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Definitive Metabolite Identification Coupled with Automated Ligand Identification System (ALIS) Technology: A Novel Approach to Uncover Structure–Activity Relationships and Guide Drug Design in a Factor IXa Inhibitor Program Ting Zhang, Yong Liu, Xianshu Yang, Gary E. Martin, Huifang Yao, Jackie Shang, Randal M. Bugianesi, Kenneth P. Ellsworth, Lisa M. Sonatore, Peter Nizner, Edward C. Sherer, Susan E. Hill, Ian W. Knemeyer, Wayne M. Geissler, Peter J. Dandliker, Roy Helmy, and Harold B. Wood J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 12 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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Journal of Medicinal Chemistry

Definitive Metabolite Identification Coupled with Automated Ligand Identification System (ALIS) Technology: A Novel Approach to Uncover Structure–Activity Relationships and Guide Drug Design in a Factor IXa Inhibitor Program , , Ting Zhang* ‡, Yong Liu* †, Xianshu Yang#, Gary E. Martin†, Huifang Yao†, Jackie Shangǁ,

Randal M. Bugianesi§, Kenneth P. Ellsworth§, Lisa M. Sonatore§, Peter Nizner§, Edward C. Sherer╚, Susan E. Hill┴, Ian W. Knemeyer┴, Wayne M. Geissler§, Peter J. Dandliker#, Roy Helmy†, and Harold B. Wood‡ ‡

Department of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box

2000, Rahway, NJ 07065, USA †

Department of Process and Analytical Chemistry, Merck Research Laboratories , Merck &

Co., Inc., PO Box 2000, Rahway, NJ 07065, USA #

Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc., 33 Avenue

Louis Pasteur, Boston, Massachusetts 02115, USA

ACS Paragon Plus Environment


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ǁ

Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research

Laboratories, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA §

Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc., 2000

Galloping Hill Road, Kenilworth, NJ 07033, USA ╚

Department of Chemistry Modeling and Informatics, Merck Research Laboratories , Merck &

Co., Inc., PO Box 2000, Rahway, NJ 07065, USA ┴

Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research

Laboratories, Merck & Co., Inc., Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115 USA

KEYWORDS: Definitive Metabolite Identification; ALIS; Factor IXa (FIXa); SAR; MicroCryoProbe NMR

ABSTRACT. A potent and selective Factor IXa (FIXa) inhibitor was subjected to a series of liver microsomal incubations, which generated a number of metabolites. Using automated ligand identification system-affinity selection (ALIS-AS) methodology, metabolites in the incubation mixture were prioritized by their binding affinities to the FIXa protein. Microgram quantities of the metabolites of interest were then isolated through microisolation analytical capabilities, and structurally characterized using MicroCryoProbe heteronuclear 2D NMR techniques. The isolated metabolites recovered from the NMR experiments were then submitted directly to an in vitro FIXa enzymatic assay. The order of the metabolites’ binding affinity to the Factor IXa


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protein from the ALIS assay was completely consistent with the enzymatic assay results. This work showcases an innovative and efficient approach to uncover structure–activity relationships (SARs) and guide drug design via microisolation-structural characterization and ALIS capabilities.

•

Introduction

Characterization of drug metabolites provides discovery scientists with information on the sites of metabolism of a structural lead series.1-5 Such knowledge is especially relevant when the pharmacokinetic (PK) profile of the molecule is suboptimal. Medicinal chemists could also benefit from such information in future analog designs intended to improve PK properties by blocking those sites of metabolism.6 In addition, metabolites sometimes are more potent and can have even better drug profiles than the parent molecule itself.

Hence, a comprehensive

understanding of the location(s) in a molecule where metabolism occurs could lead to new drug design and expanded intellectual property space.7-8 In drug discovery research, metabolites are typically generated in vitro from hepatocytes and/or microsomes, or they may be isolated in vivo from body fluids such as urine and/or blood samples from human and preclinical species. Traditionally, metabolite characterization has relied heavily on liquid chromatography–mass spectrometry (LC-MS) techniques for sensitivity reasons. With the advancement of high resolution (HR)-MS/MS capabilities, investigators can define the metabolically modified region(s) of the molecule and propose likely metabolite structures based on the molecular weight change and MS/MS fragmentation pattern.9-16 Definitive structural assignment of a particular metabolite requires a sample of the metabolite that is amenable to NMR analysis. Generally, a suitable metabolite sample can be prepared from



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large scale incubation, followed by isolation and NMR characterization. After identifying the structure of such metabolite, if the metabolite is of interest, medicinal chemists may then need to design a synthetic sequence to produce larger quantities of the material for additional in vitro and in vivo studies.6,17 Conversely, if a given metabolite is not of interest, then the cycle begins again for another metabolite.6,17 The overall process is not targeted and consequently requires a significant and continuing resource commitment. Because of these limitations and the low abundance of the minor metabolites in an incubation mixture, typically only a selected number of metabolites, usually the major ones, are isolated, fully characterized, and synthesized for further studies. However, there remains the distinct possibility that one or more of the minor metabolites could represent missed and new structure–activity relationships (SARs) for better drug design. The relatively recent development of 1.7 mm MicroCryoProbe NMR capabilities has significantly reduced the quantity of a metabolite that must be isolated for NMR characterization.18-20 This enhanced analytical capability will open new avenues for the characterization and investigation of minor metabolites that may embody potentially new SAR. Recently, the automated ligand identification system (ALIS), an affinity selection−mass spectrometry based (AS-MS) platform has been used to identify hits from mixture-based combinatorial libraries.21-26 The ALIS methodology coupled with the microisolation and structure elucidation capabilities can be applied to investigate metabolism incubation mixtures, rank order the binding affinity of metabolites, and identify metabolites that warrant structural characterization and synthetic elaboration. Ultimately, these novel analytical capabilities provide tools that can help guide medicinal chemists to synthesize only the desired compound(s) and improve the discovery time cycle for novel therapeutic leads.


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Advancements in the area of in vitro pharmacologic testing now allow biologic potency measurements to be undertaken with low microgram (µg) quantities of samples. Typical metabolite microisolation techniques afford quantities of metabolites that, when prepared for NMR characterization using 1.7 mm MicroCryoProbe techniques, allow direct utilization of the NMR sample in the biological assay following structural characterization. The metabolite in the NMR sample can be quantitated by either NMR or HPLC-UV/MS techniques. We report herein an example showcasing the power of combining the aforementioned technologies in an anti-thrombotic drug discovery program,27,28 which enabled the quick identification of the metabolites of interest and uncovered new SAR for improving potency. After microsomal incubation of parent compound, ALIS-AS technology was used to guide and prioritize definitive metabolite isolation and identification, which ultimately led to the discovery of an active minor metabolite with much higher binding affinity than parent compound. Following isolation on microgram scale and NMR structural characterization, the intrinsic activity of the µg quantity of the metabolite was confirmed in an enzymatic activity assay. •

Chemistry

Compound 7 was synthesized via the synthetic route shown in Scheme 1. Starting from a heterocyclic halide 1, Pd-catalyzed α-arylation with compound 2 led to nitrile 3.29 Compound 3 was then reduced using literature methods to give primary amine 4.30 In parallel, carboxylic acid 5 was converted to the acid chloride 6, which was then coupled with amine 4 to give the final compound as a racemate. Finally, chiral resolution afforded FIXa inhibitor 7. Titrant compound 8 was synthesized using similar methods.



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Scheme 1a

a

Reagents and conditions: (i) Pd(OAc)2, 2,8,9-triisobutyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane, NaHMDS in THF, dioxane, 100 oC, 3 h; (ii) NaBH4, NiCl2, EtOH, THF, 25 oC, overnight; (iii) oxalyl chloride, cat. DMF, DCM, 0-25 oC, 2 h; (iv) EtOH, THF, 25 oC, 1 h; (v) Chiral separation on IC column.

Results and Discussion Previously, our group reported a discovery research effort focused on the development of a novel class of anti-thrombotic Factor IXa (FIXa) inhibitors.27,28 A high-throughput screening campaign was conducted and a benzimidazole hit was identified. The discovery chemistry team developed the benzimidazole hit into a highly potent and selective FIXa inhibitor, 7, which exhibited a robust response in a pharmacodynamic (PD) assay and demonstrated the characteristics of a true FIXa inhibitor.28 As shown in Table 1, 7 has good human Factor IXa (hFIXa) potency and is highly selective against off-target human Factor Xa (hFXa). However, when subjected to rat pharmacokinetics (PK) studies, high in vivo clearance of 7 was observed (Table 1). Moreover, high in vitro intrinsic clearance (CLint) values were also observed in human and rat liver microsomal and hepatocyte incubations.31 Table 1. Potency and rat PK data of 7


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H N

O

N

Cl

N H Cl

N

N N

F 7

hFIXa Ki (nM)a

hFXa Ki (nM) a

Ratio a

T1/2 a

CL a

µM·h/mpk

h

mL/min/kg

11.1

0.0970

1.24

36.5

9.11

2798

b

N=3

N=2

N=2

N=2

N=2

SD = 737

SD = 1.2

SD = 0.010

SD = 0.30

SD = 2.1

N =3 c

SD = 1.5

308

AUCN (po) a,d

F% a

a) mean values; b) N: number of times data acquired; c) SD: standard deviation; d) AUCN: AUC normalized by dose.

XIC-HLM

7

M538d

M538c

M538b M538a

XIC-RLM

7 M538d M538c M538b M538a



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Figure 1. Extracted Ion Chromatograms (XIC) showing metabolites arising from human liver microsomal (HLM) and rat liver microsomal (RLM) incubations of 7. To better understand the high clearance of 7, the compound was exposed to human and rat microsomal incubations and the results are shown in Figure 1. Extensive oxidation of 7 was observed when incubated with both human and rat microsomes. Results from LCMS-MS analysis partially narrowed down the site of oxidation for each metabolite (Figure 2). MS/MS spectra and proposed fragmenation patterns are included in Supporting Information.

Figure 2. Proposed relative structures for major oxidative metabolites with chromatographic retention times from the chromatograms shown in Figure 1.

While the original goal of the metabolite identification study was to improve PK, we were intrigued by the extensive oxidative metabolism observed in multiple regions of the molecule. Each metabolite represents a potential direction for lead optimization. To utilize the results of


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the microsomal incubation effectively, we would need to know the exact structure of each metabolite. However, pursuing the isolation and characterization of all of the metabolites is extremely resource intensive and time-consuming. Therefore, methodology that can help prioritize the metabolites for isolation and structural characterization is highly desirable. This need prompted us to explore ALIS-AS experiments. Affinity-based competition experiments (ACE) are based on equilibrium affinity selection, whereby a fixed concentration of a mixture of compounds and serially increasing concentrations of a known competitor ligand (titrant) are incubated together with the target protein. The amount of each compound bound to the target protein is then analyzed by ALIS-AS. The MS signals of the ligands and the competitor from such experiments reflect the equilibrium concentrations of each protein-ligand complex, thereby providing information about the equilibrium dissociation constant (Kd) of each component in a complex mixture. As the titrant concentration is increased, MS signals for FIXa-bound metabolites are correspondingly reduced in a sigmoidal response. A curve-fitting algorithm is used to calculate the concentration of titrant at which metabolite MS signal is reduced by 50% compared to a control sample without titrant (ACE50). The ability of the titrant to competitively displace the target-bound ligands reveals their binding affinity to the same site of protein and allows relative binding affinity ranking of the ligands.22-26 In an ACE50 curve, a higher value indicates a ligand with higher affinity for the protein reflected by a higher titrant concentration required to displace the compound of interest from the binding site. ALIS has demonstrated its unique capability in screening label-free mixture-based libraries and finding hits that were subsequently developed into lead compounds in various classes of protein targets.32-44 The strength of ALIS in measuring binding affinity directly from a mixture of compounds can be brought into the workflow of metabolite identification. Instead of isolating



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and characterizing every metabolite and profiling them individually, one can use the FIXa protein and a crude metabolite mixture from rat liver microsomes to rank order binding affinities via ALIS screening. The binding affinity of the metabolite provides an indication of whether a metabolite is potentially active. Consequently, the ALIS screening methodology was utilized to prioritize metabolites of 7 with highest affinity for FIXa for isolation and subsequent structural characterization. FIXa is a structurally well-defined enzyme target.45 Its enzymatic activities have been characterized in several different assays.46 In ALIS screening, a truncated human FIXa protein (415 aa, E.coli derived) was used. Compound 8, bearing fluoro (F) substitution on the benzimidazole moiety, is a close analog of 7. The hFIXa Ki of 8 is 10.6 nM (N = 3, SD = 5.6), which is in the same range as that measured for 7 (Ki = 9.11 nM). Therefore, 8 was selected as the titrant in the ALIS competition assay. Based on their structures, 7, 8, and the metabolites of 7 were assumed to bind to the same site of the FIXa protein. The binding affinity of 8 can be utilized to assess the binding affinity of the metabolites of 7 for the FIXa protein in ALIS competition experiments, allowing the metabolites to be rank-ordered.

The characterization of the binding mode and binding affinity of metabolites of 7 with FIXa protein was analyzed using ACE described briefly above. In these experiments, a constant concentration of the RLM metabolite mixture derived from 7 and serially increasing


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concentrations of the titrant, 8, were incubated with the FIXa protein. Protein−ligand complexes were separated from the unbound compounds by SEC. The protein-bound complex of 7, its metabolites, and the titrant, 8, were then dissociated from the protein using reverse-phase chromatography and finally quantitated by mass spectrometry. The ACE result of 7 and its metabolites in the presence of the titrant was consistent with the assumption that they share a common binding site of the FIXa protein. As shown in Figure 3, 7 and its metabolites were displaced from the binding site of FIXa protein by the titrant. M538a has a higher ACE50 value than 7, indicating it has greater binding affinity in the RLM metabolite mixture derived from 7, and therefore requires higher concentration of the titrant, 8, for displacement from the binding site. Conversely, M538b had a lower ACE50 value than 7, indicating that M538b has a weaker binding affinity. Finally, M538c and M538d have even weaker binding affinities than M538b. Therefore, the ALIS binding affinity ranking from strongest to weakest is: M538a>7>M538b> M538c, M538d. We expected this to be consistent with their respective inhibitory activity in the FIXa enzymatic assay. The rank-ordered binding affinity provides the needed guidance for the prioritization of definitive metabolite isolation and identification work. Due to its high binding affinity to FIXa protein, M538a was considered to be a high priority metabolite for isolation and structural characterization. Due to its low abundance, which could pose isolation and characterization challenges using conventional techniques, M538a was subjected to the aforementioned microisolation methodology and MicroCryoProbe NMR characterization. In addition, M538b was selected for definitive structural elucidation due to its close binding affinity compared to compound 7. Finally, as a negative control to validate whether ALIS binding affinity is aligned with FIXa enzymatic assay potency, the structural elucidation of



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one of the weaker binding metabolites, M538c, was also undertaken. It should be noted that M538b and M538c were also selected for PK reasons. In a previous human hepatocyte incubation experiment (not discussed in this report), M538b was shown to be the major metabolite; whereas in the present human liver microsomal (HLM) incubation, M538c was shown to be the major metabolite (Figure 1). These two metabolites represent 7’s major oxidative metabolism outcome in human in vitro systems.

Figure 3. ALIS Screen using a crude RLM incubation metabolite mixture. ALIS binding affinity ranking was: M538a > 7 > M538b > M538c, M538d. At each concentration, two replicates were performed and data averaged. As was noted above, incubation of 7 with liver microsomes resulted in the formation of metabolites M538a, M538b, and M538c; M538a was a minor metabolite relative to M538b and M538c. In order to obtain sufficient quantities of M538a to allow NMR characterization,


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incubation protocols were optimized. It was found that incubations of 7 at 100 µM concentration with dog liver microsomes gave the best conversion of 7 to M538a (Figure 4). The same protocol also produced acceptable amounts of M538b and M538c. Even though dog microsomal incubation provided the highest yield of M538a, the absolute conversion was only 0.14% and only about 25 µg of M538a was formed in the large scale incubation mixture. Isolation of µg quantities of metabolites from a complex in vitro/in vivo matrix poses a significant analytical challenge and a systematic approach for microisolation must be implemented. First, due to the limited amounts of metabolites present in the matrix, an analytical scale prep-HPLC system equipped with an analytical HPLC column will need to be employed. Second, analytical control for purity evaluation and concentration of different aliquots should be in place in every step of the isolation protocol such as work-up, fraction pooling, and solvent removal to ensure best recovery possible. Next, the buffer used in the isolation (which in most cases is acidic) can cause decomposition of metabolites during the solvent removal step. A probe stability study must be conducted to determine the proper drying procedure to ensure the integrity of metabolites and minimize degradation. This microisolation approach described herein was applied in our study. The final step in the isolation sequence involved dissolving the isolated metabolite in a suitable deuterated solvent for the NMR work and evaporating the sample to dryness a final time.



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Figure 4. Dog liver microsomal incubation of 7. By applying the microisolation protocol, microgram quantities of M538a, M538b, and M538c were successfully isolated and the site of oxidation was partially defined by LC-MS. The structures were then unequivocally established by heteronuclear 2D NMR methods (Table 2).

Table 2. Isolation and the characterized structures of metabolites of 7. H N 25

20

21

N 19

23 22 18

16

14 15

17

12

NH

31

27

30

Cl

28 29

Amount (µg)

8 9

7

10

6

13 26

Metabolite name

Cl

O

24

F

11

N 1 5

N 2

3

32

N 4

Purity by LC-UV


Structure*

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M538a

12

100%

M538b

30

96%

M538c

114

95%

*Tentative stereochemistry based on the known stereochemistry of 7. Structural characterization of low microgram quantities of drug metabolites by NMR has long been a challenging undertaking due to the inherent insensitivity of NMR spectroscopy as compared, for example, to mass spectrometry.5,47 There has been a continual advancement in NMR probe design over the past several decades.18,48,49 The biggest advance insofar as using NMR to characterize drug metabolites quite likely was the development of a 1.7 mm MicroCryoProbe in 2008-2009, the performance of which has been rigorously benchmarked for small samples.19 When 1.7 mm cryoprobe technology is employed in tandem with a pure shift HSQC (PS-HSQC)50-52 NMR spectrum, samples of a few micrograms of an isolated drug metabolite can be successfully characterized in very reasonable time.20 The sensitivity of this approach can be further enhanced by intentionally folding the PS-HSQC spectrum in the F1 dimension to either acquire the data in a shorter time or with a commensurate increase in s/n for the same expenditure of spectrometer time.



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Structural characterization is greatly facilitated by the concerted utilization of accurate mass MS/MS and the acquisition of pure shift HSQC data. The MS/MS data can be utilized to localize the site of oxidative metabolism as denoted by the bracketed segments of the structures shown in Figure 2. While one-dimensional proton spectra may be severely complicated by overlapping, extraneous peaks from the chromatographic isolation, e.g. column bleed, contaminating species eluting from the column from previous chromatographic isolations, etc., separating resonances in the second frequency domain of a pure shift HSQC spectrum sidesteps many of the inherent problems of metabolite characterization when only proton homonuclear methods are employed. Fortuitously, 13C shifts are quite empirical in nature, which allows the 13

C shift perturbation of protonated carbons flanking the site of oxidation to be calculated. Using

this approach, the site of hydroxylation can be unequivocally pinpointed. When necessary, pure shift HSQC data can be augmented by long-range heteronuclear shift correlation data, e.g. LRHSQMBC53 and IDR-(Inverted Direct Response)-HSQC-TOCSY54 data when there is sufficient sample available. The sample of M538a was the smallest of the three metabolites with ~12 µg available for analysis by NMR.20 From a PS-HSQC “fingerprint”, it was obvious that one of the protonated carbons of the fluorophenyl moiety was absent from the spectrum of the metabolite. Both fluoro and hydroxyl substituents shift the carbon ortho to the substituent on a phenyl ring upfield. Hence, as shown for the potential 30-hydroxyl metabolite, the C29 resonance between the fluoro and hydroxyl groups would be expected to resonate considerably upfield near 100 ppm. Since none of the protonated aromatic carbons in the PS-HSQC spectrum of M538a resonated upfield of 115.9 ppm, 30-hyroxylation was ruled out. Hydroxylation at the 31-position (see Supporting Information) would require all three protonated carbons of the fluorophenyl


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moiety to resonate in the range of 114-117 ppm, which also allowed this possibility to be ruled out. Hence, it was necessary to differentiate between 27- and 29-hydroxylation. The structures were differentiated on the basis of an IDR-HSQC-TOCSY spectrum54 acquired using a 20 ms mixing time. 27-Hydroxylation would lead to three contiguous protonated carbons whereas 29hydroxylation would only require two adjacent protonated carbons. The latter case was confirmed with the IDR-HSQC-TOCSY data, establishing the structure of M538a as the 29hydroxy, which was also consistent with the chemical shifts of the protonated aromatic carbon resonances in the PS-HSQC spectrum. Similarly, the more abundant metabolites, M538b and M538c, were characterized via PSHSQC and LR-HSQMBC54 (detailed discussions in Supporting Information).



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Lastly, the three isolated and structurally characterized µg-quantity metabolites were submitted to the FIXa enzymatic assay at a modified low-concentration titration range in order to accurately assess the intrinsic potencies of each metabolite. As shown in Figure 5, the FIXa enzymatic potency rank-ordering of M538a, 7, M538b, and M538c aligns remarkably well with ALIS FIXa binding affinity ranking shown in Figure 3.55 This observation further validated the premise that using the resource-sparing ALIS-AS alone on a metabolite mixture can directly rank-order metabolite potency against the target protein. The ALIS-AS assay results can then be used to prioritize further work on the isolation, structural characterization, and synthesis of potentially active metabolites, as well as additional SAR study follow-up on the metabolite(s) of interest. This approach circumvents tedious isolation and structural characterization of every metabolite and instead directs the discovery chemistry effort more effectively. In this particular case, for example, the more potent metabolite, M538a, became a new opportunity for lead optimization.


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Figure 5: FIXa enzymatic assay titration of 7 and its metabolites. *IC50 values represent single determinations performed with two replicates per concentration. Conclusions In conclusion, we have showcased a novel and efficient approach of coupling definitive metabolite isolation/identification and ALIS technology as a means of identifying new SARs in an anti-thrombotic drug discovery research program. The ALIS-AS technology enabled preliminary affinity ranking using a microsomal incubation mixture without the need of isolating each metabolite. The affinity ranking results were then used to guide and prioritize definitive metabolite isolation and subsequent structural characterization. The isolation of pure metabolites



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in microgram quantities was made possible by microisolation capabilities; and the structure elucidation of µg scale metabolites was enabled by MicroCyroProbe NMR. Lastly, the intrinsic potencies of the µg quantities of metabolites were confirmed in a FIXa enzymatic assay and proven to be consistent with their ALIS binding affinities towards the FIXa protein. These results demonstrate that ALIS-AS binding affinity ranking can be used to predict the intrinsic potencies of metabolites in an incubation mixture without isolation and characterization as prerequisites. With the intrinsic potency ranking from an ACE experiment as an initial guide, a more focused effort in definitive metabolite identification can then ensue, leading to quick identification of region(s) on the parent molecule as a lead optimization focus. This approach avoids the traditional tedious isolation, characterization, resynthesis and in vitro potency assessment for each metabolite in order to identify metabolite(s) of interest. Moreover, as demonstrated herein, ALIS can also reveal highly potent metabolites, some of which could be missed by traditional approaches if such metabolites are present at very low abundance. It is in those highly potent but otherwise overlooked metabolites where potentially new structure–activity relationships that can lead to better drug design may be uncovered. Ultimately, we have demonstrated for the first time an end-to-end work flow using microgram quantities of materials, from microsomal incubation/isolation, and NMR structure elucidation, to in vitro enzymatic assay. This practice eliminates the lengthy and expensive synthetic steps in making pure metabolites for biological assay, which has considerable potential to speed up the process of drug discovery.

Experimental Section

Chemistry:


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All reactions were carried out employing standard chemical techniques in ambient atmosphere unless otherwise noted. Solvents used for extraction, washing, and chromatography were HPLC grade. Analytical HPLC was performed on a Waters Acquity Ultra Performance LC with UV detection at 230 and 254 nm along with ELSD and ESI-MS, with all final compounds showing >95% purity and a parent mass ion consistent with the desired structure. (HPLC conditions: Column: Supelco Ascentis Express C18, 2.7 µM, 3.0 × 100 mm. Sovents: A = water/0.05% TFA; B = Acetonitrile/0.05% TFA. Gradient: 10-99% B in 4 min. Flow: 1.0 mL/min, 2 µL injection. UV: 200-400 nm. MS Conditions: Scan Range: 170-900 amu. ESI positive. Run time 5 min.) All routine NMR spectra were recorded on a 500 MHz Varian INOVA 500 instrument unless noted otherwise. 1H chemical shifts are reported as δ values in ppm relative to the residual solvent peak (CD3OD = 3.30, acetone-d6 = 2.04). Automated flash column chromatography was performed on a Teledyne ISCO Combiflash Rf system.

2,6-Dichloro-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzoic acid (5) Step A: Methyl 2,4,6-trichlorobenzoate (10) Into a 5000 mL 3-necked round-bottom flask was placed 2,4,6-trichlorobenzoic acid 9 (200 g, 887 mmol), N,N-dimethylformamide (2000 mL), potassium carbonate (368 g, 2.66 mol), and CH3I (379 g, 2.67 mol). The resulting solution was stirred for 12 h at 25 oC. It was then diluted with 3000 mL of water and extracted with 3 × 1500 mL of ethyl acetate. The organic layers were combined, washed with 1 × 2000 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 192 g (86%) of methyl 2,4,6-trichlorobenzoate 10 as a light yellow oil.



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Step B: Methyl 2,6-dichloro-4-((diphenylmethylene)amino)benzoate (11) Into a 5000-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed methyl 2,4,6-trichlorobenzoate 10 (192 g, 802 mmol), toluene (2000 mL), Cs2CO3 (789 g), Xantphos (46.7 g, 80.7 mmol), diphenylmethanimine (173 g, 956 mmol) and Pd(OAc)2 (9.1 g, 40.5 mmol). The resulting solution was heated to reflux for 12 h in an oil bath. The reaction mixture was cooled to 25 oC with a water/ice bath. The solid was filtered out and washed with toluene. The combined filtrate was concentrated under vacuum. The residue was applied onto a silica gel column and eluted with ethyl acetate/petroleum ether (1:20). This resulted in 208.1 g (64%) of methyl 2,6-dichloro-4-[(diphenylmethylidene)amino]benzoate 11 as a colorless oil. Step C: Methyl 4-amino-2,6-dichlorobenzoate (12) Into a 3000-mL 3-necked round-bottom flask was placed methyl 2,6-dichloro-4[(diphenylmethylidene)amino]benzoate 11 (208 g, 542 mmol), tetrahydrofuran (1250 mL), and 2N hydrogen chloride (504 mL). The resulting solution was stirred for 3 h at 25 oC. The pH value of the solution was adjusted to 9 with aq. sodium bicarbonate and extracted with 3 × 1000 mL of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was applied onto a silica gel column and eluted with ethyl acetate/petroleum ether (1:20). This resulted in 87 g (69%) of methyl 4-amino-2,6dichlorobenzoate 12 as a white solid. Step D: Methyl 2,6-dichloro-4-hydrazinylbenzoate hydrochloride (13)


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Into

a

2000-mL 3-necked

round-bottom

flask

was

placed

methyl

4-amino-2,6-

dichlorobenzoate 12 (87 g, 395 mmol), and HCl (37%, 870 mL). This was followed by the addition of a solution of NaNO2 (27.3 g, 396 mmol) in water (30 mL) dropwise with stirring at 0 o

C over 15 min. To this was added a solution of SnCl2 (210 g, 1.11 mol) in HCl (37%, 200 mL)

dropwise with stirring at 0 oC over 30 min. The resulting solution was stirred for 2 h at 0 oC in a water/ice bath. The solid was filtered out and washed with 100 mL of DCM. The combined filtrate was concentrated under vacuum. This resulted in 85.5 g (76%) of methyl 2,6-dichloro-4hydrazinylbenzoate hydrochloride 13 as a white solid. Step E: Methyl 2,6-dichloro-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzoate (14) Into a 2000-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed ethyl ethanecarboximidate hydrochloride (39.0 g, 315 mmol) and dichloromethane (1000 mL). This was followed by the addition of triethylamine (38.2 g, 378 mmol) dropwise with stirring at -40 oC over 30 minutes. The resulting solution was stirred for 4 h at 0 oC in a water/ice bath. To this was added methyl 2,6-dichloro-4-hydrazinylbenzoate hydrochloride

13

(85.5

g,

315

mmol),

pyridine

(99.69

g,

1.26

mol),

and

(diethoxymethoxy)ethane (934 g, 6.30 mol). The resulting solution was concentrated under vacuum. The residual solution was stirred for 16 h while the temperature was maintained at 80 oC in an oil bath. The reaction mixture was cooled to 25 oC with a water/ice bath and then poured into 1000 mL of EtOAc/water (1:1). The aqueous phase was extracted with 3 x 1000 mL of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product (80 g) was purified with Flash-Prep-HPLC with the following conditions: column, C18-SG; mobile phase, AcCN/aq. NH4HCO3 (0.1%) = 40/60 increasing to AcCN/aq. NH4HCO3 (0.1%) = 55/45 within 25 min; detector, UV 254 nm. This



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resulted in 35.7 g (38%) of methyl 2,6-dichloro-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzoate 14 as a white solid. Step F: 2,6-Dichloro-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzoic acid (5) Into a 500-mL 3-necked round-bottom flask was placed methyl 2,6-dichloro-4-(3-methyl-1H1,2,4-triazol-1-yl)benzoate 14 (14.7 g, 51.4 mmol), MeOH/H2O/THF (1:1:4, 240 mL) and LiOH·H2O (10.8 g, 257 mmol). The resulting solution was stirred for 12 h at 60 oC in an oil bath. The reaction mixture was cooled to 25 oC, then concentrated under vacuum. The residual solution was diluted with 100 mL of water, then washed with 200 mL of ethyl acetate. The aqueous layer was adjusted to pH 3 with HCl (2 M). The solid was collected by filtration and dried. This resulted in 9.8 g (67%) of 2,6-dichloro-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzoic acid 5 as a white solid. LC-MS: m/z = 272 [M+H]+. 1H NMR (Bruker 400 MHz, DMSO-d6, ppm): δ 14.29 (s, 1H), 9.31 (s, 1H), 8.06 (s, 2H), 2.38 (s, 3H). (R)-2,6-dichloro-N-(2-(3-fluorophenyl)-2-(2-methyl-1H-benzo[d]imidazol-5-yl)ethyl)-4-(3methyl-1H-1,2,4-triazol-1-yl)benzamide (7). Step A: (±)-2-(3-Fluorophenyl)-2-(2-methyl-1H-benzo[d]imidazol-5-yl)acetonitrile (3). The reactor was charged with palladium(II) acetate (0.160 g, 0.711 mmol), 2,8,9-triisobutyl2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane (0.507 mL, 1.42 mmol), and 5-bromo-2methyl-1H-benzo[d]imidazole 1 (1.5 g, 7.11 mmol). The vessel was evacuated and refilled with N2 for three cycles and degassed dioxane (14.2 mL) was added, followed by NaHMDS (28.4 mL, 28.4 mmol). The reaction was stirred at 25 °C for 20 min before 2-(3fluorophenyl)acetonitrile 2 (73.3 mg, 0.543 mmol) was added. The reaction was then capped and heated at 100 °C for 3 h. More NaHMDS (6 mL) was added and the reaction was stirred for


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another 1 h. Additional NaHMDS (4 mL) was added and the reaction was stirred for another 1 h before it was cooled to room temperature, and quenched with water. The aqueous layer was extracted with EtOAc (3 × 300 mL), washed with brine (300 mL), dried over MgSO4, filtered, and the filtrate was concentrated in vacuo. The crude material was purified by chromatography on silica gel (eluent: 0-100% A in B. A: 10% MeOH in DCM; B: DCM.) to give 1.62 g (±)-2-(3Fluorophenyl)-2-(2-methyl-1H-benzo[d]imidazol-5-yl)acetonitrile 3 as a tan gue. LC-MS: m/z = 266.0 [M+H]+. Step B: 2,6-Dichloro-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzoyl chloride (6). At 0 °C, in a pre-weighed flask containing carboxylic acid 5 (2.28 g, 8.38 mmol) in 3 mL DCM, was added oxalyl chloride (1.50 mL, 16.8 mmol), followed by one drop of DMF. After 0.5 h, the reaction was warmed to 25 °C and stirred for 2 h. The reaction mixture was concentrated and the excess oxalyl chloride was azeotropically removed with toluene. LC-MS: m/z = 289.9 [M+H]+. Step C: (±)-2-(3-Fluorophenyl)-2-(2-methyl-1H-benzo[d]imidazol-5-yl)ethanamine (4). Under N2, to the step A product 3 (1.62 g, 6.11 mmol) was added anhydrous nickel(II) chloride (0.791 g, 6.11 mmol) in anhydrous ethanol (15.3 mL) and THF (15.3 mL) was added sodium borohydride (1.39 g, 36.6 mmol). The reaction was stirred at 25°C overnight. LC-MS: m/z = 270.1 [M+H]+. Step

D:

(R)-2,6-Dichloro-N-(2-(3-fluorophenyl)-2-(2-methyl-1H-benzo[d]imidazol-5-

yl)ethyl)-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzamide (7)



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Page 26 of 49

The reaction mixture from step C was added to the step B product 6 (2.66 g, 9.16 mmol). The vial containing the step C reaction was rinsed with a total of 5 mL THF that was also added to the step B product 6. The mixture was stirred at 25 °C for 1 h. The reaction was then quenched with a saturated NaHCO3 aqueous solution (200 mL), diluted with 300 mL EtOAc, agitated, and filtered. The filtrate was collected, and the phases separated. The aqueous phase was extracted with EtOAc (3 × 300 mL). The combined aqueous layers were washed with brine (300 mL), dried over MgSO4, filtered, and concentrated in vacuo. The crude material was purified by chromatography on silica gel (eluent: 0-100% A in B. A: 10% MeOH in DCM; B: DCM.) to yield 0.88 g racemic product. LC-MS: m/z = 523.0 [M+H]+. ¹H NMR (500 MHz, CD₃OD): δ 8.97 (s, 1 H); 7.79 (s, 2 H); 7.46 (s, 1 H); 7.39 (t, J = 8.3 Hz, 1 H); 7.33-7.25 (m, 1 H); 7.22-7.09 (m, 3 H); 6.91 (td, J = 8.5, 2.4 Hz, 1 H); 4.55 (t, J = 8.1 Hz, 1 H); 4.16-4.00 (m, 2 H); 2.53 (s, 3 H); 2.39 (s, 3 H). Chiral separation: IC column (2 × 15 cm), 35% ethanol (0.1% DEA)/CO2, 50 mL/min, 100 bar, 220 nm, injection volume: 1 mL; 9 mg/mL in methanol. Fast-eluting enantiomer A, 378 mg; slow-eluting enantiomer B, 379 mg. The title compound 7 is enantiomer B. (The assignment of stereochemistry was based on known X-Ray co-crystallization data between FIXa enzyme and analogs of 7)27,28 (S)-2,6-Dichloro-N-(2-(7-fluoro-2-methyl-1H-benzo[d]imidazol-5-yl)-2-(3fluorophenyl)ethyl)-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzamide (8) Step A: 5-Bromo-7-fluoro-2-methyl-1H-benzo[d]imidazole (16) A solution of 5-bromo-3-fluorobenzene-1,2-diamine 15 (1 g, 4.88 mmol), acetic acid (2.93 g, 48.8 mmol) in PPA (polyphosphoric acid) (4.88 mL) was heated to 135-140 oC for 4 h under N2.


Page 27 of 49

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The reaction was then cooled to 25 oC and transferred into 25 mL of ice cold water. Charcoal (about 1 g) was added and the mixture was filtered over a Celite bed, which was rinsed with water. The filtrate was basified with aq. ammonia solution until pH = 10~12. The basified reaction mixture was extracted with ethyl acetate (3 × 100 mL), and the combined ethyl acetate layers were dried over MgSO4 and concentrated to a yellow solid residue. To the residue was added hexanes (5 mL) and the mixture was filtered to reveal 0.93 g 5-bromo-7-fluoro-2-methyl1H-benzo[d]imidazole 16 as a white solid. LC-MS: m/z = 228.9 [M+H]+. Step B: 2-(7-Fluoro-2-methyl-1H-benzo[d]imidazol-5-yl)-2-(3-fluorophenyl)acetonitrile (17) The reactor was charged with palladium(II) acetate (0.037 g, 0.166 mmol), 2,8,9-triisobutyl2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane (114 mg, 0.332 mmol), and 5-bromo-7-fluoro2-methyl-1H-benzo[d]imidazole 16 (380 mg, 1.66 mmol). The vessel was evacuated and refilled with N2 for 3 cycles and degassed dioxane (3.32 mL) was added, followed by NaHMDS (6.64 mL, 6.64 mmol). The reaction was stirred at 25 °C for 20 min before 2-(3fluorophenyl)acetonitrile (269 mg, 1.99 mmol) was added. The reaction was then capped and heated at 100 °C for 6 h. The reaction was cooled to room temperature, and quenched with water. The aqueous layer was extracted with EtOAc (3 × 100 mL), washed with brine (100 mL), dried over MgSO4, filtered, and the filtrate was concentrated in vacuo. The crude material was passed through a silica short column using eluent 0-100% A in B (A: 10% MeOH in DCM; B: DCM.) to yield a crude product, which was used directly in the next step. LC-MS: m/z = 284.1 [M+H]+. Step C: 2-(7-Fluoro-2-methyl-1H-benzo[d]imidazol-5-yl)-2-(3-fluorophenyl)ethan-1-amine (18)



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Page 28 of 49

Under N2, to product 17 (180 mg, 0.635 mmol), anhydrous nickel(II) chloride (82 mg, 0.635 mmol) in anhydrous ethanol (1.6 mL) and THF (1.6 mL) was added sodium borohydride (144 mg, 3.81 mmol). The reaction was stirred at 25 °C overnight to produce 18. Step

D:

(S)-2,6-Dichloro-N-(2-(7-fluoro-2-methyl-1H-benzo[d]imidazol-5-yl)-2-(3-

fluorophenyl)ethyl)-4-(3-methyl-1H-1,2,4-triazol-1-yl)benzamide (8) The reaction mixture containing 18 from above was then transferred to a vial containing compound 6 (119 mg, 0.318 mmol). The vial containing the reaction mixture from above was rinsed with a total of 2 mL THF and then added to the vial containing compound 6. The mixture was stirred at 25 °C for 1 h. The reaction was then quenched with saturated NaHCO3 aqueous solution, diluted with 10 mL EtOAc, agitated, and filtered. The filtrate was collected, and phases were separated. The aqueous phase was extracted with EtOAc (3 × 10 mL). The combined aqueous layers were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude material was purified by chromatography on silica gel (eluent: 0-100% A in B. A: 10% MeOH in DCM; B: DCM.) to yield 90 mg racemic product containing impurity. This crude product was subjected to chiral resolution and purity upgrade: IC column (2 × 15 cm), 45% 2:1 ACN:isopropanol(0.1% DEA)/CO2, 60 mL/min, 100 bar, 220 nm, inj vol.: 0.5 mL; 8 mg/mL 1:2 DCM:methanol. Faster eluent, enantiomer A, 25 mg; Slower eluent, enantiomer B, 22 mg. The title compound 8 is enantiomer B. LC-MS: m/z = 541.1 [M+H]+. ¹H NMR (500 MHz, acetoned6): δ 11.8 (brs, 1 H), 9.02 (s, 1 H); 8.07 (s, 1 H); 7.85 (s, 2 H); 7.37 (t, J = 5.5 Hz, 2 H); 7.317.24 (m, 2 H); 6.99 (m, 2 H); 4.62 (t, J = 7.9 Hz, 1 H); 4.25-4.12 (m, 2 H); 2.55 (s, 3 H); 2.38 (s, 3 H). FIXa/FXa Enzymatic Assay Protocols:


Page 29 of 49

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•

Materials:

Buffer components PEG-8000, HEPES, and CaCl2•H2O were obtained from Hampton Research.

Sodium

chloride

and

n-Acetyl-KPR-AFC

(AFC

=

7-amido-4-trifluoro-

methylcoumarin) were obtained from Sigma. CH3SO2-D-cyclohexylGly-Gly-Arg-AFC·AcOH), was obtained from CPC Scientific Custom Peptide. Human FXa was purchased from Sekisui Diagnostics. Recombinant human Factor IXa enzyme was purchased from Haemtech Technologies and further purified via a ResS column. •

Methods: Test compounds were titrated with DMSO as the diluent in a 10-point dose response in a

separate step followed by a 60-fold dilution into the reaction. Assay buffer contained 50 mM HEPES, pH 7.4, 5 mM CaCl2•2H2O, 150 mM NaCl, 0.1% PEG-8000. Assay pipetting steps to 384 well microplates were performed with a Hamilton Microlab Star robotic platform. Enzyme activity was measured by recording the increase in fluorescence signal after cleavage of a labeled tripeptide and subsequent release of the free fluorophore AFC as follows. Human Factor IXa protein was incubated with test compound for 30 minutes followed by the addition of peptide substrate CH3SO2-D-CHG-Gly-Arg-AFC·AcOH, resulting in a 60-fold dilution of compound and final concentration of 2 nM FIXa and 250 µM substrate. After incubation at ambient temperature for 1 h, the fluorescence was measured on an Envision Multilabel reader with 405 nm excitation and 510 nm emission filters. FXa activity was determined using the same methodology with final concentrations of 15 nM FXa and 100 µM n-Acetyl-KPR-AFC. Inhibitory activity (IC50) of test compounds was determined from a dose titration using a four parameter logistic fit, and subsequently converted to Ki using the Cheng-Prusoff equation (Ki =



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Page 30 of 49

IC50 / ((1+(S/KM))) for competitive inhibition.56 Selectivity of the compounds for Factor IXa is represented as a ratio of Ki values.

Rat PK protocols: PK studies were conducted in fasted male Hans-Wistar rats (Charles River Laboratories, Inc.), administering a dose of 0.5 mg/kg bolus intravenously (IV) and 2 mg/kg orally (PO).

Metabolite Incubation, Isolation, and Analysis: •

Materials:

Reduced nicotinamide adenine dinucleotide phosphate (NADPH), acetonitrile, labetalol, water, methanol, and formic acid were purchased from Sigma-Aldrich Co. (St. Louis, MO), and were the highest grade available. All other reagents and solvents were obtained from Thermo Fisher Scientific Inc. (Waltham, MA). Rat liver microsomes (male sprague dawley with cat#452501), dog liver microsomes (male beagle cat#452601) and human liver microsomes (cat#452117) were purchased from BD Gentest (San Jose, CA). •

Incubations:

Dog liver microsomes (1 mg protein/mL) were suspended in phosphate buffer (100 mM, pH 7.4) containing MgCl2 (6 mM). Substrate was dissolved in DMSO and was added to a final concentration of 100 µM, such that the concentration of organic solvent in the incubation mixture did not exceed 0.2%. The reactions were initiated at 37 °C by the addition of 1 mM NADPH and terminated at 4 h by the addition of two volumes of acetonitrile. The precipitated proteins were removed by centrifugation and the resulting supernatant was subjected to Genevac dry-down and reconstitution for microscale isolation/purification.


Page 31 of 49

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•

LC-MS/MS:

Liquid chromatography separations were performed on a Waters Acquity UPLC system (Waters, Milford, MA) equipped with a photodiode array detector monitored in wide band from 190 to 400 nm. The mobile phase program consisted of initial linear gradient from 5% B to 50% B in 10 min, then to 95% B in 3 min and held for 4 min with post time for 4 min (A = 0.1% formic acid in water; B = 0.1% formic acid in acetonitrile). The column used was a Phenomenex Kinex C18 (50 x 2.1mm, 2.6 µm) operated at a flow rate of 0.6 mL/min at 40 °C. Waters QTOF Premier mass spectrometer (Waters, Milford, MA) was operated in the positive ion, V-mode at a mass resolution of 9000 (FWHM) at m/z 556. For the Premier, the source temperature was set at 110 °C, the capillary voltage was set at 3.0 kV, the sample cone voltage was set at 30 V, and the desolvation temperature was set at 250 °C and MCP plate was optimized at 1900 V. Nitrogen was used for desolvation and cone and gas was set at 800 and 20 L/h, respectively. The collision gas was argon at cell pressure of 12 psi. Scans were at 0.2 s duration with a 0.02 s interscan delay. For MS/MS measurements, the collision energy was set at 30 V. For MSE experiments, a collision energy ramp from 15 to 45 V was used. The data acquired in a centroid mode were collected from 50 to 1250 amu and MS/MS data collected from 50 to 700 amu. The lock mass was leucine enkephalin (m/z 556.2771) and was infused at 10 µL/min at a concentration of 500 pg/µL. •

Metabolite isolation:

Instrumentation: An Agilent 1260 analytical scale preparative HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an auto sampler (G1367E), a binary pump (G1312B), a column oven (G1316C) and a diode array detector (G1315D) were employed for the isolation of metabolites



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Page 32 of 49

from microsomal incubation mixture. The system was operated using the Chemstation software (version 5.5.2). Procedure: A RPLC method was developed to isolate metabolites from the microsomal incubation of compound 7. The conditions are as follows: column, XBridge C18 column ( 4.6 x150 mm, 3.5 µm; Waters, Milford, MA); flow rate 1.5 mL/min; column temperature, 25 °C; mobile phase, A, 0.1% formic acid in water(v/v), B, 0.1% formic acid in acetonitrile; gradient, A/B, 95/5, V/V, to A/B, 50/50, V/V in 13 min, then to A/B, 5/95 in 1 min and hold for 1 min; post time 6 min; detection, UV at 210 nm; injection volume, 40 µL. Fractions were collected at desired time intervals during the run. The collected fractions were pooled and subjected to solvent removal using a Genevac system (HT-4XHCl) (Genevac Inc., Stone Ridge, NY, USA). See Supporting Information for preparative HPLC chromatogram. •

Metabolite amount and purity assessment:

The amount and purity of isolated metabolites were determined by liquid chromatography using a Waters Acquity UPLC system (Waters, Milford, MA) equipped with a photodiode array detector monitored in wide band from 210 to 400 nm. The mobile phase program consisted of initial linear gradient from 5% B to 40% B in 10 min, then to 95% B in 3 min and held for 4 min with post time for 4 min (A = 0.1% formic acid in water; B = 0.1% formic acid in acetonitrile). The column used was a Phenomenex Kinex C18 (50 x 2.1mm, 2.6 µm) operated at a flow rate of 0.6 mL/min at temperature of 40 °C. Purity of each metabolite is assessed by UV at 218 nm (Figure S3 in Supporting Information). The concentration of isolated metabolite is based on its UV response comparing to compound 7 as standard with concentration at 0.052 mg/mL and


Page 33 of 49

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detection wavelength at 218 nm. The amount of metabolite is then estimated by its concentration times the volume. Metabolite NMR Data Acquisition: All metabolite NMR data were acquired using a Bruker AVANCE III, three channel 600 MHz NMR spectrometer equipped with a 1.7 mm TXI MicroCryoProbe. Following chromatographic isolation, metabolite samples were finally dried down from 1-2 mL of either acetonitrile-d3 or DMSO-d6 (Cambridge Isotope Laboratories, Andover, MA) in a 2 mL conical bottom glass vial that had been pre-rinsed with deuterated solvent and then dried. The metabolite sample was dissolved in 35 µL DMSO-d6 (99.96% D) and then transferred to a 1.7 mm NMR tube (Bruker BioSpin, Billerica, MA) using a 24G flexible Teflon needle (Wilmad Glass Co., Vineland, NJ) attached to a 100 µL Hamilton gas-tight syringe (Hamilton Company, Reno, NV). A typical metabolite data acquisition protocol employed the acquisition of a proton reference spectrum with water suppression, the data were acquired with the transmitter centered on the water resonance at ~3.34 ppm. Proton chemical shifts were reference to the center line of the residual protio DMSO multiplet at 2.50 ppm. A proton spectral width of 16 ppm digitized with 64K data points was typically employed with the number of transients accumulated dependent on the amount of metabolite available for analysis. Pure shift HSQC (PS-HSQC) spectra were acquired using the pulse sequence previously described.20 Data were acquired with water suppression and multiplicity-editing; data were phased so that CH/CH3 resonances exhibited positive phase while CH2 resonances were inverted. When the focus of the PS-HSQC experiment was aliphatic correlations, the 1JCH coupling constant-based delays were optimized for 145. For experiments focused on aromatic correlations, the delay for 1JCH couplings was



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Page 34 of 49

optimized for 165 Hz. The loop counter for application of the BIRD pulses during acquisition was set to l0=8 for all experiments performed. Long-range heteronuclear correlations were observed using the recently developed LR-HSQMBC experiment54 (detailed structure elucidation discussions in Supporting Information).

ALIS Experiment: •

Materials:

Buffer reagents and β-lactoglobulin were purchased from Sigma-Aldrich (St. Luis, MO) and used as received. HPLC solvents were purchased from Fisher Scientific (Fair Lawn, NJ). Factor IXa protein (415 amino acids) was prepared according to literature by the SPRI Protein Science Group and was purified to apparent homogeneity as assessed by SDS-PAGE, SEC, LC-MS, and N-terminal sequence. •

Sample preparation:

Metabolite solution preparation: 500 µL of RLM microsomal incubation solution was mixed with 500 µL of acetonitrile to precipitate proteins. After centrifugation at 14,000 rpm for 10 min, the supernatant was dried using a speed vac. The residue was re-suspended in 75 µL of Tris buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 0.1 mM DTT) to yield an aqueous metabolite mixture. Metabolite sample for affinity competition assay: A mixture containing metabolites and FIXa protein was prepared by combining 6 µL of metabolite solution described above with 34 µL of 23.5µM of FIXa protein. A 1 µL DMSO aliquot of a serially diluted stock solution of titrant 8 (10000, 5000, 2500, 1250, 624, 312, 156, 78, 38.8, 19.6, 10, 5 and 2.4 µM) was dissolved in 19 µL of above Tris buffer. The resulting solutions were mixed by repeated pipetting and clarified


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by centrifugation at 10,000 g for 10 min. The FIXa / metabolite solution (6 µL) and titration solutions (6 µL) were combined to yield a series of 12 µL experimental samples containing 10 µM FIXa protein, metabolites, and varying concentration of the titrant 8 (250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95, 0.97, 0.49, 0.25, 0.12, and 0.06 µM). These prepared samples were incubated at room temperature for 30 min, and then chilled to 4 °C prior to ALIS-AS analysis. •

ALIS-AS Data Acquisition:

Size-Exclusion Chromatography (SEC) separation: The ALIS-AShardware configuration used in this study has been described in previous publications.22-26 Briefly, SEC was performed at 4 °C using 700 mM of ammonium acetate (NH4OAc) buffer of pH 7.5. SEC columns were prepared in-house by proprietary methods.

An Agilent (Palo Alto, CA) isocratic pump

(G1310A) fitted with an Agilent online degasser (G1322A) was used for eluant delivery at 300 µL/min. Using this configuration, the SEC retention times were reproducible to better than ±2 s for a ~20 s chromatography run. With reproducible SEC chromatography, the slight variations in retention time from sample-to-sample were not a significant source of error in this method. Sample injection volume was 4.0 µL/injection; each sample was injected twice. RHPLC conditions: The eluant from the SEC column was passed through a UV detector (Agilent G1314A using a G1313 micro flow cell) where the band containing the protein-ligand complexes was identified by its native UV absorbance at 230 nm. After a pause to allow the band to leave the first detector and enter a valving arrangement, the protein-ligand complex peak was automatically transferred to an RPC column (Higgins Targa-C18, Higgins Analytical Inc., Mountain View, CA). Ligands were dissociated from the complex, trapped, and desalted at the head of the RPLC column with 12% acetonitrile in water (0.1% formic acid) for 3 min. The ligands were eluted into the mass spectrometer using a gradient of 12-27% acetonitrile (0.1%



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formic acid) in water (0.1% formic acid) over 22 min using an Agilent capillary binary pump (G1376A) for eluant delivery at 20 µL /min. To promote dissociation of ligands from the protein-ligand complex, the RPLC column was maintained at 40°C using an Agilent G1316A column compartment. MS conditions: In this study, MS analysis was performed using a Waters LCT Premier highresolution time-of-flight mass spectrometer (Milford, MA) with positive mode ionization occurring from a standard nebulized ESI source with the capillary set at 2.7 kV, a desolvation temperature of 250 °C, a source temperature of 100 °C, and 30 V “cone”. Raw data collection: Based on the metabolic profile of 7, several ions are monitored such as (M), (M+16), (M+32), along with titrant (8) ions. The positional isomers of some ions were well separated. The Waters Masslynx program was used to calculate the peak areas. The areas underlying the extracted ion chromatograms (XICs) for the singly protonated, doubly protonated, and monosodiated ([M+H]+, [M+2H]2+, [M+Na]+) species were summed. As binding control experiments, the RLM metabolite mixture from 7 was incubated with 10 µM of β-lactoglobulin. The sample was analyzed with the same procedure as above. No βlactoglobulin-bound RLM metabolites of 7 were detected, which indicated that RLM metabolites of 7 specifically bind with FIXa protein. •

Data Analysis:

For each AS-MS experiment representing a single data point in an Affinity Competition Experiment 50% titration (ACE50), the peak area of the ions of interest for each data point was normalized for the entire titration curve by dividing each ion’s raw response by its highest response in the titration experiment (typically a data point where the titrant concentration was lowest). The titration data were then fitted to a variable slope sigmoidal dose-response using


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GraphPad Prism (version 6.0, GraphPad Software, La Jolla, CA, www.graphpad.com) with a maximum normalized value of 1.0. The titrant concentration at which the fit curve passed through 0.5 represents the ACE50 value. Because the concentration of competitor necessary to reduce the ligand response to one-half its value in the absence of competitor defines the affinity ranking, normalized responses are a more readily interpretable way to represent the data than raw responses.

No calibration curves are necessary to determine the affinities of the mixture

components.

Supporting Information. Experimental procedures and additional results and discussions. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors: * T.Z.: Phone: +1-732-594-3031; e-mail: [email protected]; Y.L.: Phone: +1-732-5949040; e-mail: [email protected] ACKNOWLEDGMENT The authors would like to acknowledge Merck Discovery Process Chemistry group for providing synthetic intermediates, Merck Department of Pharmacokinetics for preliminary PK studies, and Drs. Kevin Bateman and Paul O’ Shea for their encouragment and invaluable discussions. ABBREVIATIONS AS-ALIS, affinity selection−mass spectrometry-based Automated Ligand Identification System; ACE, affinity-based competition; AUC, area under the curve; CL, clearance; ELSD, evaporative



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light scattering detectordynamic light scattering; ESI-MS, electrospray ionization mass spectrometer; F%, bioavailability; FIXa, Factor IXa; h, hour(s); hFIXa, human Factor IXa; FXa, Factor Xa; hFXa, human Factor Xa; IDR, inverted direct response; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy; HLM, human liver microsome; HPLC, high-performance liquid chromatography; HR-MS, high-resolution mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; LR-HSQMBC, long-range heteronuclear single quantum multiple bond correlation; min, minute(s); NMR, nuclear magnetic resonance; PD, phramacodynamics; PK, pharmacokinetics; PS-HSQC, pure shift heteronuclear single quantum coherence; RLM, rat liver microsome; RPLC, reverse-phase liquid chromatography; s, second(s); SAR, structure–activity relationship; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography; s/n, signal-to-noise; T1/2, half-life. REFERENCES (1) Pelkonen, O.; Turpeinen, M.; Uusitalo, J.; Rautio, A.; Raunio, H. Prediction of drug metabolism and interactions on the basis of in vitro investigations. Basic Clin. Pharmacol. Toxicol. 2005, 96, 167–175. (2) Plant, N. Strategies for using in vitro screens in drug metabolism. Drug Discovery Today 2004, 9, 328–336. (3) Brandon, E. F. A.; Raap, C. D.; Meijerman, I.; Beijnen, J. H.; Schellens, J. H. M. An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons. Toxicol. Appl. Pharmacol. 2003, 189, 233–246.


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Graphical Abstract 296x139mm (96 x 96 DPI)



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Figure 3 136x74mm (120 x 120 DPI)


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Figure 5 145x127mm (96 x 96 DPI)


Definitive Metabolite Identification Coupled with ... - ACS Publications

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