

HN

N

N

O O

N

CH3

5a HN

HN

N

O

CH3

N

5b

HN

HN

F N

N

O O

N

H

5c HN

HN

N

N

O O

N

CH3

5d HN

HN

N

N

O O

CH3

N

5e

HN

HN

N

N

O O

CH3

N

5f

HN

HN

N

N

O O

N

H

5g HN

HN

N

N

O O

N

H

5h

O

HN

HN

N O

5i

H

N

O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




HN

HN

N

O

O O

N

H

5j

1200

0.197

>10000

-

>10000

-

O

HN

HN

N O

N

O

CH3

5k

O

HN

HN

N O

a

H

N

5l

N

O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Values are expressed as the mean of at least three independent experiments and are within

±15%. b LE= -1.4*log(IC50)/HAC.45 HAC is the number of non-hydrogen atoms of the ligand.


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Table 2. Structure data collection and structure refinement statistics of ALK complex structure.

apo ALK

ALK/5a

ALK/4

ALK/5d

19.69 - 1.5

29.29 – 1.92

23.63 - 1.87

19.53 - 2.09

P212121

P212121

P212121

P212121

a=51.768

a=51.913

a=51.851

a=52.29

b=57.999

b=56.495

b=57.432

b=56.575

c=104.691

c=102.710

c=105.017

c=103.049

50680 (4931)

23336 (2256)

25768 (2543)

17835 (1618)

99.17 (97.92)

99.31 (98.47)

97.93 (98.07)

96.10 (90.29)

Mean I/sigma(I)

16.08 (2.92)

15.82 (3.62)

12.50 (3.04)

15.66 (3.81)

Wilson B-factor

19.74

23.91

23.45

28.97

R-work/R-free (%)

18.18/20.07

18.29/21.12

17.94/20.51

18.11/22.08

RMSD (bonds)

0.006

0.004

0.007

0.005

RMSD (angles)

0.96

0.93

1.07

0.88

Ramachandran favored (%)

99

97

97

99

1.49

0.45

2.33

1.12

0.89

0.77

1.19

0.89

Resolution range (Å) Space group

Unit cell (Å) (α=β=γ=90°)

Unique reflections Completeness(%) (outer shell)

MolProbity Clashscore MolProbity Score



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Figure 1. (A) The pharmacophore of 5a, the type-II ALK inhibitor. 5a was divided into five different portions, including the water-exposed head group (yellow); hinge binding group (cyan); aromatic linker (green); hydrogen bonding motif (purple); and tail group (purple). (B) Crystal structure of ALK in complex with 5a (yellow stick). Hydrogen bond and charge-charge interaction are shown with red and green dashed lines, respectively.


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Figure 2. Superposition of the crystal structure of ALK/5a with ALK/5d gave a RMSD of 0.354 Å for 284 Cα atom. Both the tert-butyl group of 5a (yellow) and the iso-propyl group of 5d (cyan) extended into the hydrophobic pocket. As compared with the structure of ALK/5a, the iso-propyl group of 5d loses the interactions with Phe1174, Ile1179 and Phe1245 (marked in green).



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Figure 3. (A) The spatial arrangement of the αC-helix (cyan), α-AL helix (purple) and JM domain (green) in apo ALK structure. (B) The spatial arrangement of the αC-helix, α-AL and JM domain in ALK/5a structure. (C) Comparison of the structures of apo ALK (pale purple) and ALK/ 5a (pale pink). Binding of 5a unfolds the α-AL to a non-structural loop and moves α-AL away from the αC-helix, which consequently shifts the αC-helix away from C-lobe to an open state. In addition, the JM domain concurrently transforms from the anti-parallel β-turn conformation to the helix conformation. Red arrow indicates that αC-helix shifts outward to an open state upon the binding of 5a. (D) A 60° rotated view of (C). Binding of 5a induces the dramatic conformation change of JM domain. Re-orientation of αC-helix to an open state upon the binding of 5a would clash with JM domain (explosion drawing represents the vdw overlapping and steric clashing).


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Figure 4. (A) Catalytic spine (C-spine, pale blue) and regulatory spine (R-spine, pale green) coordinated the internal architecture of ALK. Both hydrophobic spines reached from the N-lobe to the C-lobe of the kinases, and anchored to the F-helix. (B) Incorporation of 5a completed the C-spine and R-spine.


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Figure 5. (A) Superposition of the crystal structure of ALK/5a with ALK/4. Binding of 5a induces the conformational changes of αC-helix, A-loop and JM domain. In the absence of the substituted-urea tail moiety, no ligand-induced conformational changes were observed in ALK/4 structure. (B) Interaction network of 4 with ALK. (C) The P-loop of ALK/4 adopts a distinct conformation among the different ALK structures. Bending of the P-loop oriented Phe1127 into the ATP binding site and directly stacked to the tolyl group of 4.



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Figure 6. (A) Schematic representation of type-I inhibitor crizotinib and type-II inhibitors, 6 and 5a. (B) & (C) Superimposition of the ALK/5a structure with ALK/crizotinib structure. (D) & (E) superimposition of the ALK/5a structure with ALK/6. Hydrogen bonds between ALK/5a and ALK/crizotinib (or ALK/6) are shown with red and green dashed lines, respectively.


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Scheme 1. Reagents and conditions: (a) (i) Pyridine, rt, (ii) H2, Pd/C, MeOH/HCl (aq); (b) pyridine, phenyl isocyanate or (5-alkyl-isoxazol-3-yl)-carbamic acid 4-nitro-phenyl ester, rt. H N N R'

O NH2

+

R 1a (R = 4-NO2, R' = H or CH3 ) 1b (R = 4-CH2NHCbz)

N a

Cl R"

R'

N NH

H N

NH

R'

NH R"

b

O R

R1

H N

H N

O n

O

2 3 (R = 4-NH2, R' = H or CH3 ) 4 (R = 4-CH2NH2 , R' = H)


5 (n = 0 or 1)

R"


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Table of Contents Graphic


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