High Throughput Method for the Indirect Detection of Intramolecular

Article pubs.acs.org/jmc

High Throughput Method for the Indirect Detection of Intramolecular Hydrogen Bonding Gilles H. Goetz,*,† William Farrell,‡ Marina Shalaeva,† Simone Sciabola,§ Dennis Anderson,† Jiangli Yan,† Laurence Philippe,† and Michael J. Shapiro† †

Groton Laboratories, Worldwide Medicinal Chemistry, Pfizer Global Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States ‡ La Jolla Laboratories, Worldwide Medicinal Chemistry, Pfizer Global Research & Development, 10770 Science Center Drive, San Diego, California 92121, United States § Neuroscience, Worldwide Medicinal Chemistry, Pfizer Global Research & Development, 610 Main Street, Cambridge, Massachusetts 02139, United States S Supporting Information *

ABSTRACT: A supercritical fluid chromatography method was developed for the detection of intramolecular hydrogen bonds in pharmaceutically relevant molecules. The identification of compounds likely to form intramolecular hydrogen bonds is an important drug design consideration given the correlation of intramolecular hydrogen bonding with increased membrane permeability. The technique described here correlates chromatographic retention with the exposed polarity of a molecule. Molecules that can form an intramolecular hydrogen bond can hide their polarity and therefore exhibit lower retention than similar compounds that cannot. By use of a pairwise analysis strategy, intramolecular hydrogen bonds are identified within a test set of compounds with diverse topologies. The chromatographic results are confirmed by NMR chemical shift and temperature coefficient studies. stant4 or experimentally highlighted by laborious spectroscopic methods. For example, Winningham and Sogah5 used the Fourier transform infrared (FTIR) spectroscopy to determine the conformation of β-turn nucleators and within the resultant structures identified the presence of hydrogen bonding. Nuclear magnetic resonance (NMR) has also been used for the detection of hydrogen bonding, through the comparison of chemical shifts in different solvents,6 deuterium exchange7 rates, and temperature coefficients.8 The throughput of such spectroscopic methods is limited and is a challenge for their use in understanding structure−activity relationships (SAR) and driving rapid cycles of compound optimization. Recently, Shalaeva et al.9 introduced a method to characterize IMHBs by measuring Δlog P, which is defined as the difference in a compound’s distribution between a protic and an aprotic

1. INTRODUCTION Polarity is a fundamental physicochemical property of molecules. It contributes to molecular geometry, shape, and conformation and impacts both chemical and biological properties, such as solubility and permeability. Charge−dipole and dipole−dipole interactions occur through both intermolecular and intramolecular forces.1 Intramolecular hydrogen bonds (IMHBs) are noncovalent interactions whose formation depends greatly upon the conformational accessibility of an interaction and the donor and acceptor characteristics of polarizable functionalities. IMHBs are ubiquitous among organic molecules and play a critical role in defining the secondary structures of proteins2 and peptides, as well as the conformations of small molecules.3 Historically the identification of IMHBs either computationally or by experiment has been challenging. Theoretically they can be characterized through ab initio calculations that generate parameters such as interaction energy, electrostatic charge transfer, polarization, exchange repulsion, and coupling con© 2014 American Chemical Society

Received: December 3, 2013 Published: March 18, 2014 2920

dx.doi.org/10.1021/jm401859b | J. Med. Chem. 2014, 57, 2920−2929

Journal of Medicinal Chemistry

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solvent, respectively octanol and toluene. The Δlog P values of pairs of compounds are compared to predict their propensities to form IMHBs. The key advantage of the Δlog P method over FTIR and NMR is that it uses a physiologically relevant environment that is conducive to IMHB formation. While an improvement over previous methods, the Δlog P technique remains low throughput and laborious for evaluating the impact of IMHBs on the SAR within a series of compounds. A chromatographic system, where the mechanism of retention is predominantly driven by polarity, has the potential to be very high throughput and can be used to identify IMHB differences between matched pairs of compounds. Higher throughput methods based on reversed-phase liquid chromatography have been used to study heteroarylamides10 and isomeric benzamides11 by tracking the retention time (tR) of compounds predicted to form IMHBs. However, reversed-phase chromatography involves a biased environment due to the use of water as a solvent, which has the ability to significantly disrupt any IMHB within the analyte. For these reasons we sought to develop a high throughput method that retained the appropriate environment for the formation of hydrogen bonds. Supercritical CO2 was used as a nonpolar eluent with the addition of methanol as a modifier, resulting in a system in which the partition of compounds between a polar stationary phase and a nonpolar mobile phase can be measured. Specifically, the rationale for the use of supercritical fluid chromatography (SFC) is that it uses inherently normal phase conditions; thus, the solvents have low dielectric constants, unlike aqueous solvents used in reversed-phase chromatography.12 Intact IMHBs are thought to reduce the overall polarity of a molecule relative to an analogous compound that cannot form an IMHB. Therefore, compounds can be distinguished from one another by comparing their relative retention under SFC conditions. Molecules capable of forming an IMHB will display a shorter retention time (tR) than similar molecules incapable of forming IMHBs. To verify this hypothesis on druglike compounds, a collection of over four million compounds with available samples was searched for structures predicted to be capable of forming IMHBs. Previous studies by Etter13 and Bilton14 have shown that mining crystallographic databases is a powerful resource to discern potential IMHB patterns. Bilton et al.14 published 50 IMHB topologies, and their corresponding propensity of formation, by analyzing small molecule crystal structures in the Cambridge Structural Database (CSD). Kuhn et al.3 mined both the CSD and the Protein Data Bank (PDB), focusing their search on motifs of pharmaceutical interest. Key in their approach was the differentiation between sp2 and sp3 hybridized carbons, nitrogens and oxygens, as well as cyclic and acyclic bonds. They reported the probability of IMHB formation for 42 unique topologies. Here the 42 substructures published by Kuhn3 were used to identify matched compound pairs from the four million available compounds. Matched pairs refer to a compound with a substructure prone to IMHB formation along with a control compound incapable of forming that bond, typically a regioisomer. The matched pairs of compounds were evaluated in the SFC method described here. This method is referred to henceforth as EPSA determination. The results demonstrate the ability of the EPSA methodology to identify the potential for a compound to form IMHBs and as a result hide polarity that might otherwise reduce the permeability of the compound.1,15,16

2. RESULTS AND DISCUSSION 2.1. EPSA Method. Medicinal chemists often use computational estimations to assess compound polarity. The topological polar surface area (TPSA) of a molecule is defined as the sum of all polar atom surfaces, primarily oxygen and nitrogen, including their attached hydrogen atoms.17 Calculated in this way, TPSA is conformationally independent and does not account for any potential intramolecular hydrogen bond. A more accurate estimation of polar surface may be achieved through a conformational analysis and quantum mechanical calculation; however, flexible compounds can make the identification of global minima and IMHBs difficult18 and the methodology is computationally expensive. The balance of accuracy and computational expense leads to the question of whether polarity is a physical property that can be evaluated more rapidly and accurately using experimental rather than computational methods. In SFC chromatographic systems the retention times of closely related compounds can differ significantly. The presence of an IMHB can therefore be identified, as it can result in a significant change in polarity. This is demonstrated in the example of two regioisomers shown in Figure 1. The separation of these two compounds in the EPSA method is in stark contrast to their TPSA values which are identical.

Figure 1. Superimposed chromatograms of compounds 1 capable of IMHB formation (red trace) and 2 incapable of IMHB formation (blue trace).

The compound separation in Figure 1 was achieved using an optimized SFC system. To identify IMHBs, it was critical that the chromatographic conditions not only separate compounds based on their polarity but also provide an environment in which the bonds are maintained. A stationary phase was needed with a balance of lipophilic and polar attributes and the ability to discriminate analytes with a wide array of polarities. After evaluation of several options, the Pirkle chiral stationary phase Chirex 3014, a silica bonded (S)-valine and (R)-1-(α-naphthyl)ethylamine with a urea linkage, was selected. A mobile phase of 15% methanol in supercritical CO2 allowed for partial separation, and with the addition of a low-slope gradient adequate separation was achieved. High retention times were mitigated by increasing the percentage of methanol in the eluent and the addition of the relatively weak salt pair ammonium formate. In the resulting chromatographic system, differences in retention depend upon hydrophobic, hydrogen bonding, dipolar, and polarizability interactions of the solutes with the stationary phase.19 2.1.1. Correlation of EPSA and TPSA. A set of 118 compounds was used to establish a correlation between retention times and TPSA values. The compounds were selected with a distribution of polar surface area (40−130 Å2) based on 2921



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typical retention times, and assigned EPSA values are given in Supporting Information. 2.1.2. Evaluation of the EPSA−IMHB Relationship. As an initial test of the methodology, the EPSA values for a set of ortho, meta, and para substituted benzene analogues were determined. Methylbenzoic acid, methylphenol, and tert-butylphenol are all incapable of forming IMHBs in any regioisomer (ortho, meta, and para) because of their functionalization. As shown in Figure 3, the three regioisomers of each compound have essentially

calculated TPSA. In addition, the 118 compounds were selected such that conformational restrictions prevented the formation of IMHBs. Figure 2A shows the resulting linear relationship (R2 = 0.94) between TPSA values and the SFC retention times.

Figure 3. Differences in retention times between selected phenols, benzaldehydes, benzoketones, and benzoic acids. For each compound, the corresponding regiosiomers are color coded as ortho (yellow), meta (red), and para (blue).

equivalent EPSA. In contrast, when the regioisomers of hydroxybenzaldehyde, 1-hydroxyphenylethanone, methoxyphenyl, hydroxyphenol, and nitrophenol are evaluated, the ortho analogues have a significantly reduced retention. This can be explained by the conformational accessibility of IMHBs within these isomers, resulting in a reduced polarity and retention. Aminobenzoic acid stands as an outlier within the compounds predicted to have a conformationally accessible IMHB within the ortho isomer. Aminobenzoic acid shows an increased EPSA for the meta analogue. We interpret this as demonstrating the dominance of carboxylic acids in the chromatographic interaction. This behavior was seen for almost all carboxylic acids evaluated with the EPSA method and indicated that the methodology, in its current state, is not optimized for this compound class. Carboxylic acids require interpretation as a separate class. 2.2. Data Set. A larger test set of druglike compounds with the potential to form IMHBs was identified through the systematic mining of a collection of 4 million compounds in the Pfizer corporate database. This search identified a data set of matched molecular pairs, one conformationally capable and one incapable of forming an IMHB. The data set of matched pairs was further filtered using cutoffs for cLogP, TPSA, and molecular weight, as well as Kuhn’s topologies3 (Figure 4). To identify the matched pairs, a fully automated search was implemented to quickly highlight substructures in small molecules found to match any of the topologies previously described by Kuhn et al.3 The 42 topologies were converted into SMARTS (SMILES arbitrary target specification) patterns and annotated with the corresponding parameters: (1) propensities to form IMHBs, (2) the Cambridge Structural Database (CSD) and Protein Data Bank (PDB) entry codes for the reference structures, (3) the type of hydrogen bond donor (HBD)/ hydrogen bond acceptor (HBA) groups, and (4) the size of the transient ring created.

Figure 2. (A) Linear relationship between TPSA and SFC retention time (minutes) for the 118-compound set. (B) Determination of EPSA values for the 6 calibration standards.

Assuming the absence of hydrophobic collapse, compounds without IMHB have their polar groups (summed up in TPSA) exposed to the environment. For this data set, retention times correlate reasonably well with TPSA, and SFC could be an experimental measurement of the polar surface area (a TPSA surrogate) or, at the very least, of the apparent polarity of the molecule. Since TPSA is widely used as a preferred tool for molecular evaluation of lead compounds by the medicinal chemistry community, a related term for the values measured by this SFC method, which could be readily interpreted and applied, is likely to be desired. Therefore, the retention-time-derived value was termed EPSA, in analogy to the ELogP/ELogD nomenclature introduced by Lombardo et al.20,21 Like any chromatographic method the retention times are the results of the interaction of a ligand with a specific chromatographic solid support, under controlled conditions. It was therefore important to normalize across runs and establish standards and calibrate the results. Six compounds were selected as calibration standards based on their retention times. By use of the linear relation established in Figure 2A, these compounds were assigned EPSA values (Figure 2B) and used to adjust for inter-run variability. The calibration standards were combined into a calibration mixture, which was injected at regular intervals between measurements, accounting for variation in pressure, temperature, and flow, as well as column deterioration. EPSA values are rounded and reported as whole numbers, without units. More details, including calibration standards structures, 2922



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Table 1. Summary of Distributions between Topologiesa

Figure 4. Simplified view of the successive filtering steps applied to the Pfizer compound database in order to create the final data set utilized in this study.

This strategy allowed an internal database of 4 million available compounds to be searched against the 42 topologies in 3−5 min, including a prefiltering step to remove compounds outside a conservative druglike chemical property space (CNS MPO ≥ 3, 0 ≤ cLogP ≤ 4, 55 ≤ TPSA ≤ 110).22 The search resulted in ∼390K matches, annotated with their ring size and HBD/HBA combination. Each of the 390K compounds with the potential to form an IMHB was used as a query in a second search to identify structural similar controls incapable of forming IMHBs. Figure 5

a

Topology Information: occurrence of topology as reported by Kuhn3 (shown in bold text); number of matched pairs before clustering (shown in italic text). The asterisk (∗) indicates use of clustering to reduce numbers. The plus character (+) indicates use of loosened inventory threshold to increase numbers. Number of matched pairs passing final QC filtering is shown as underline text.

carbon-bonded protons was accomplished using C−H HSQC experiments, which helped confirm the assignment of the non C−H proton signals. Proton spectra were also recorded for a subset of these compounds at low concentration, at temperatures ranging from 25 to 45 °C, to minimize the possible effect of intermolecular hydrogen bonding. Where the hydrogen bond donor proton could be positively assigned, the temperature coefficients (TCs), calculated by linear fitting of chemical shift vs temperature response, of all the compounds were determined from these data. Comparison of the TCs at higher and lower concentration is shown in Table 2. NMR is very sensitive to structural as well as environmental effects, and chemical shifts can often be measured with very high precision. For example, the amide proton chemical shift depends upon many factors, including hydrogen bonds. In general, a stronger hydrogen-bonded amide proton has a decreased shielding and therefore tends to move downfield in an NMR spectrum. It has been suggested that a direct hydrogen bond has a dominant effect on the chemical shift of amide protons.23 When protons in similar chemical environments are compared, the chemical shift provides useful information for studying IMHB. As the observed chemical shifts reflect all structural and environmental effects, corroborating evidence may be required to make definitive assignments.

Figure 5. Examples of appropriate controls. Regioisomers b and c are acceptable as controls because they are unable to form IMHB. Compounds d and e were rejected because they can also form IMHB. The amino groups of isomers e, f, and g are on the wrong ring and make poor comparators.

shows examples of acceptable regioisomer controls. This second search resulted in 574 matched pairs representing 23 IMHB topologies after additional restrictions were placed on (1) carboxylic acids, (2) the presence of only one IMHB topology, (3) isomerism in the IMHB region of the molecule, and (4) 20 mg of compound being available for both members of the matched pair. The final 84 matched pairs used in this study were obtained by focusing on the most common IMHB topologies as reported by Kuhn.3 For topologies that were over-represented, clustering was used to select a set of diverse representatives. Table 1 summarizes the data set obtained after QC validation. 2.3. Detection of IMHB by NMR. The proton spectra of the 137 compounds from the 84 matched pairs in the final data set were acquired in deuterated DMSO at three temperatures, 25, 35, and 45 °C. The chemical shifts of each hydrogen bond donor were recorded whenever possible. Assignment of the signals for 2923



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The formation of IMHBs also changes the temperature dependence of amide protons’ chemical shifts, and the TC can therefore be used to evaluate IMHBs.8,24 Amides involved in strong IMHB tend to have less negative temperature coefficients than those that are not H-bonded. Even though this mechanism is not well understood,25,26 it was confirmed that the TC of amide proton chemical shifts and J-couplings through H-bond (or Hbond strength) are strongly correlated in proteins.25 The TCs of all compounds in this study were measured, and six matched pairs in the T2 topology were evaluated. Five of these pairs showed the predicted result: the N−H, which is potentially H-bonded, had higher chemical shifts and less negative TCs relative to the N−H of the control compounds. In this topology, both NMR results of chemical shift and TC are consistent with the EPSA results, indicating the presence of an IMHB from N−H to CO through a transient six-membered ring (Table 2). In other topologies, such as T3, evidence of IMHBs was found neither by chemical shift nor by TC under the testing conditions. This is in contrast to what is observed in the EPSA measurements where all eight matched pairs are consistent with hidden polarity. One possible explanation for these data may lie in the nature of the R group substituents near the hydrogen bond donor and acceptor. In compounds with the T3 topology, steric effects of bulky R-groups located near either the donor or acceptor may disrupt the ability of the donor−acceptor pair to approach the preferred geometry for the hydrogen bond. Yet the bulky R-groups may also act to hide the polar donor and acceptor groups in the interior of the molecule and shield them from bulk solvent interaction, accounting for the apparent differences in polarity between the potentially hydrogen-bonded compounds and their controls. Where weak intermolecular hydrogen bonds potentially may have competed with the IMHBs and confounded the results, NMR experiments were repeated at 0.1 mM to avoid said intermolecular hydrogen bonds. As seen in Table 2, both chemical shift and TC data do not point to the existence of intermolecular hydrogen bonds, being consistent with data generated under more concentrated conditions (50 mM) required for opimal flow NMR automation. 2.4. Methodology for Data Interpretation. Every matched pair was assessed for IMHBs by comparing the difference in EPSA for the compounds in the pair (ΔEPSA). On the basis of the hypothesis that an IMHB reduces the polarity of a compound compared to its control, when the EPSA value of the control is larger than the EPSA value of the sample, the presence of an IMHB is predicted. If the EPSA value of the control is equal to or smaller than the EPSA value of a sample, an IMHB is not apparent. Data for standards collected over the course of a year in multiple laboratoties showed EPSA values to be measured consistently within ±1 unit, resulting in error bars of ±2 units for the ΔEPSA. ΔEPSA values of greater than +3 units were therefore considered significant and indicative of the presence of an IMHB. Trends within and across topologies were studied using ΔEPSA values for the matched pairs (Table 1 and Figure 6). ΔEPSA values were deemed significant for 63 out of 84 matched pairs and therefore highlight the presence of IMHBs, the distribution of which are summarized in Table 1. 2.5. Examples of EPSA Highlighting Presence of IMHB. Significant differences were measured by EPSA between members of each matched pair identified from the following topologies: T2, T3, T6, T7, T10, and T11 (pyridine). Noticeably, three of the six topologies feature a N−H to C

Figure 6. IMHB demonstrated by ΔEPSA for the matched pairs. Each topology chart is divided into two parts, ΔEPSA > 3 (green) and ΔEPSA ≤ 3 (red), and annotated with the pairs count and percentage.

O IMHB and are among the most prominent topologies in the CSD.3 Figure 7 depicts typical representative matched pairs of compounds for these topologies. Topology T2 is represented by matched pairs 1−2 and 3−4 whose EPSA data were measured respectively at 108, 119 and 59, 77. The significant difference of polarity between those two matched pairs is due to the presence of the amidine moiety in 1−2. However, EPSA still displays a significant difference within the 3−4 matched pair, highlighting the IMHB in 3. NMR chemical shifts and TC differences corroborate the EPSA results for most compounds in the T2 topology. For example, the chemical shift of the N−H in 1 is 10.1 ppm and in 2 it is 9.0 ppm. As shown in Figure 7, the N−H TC for 1 is −1.4 ppb/K and that for 2 is −4.4 ppb/K. The only difference between the two molecules is the position of the C O relative to the amide function. The CO and amide N−H in 1 can form an IMHB through a six-membered ring, while an IMHB is impossible for 2, indicating that a significant contribution to the N−H chemical shift difference of 1.2 ppm between the two molecules is caused by the presence of an IMHB in 1. The T3 topology is represented by pair 5−6 whose EPSA data were measured respectively at 52 and 65. The chemical shift of the N−H in 5 and 6 is 9.0 and 5.5 ppm, respectively, in line with expectations, but the TC of 5 is −2.5 ppb/K, which is on the high end of IMHB-indicating TCs. Compounds 8 and 9 are both controls for 7, creating matched pairs 7−8 and 7−9, representatives of T6. The EPSA value for 8 was 106 and 9 had a value of 94, compared to 75 obtained for compound 7. The chemical shift differences are as expected for presence/absence of IMHB in matched pairs, but the TC of 7 is −5.9 ppb/K, a value incompatible with the presence of an IMHB under our NMR conditions. When recorded in dilute conditions, chemical shifts stayed constant but TCs for both 7 and 8 became less negative: −3.4 and −3.7 ppb/K, respectively. Both matched pairs of T7 show significant differences between measured EPSA values, as illustrated by pair 10−11; unfortunately significant signal broadening precluded us from generating reliable NMR data for this pair, even in dilute conditions. A clear difference was observed within topology T11 between compounds where the HBAs were pyridine N or pyrazole N 2924



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Figure 7. Examples of matched pairs of compounds where IMHB is highlighted by EPSA. NMR chemical shifts and TCs were recorded at 50 and 0.1 mM.

(Figure 6). ΔEPSA values were significant and elevated for pyridine containing compounds and reduced for N-alkylated pyrazoles. The strength of the HBA is a clear contributor to the existence of the IMHB in this particular case. 2.6. Examples of EPSA Highlighting the Presence of Multiple IMHBs. Several of the 84 studied matched pairs gave near zero ΔEPSA where we expected to observe an IMHB. The 12−13 pair is a member of the topology T2 which was identified by Kuhn3 as prone to IMHB (93% occurrence and consistent ΔEPSA). An IMHB is expected in 12; however, the measured EPSA values for both compounds in this matched pair are identical. Upon careful inspection of the structures, it appears that the control compound 13 could form an IMHB as well, between the O of the lactone and the N−H of the amide.

While this topology was not described by Kuhn,3 it appears to represent a valid intramolecular hydrogen bond (C−O···H−N, five-membered ring). Molecular modeling studies confirmed such five-membered rearrangement as a low energy conformation (Figure 9). The matched pair 14−15 from topology T11 shows yet another example of an unexpected topology highlighted through EPSA results. The measured EPSA values for this matched pair are nearly identical. As illustrated in Figure 10, the control compound 15 can form an IMHB between the N−H of the amide and the nonbridge N in the pyrimidine ring (N···H−N, six-membered ring). This is in line with the observed TC data recorded in both concentration conditions, an IMHB being detected for 15 but not for 14. 2925



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Table 2. EPSA and NMR Data for Selected Topologiesa

a

The asterisk (∗) indicates that NMR signals were too broad for accurate assignments.

recognize new topologies in our database and integrate them in our list of IMHB motifs. While EPSA appears to be effective at discriminating IMHB forming compounds from their controls in topologies featuring six-or seven-membered ring IMHBs from N−H to CO or from N−H to aromatic N, the differences between matched pairs in topologies featuring five-or six-membered ring IMHBs from C−O to N−H, from CO to O−H, or from N−H to aromatic N were less obvious. Understanding the mechanism of retention and which properties impact that retention, in addition to IMHB-

Further examination of the structures of other matched pairs with near zero ΔEPSA brought to our attention that this very same topology was present in both compounds of the only matched pair of the T1 topology. The difference in EPSA values was not significant, with the control 17 eluting ahead of the IMHB-prone 16. Indeed, 17 and 16 form an IMHB between the N−H of the amide and the N of the quinazoline, as depicted in Figure 8. As it turned out, this IMHB had been specifically designed by K. Liu et al. 27 and confirmed by X-ray crystallography. NMR data recorded in dilute conditions indicate similar TC behavior for both 16 and 17. This study allowed us to 2926



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Figure 8. Examples of significant exceptions where EPSA data highlighted the presence of an additional IMHB in the control compound.

Figure 9. Removal of the T2 topology in 12 led to compound 13 which does not share any of the previously reported topologies.3 Conformational analysis of 13 produced an energy minimum structure with an IMHB between the endocyclic oxygen atom and the amide NH. Figure 10. Removal of the T11 topology in 14 led to compound 15. Conformational analysis of 15 produced an energy minimum structure with an IMHB between the pyrazolopyrimidine N atom and the amide NH.

hidden polarity, is key to further understanding these observations.

3. CONCLUSION A new chromatographic method to identify IMHBs with increased throughput has been developed and successfully tested on known six- to seven-membered ring IMHB motifs. The hypothesis that IMHBs cause shorter retention in our system has been verified through a pairwise analysis. Many of the observed exceptions can be explained by additional IMHB topologies interfering with expected behavior or by other strong interactions formed with the solid support competing with the IMHB. NMR

data were successfully used to corroborate EPSA data in most cases. EPSA has the potential to be applied in medicinal chemistry programs as an indicator of the presence of IMHBs and as a tool for indirect polarity measurement.28 Since it has been shown that formation of IMHBs increases potential for membrane permeability, the EPSA property promises to have significant utility in drug design.15,16 2927



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4.3. Computational Methods. The Python scripting language (version 2.7.3) was adopted to implement and automate the different mining stages discussed in the paper. The multiprocessing API within Python was applied to fully leverage the multiple processors available on our high performance computing infrastructure and speed up the calculations. For all compounds in the database, the RDKit 2011.12.1 cheminformatics library for Python was used to handle all the data mining automation: (1) SMARTS search, (2) fingerprint annotation, (3) similarity search. Additionally, the MDL 166-bit keyset was used as preferred chemical fingerprint for the similarity search, and an in-house molecular properties calculator was used to compute simple properties (MW, PSA, cLogP, H, C, N, O, F, P, S, and Cl count, number of atoms, bonds, hydrogens, explicit atoms, explicit bonds, positive atoms, negative atoms, ring bonds, rotatable bonds, aromatic bonds, bridge bonds, rings, aromatic rings, ring assemblies, rings 3, rings 4, rings 5, rings 6, rings 7, rings 8, chains, chain assemblies, fragments, complexed fragments, single bonds, double bonds, triple bonds, aliphatic single bonds, aliphatic double bonds). CORINA29 was used to generate the initial 3D structures of compounds 12, 13, 14, and 15. A conformational analysis was performed using the software OMEGA30 with a dielectric constant of 4 and the MMFF94s force field (all the other command line options were kept as default).

Ultimately, the ability to predict the EPSA values of virtual compounds will enable design of analogues using polarity-based prioritizations. The development and implementation of our EPSA prediction tool are in progress and will be reported in due course.

4. EXPERIMENTAL SECTION 4.1. EPSA Method. 4.1.1. Chemicals and Reagents. The mobile phase modifier consists of ammonium formate (98%) (Acros Organics, Geel, Belgium) diluted to 20 mM in HPLC grade methanol (J. T. Baker, Center Valley, PA). Carbon dioxide and nitrogen are bulk grade and were purchased from AirGas. The CO2 was purified and pressurized to 1500 psi using a custom booster and purifier system from Va-Tran Systems Inc. (Chula Vista, CA) and supplied to all SFC instruments in the lab. 4.1.2. Sample Preparation. Each sample was dissolved to approximately 3 mM in DMSO, and an amount of 100 μL was plated in 96 V-bottom polypropylene plates. 4.1.3. Instrumentation. The SFC/MS instrument used in this experiment was an Agilent-Aurora supercritical fluid chromatograph system. The mass spectrometer used was a single quadrupole LC/MSD with an ESI source (Agilent, Palo Alto, CA). All data were acquired using Agilent 32-bit ChemStation (version B.03.01 [317]) The effluent of the SFC was split to the MSD using a tee (Valco, Houston, TX) and PEEKsil capillary tubing (Upchurch Scientific, Oak Harbor, WA), 50 cm long with an internal i.d. of 50 μm. 4.1.4. Analysis Conditions. Analysis was performed using a 4.6 mm × 250 mm Chirex 3014 column (Phenomenex, Torrance, CA) with 5 μm particle and 100 Å pore size. The flow rate was 5 mL/min with the outlet backpressure set to 140 bar. The injection volume was 5 μL. The mobile phase composition was varied from 5% to 50% modifier at 5%/min in a linear gradient, holding at 50% for 1 min and reverting to the original 5% until the end of the run. Data are acquired during 12 min, which, including rinsing and equilibration time, results in an injection to injection time of 13 min. Each sample is analyzed in duplicate, resulting in a sample to sample turnaround time of 26 min. The column temperature was set to 40 °C. Prior to reaching the UV detector, the eluent was heated to 60 °C, and after the split the portion destined to the MSD was heated to 76 °C. The MSD was used in single ion monitoring mode along with scanning. 4.2. NMR Methods. NMR data were collected under two different conditions. Data for the full set of compounds were collected on an Agilent DD2 600 MHz spectrometer using a Protasis microflow cell probe with a 15 μL flow cell. Samples were prepared in 96-well plates using stock solutions from the company’s internal sample bank at a nominal 30 mM concentration in DMSO. The stock solution was evaporated to dryness using a Genevac evaporator, reconstituted in deuterated DMSO, and re-evaporated. The samples were then reconstituted in DMSO-d6 to a nominal concentration of 50 mM. After each sample was pumped into the microflow cell probe, the sample was allowed to equilibrate at the probe temperature for 5 min prior to data acquisition. Temperatures of 25, 35, and 45 °C were used for each sample. The proton experiments used 45° pulses, 64K complex data points for acquisition, 128 transients, and an interpulse cycle time of 5 s. An edited CRISIS type HSQC with sensitivity enhancement was used to determine single bond correlations. Experiments were acquired using 2K complex points in F2 and 64 complex points in F1. The F1 points were linear predicted to 256 points and zero-filled to 1K points. Gaussian window functions were used in both dimensions. A subset of the compounds, representing many different topologies, was used for study at a lower concentration (0.1 mM). Aliquots of the reconstituted solutions used for 50 mM samples were diluted to a concentration of 0.1 mM in DMSO-d6. Data for these samples were collected on a Bruker Avance III 600 MHz spectrometer using a TCI cryoprobe. Proton experiments were conducted at temperatures of 25, 30, 35, 40, and 45 °C for each sample, with a 10 min equilibration time for each temperature point. Experiments were run using 30° pulses, 64K complex data points for acquisition, 256 transients, and an interpulse delay of 4 s.

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ASSOCIATED CONTENT

S Supporting Information *

Structures, retention times, and assigned EPSA values of calibration standards. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 1-860-715-6311. E-mail: gilles.h.goetz@pfizer.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Robert Stanton for critical reading and editing of the manuscript. ABBREVIATIONS USED SFC, supercritical fluid chromatography; NMR, nuclear magnetic resonance; SAR, structure−activity relationship; HPLC, high performance liquid chromatography; DMSO, dimethyl sulfoxide; LC/MSD, liquid chromatography/mass spectrometry detector; HBD, hydrogen bond donor; HBA, hydrogen bond acceptor; IMHB, intramolecular hydrogen bond; ΔEPSA, difference in EPSA; Δlog P, difference in log P; cLogP, calculated log P; CNS MPO, central nervous system multiparameters optimization; CRISIS, compensation of refocusing inefficiency with synchronized inversion sweep; HSQC, heteronuclear single quantum coherence; PDB, Protein Data Bank; QC, quality control; CSD, Cambridge Structural Database; FTIR, Fourier transform infrared; SMILES, simplified molecular-input line-entry system; SMARTS, simplified molecular-input line-entry system arbitrary target specification; tR, retention time; TC, temperature coefficient; TPSA, topological polar surface area

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REFERENCES

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