Beyond the Rule of 5: Lessons Learned from AbbVie's Drugs and

Sep 19, 2017 - Recently, there has been an increasing focus on the pursuit of targets considered to be less-druggable that offer potential for develop...
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Beyond the Rule of 5: Lessons Learned from AbbVie’s Drugs and Compound Collection Miniperspective David A. DeGoey,* Hui-Ju Chen, Philip B. Cox, and Michael D. Wendt Research and Development, AbbVie Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States S Supporting Information *

ABSTRACT: Recently, there has been an increasing focus on the pursuit of targets considered to be less druggable that offer potential for development of promising new therapeutic agents for the treatment of diseases with large unmet medical need, particularly in the areas of oncology and virology. However, conducting drug discovery campaigns in “beyond rule of 5” (bRo5) chemical space presents a significant drug design and development challenge to medicinal chemists to achieve acceptable oral pharmacokinetics. Retrospective analysis of past successes and failures in drug discovery bRo5 may shed light on the key principles that contribute to the oral bioavailability of successful bRo5 compounds and improve the efficiency of drug design for future projects. We present here highlights and case studies of lessons learned from discovery of bRo5 compounds. A simple multiparametric scoring function (AB-MPS) was devised that correlated preclinical PK results with cLogD, number of rotatable bonds, and number of aromatic rings.

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INTRODUCTION It has been 20 years since the publication of Lipinski’s influential analysis of the physicochemical property space occupied by phase II clinical candidates.1 While Lipinski’s rule of 5 (Ro5) described the property space with the highest probability of achieving good oral absorption, today there is an increasing focus on the pursuit of less druggable targets, such as the disruption of protein−protein interactions (PPI), that offer high potential for the development of new therapeutic agents and may require beyond rule of 5 (bRo5) chemical matter in order to take advantage of these opportunities. The fact that approximately 6% of oral drugs are outside of Lipinski space suggests that there are ample opportunities for exploration and exploitation bRo5. Historically, bRo5 drugs have played a large role in immunosuppression (e.g., cyclosporine A, CsA), in the treatment of infectious and viral diseases (e.g., antibacterial agents, HIV protease inhibitors), and in the areas of oncology (e.g., taxanes) and cardiovascular (e.g., digoxin), and that trend has continued in recent years with new approvals of directacting antivirals (DAA) including hepatitis C (HCV) NS5A and NS3/4A protease inhibitors as well as oncology drugs, such as Bcl-2 inhibitors. In fact, new U.S. FDA drug approvals in the past 3 years have yielded 12 new oral bRo5 drugs, accounting for 21% of new oral drug approvals in that time period, while having a tremendous impact on the treatment of illnesses with high unmet medical needs such as HCV and cancer (Table 1). Thus, there has been significantly increased interest in the discovery and development of bRo5 drugs with a number of excellent reviews and perspectives published in recent years.2−6 However, it is clear that our understanding of the principles that govern success and failure for designing oral bRo5 drugs is © XXXX American Chemical Society

only partial at best. Investigation of bRo5 chemical matter remains higher risk and is fraught with uncertainties that hinder progress. Frequently, bRo5 compounds have poor drug metabolism and pharmacokinetic (DMPK) properties, such as low permeability, low solubility, and high metabolic clearance. In addition, poor properties such as low solubility can limit the utility of in vitro data for establishing in vitro/in vivo correlations (IVIVC) for the key drivers of achieving acceptable oral pharmacokinetics (PK), such as correlation of in vitro permeability with in vivo oral absorption. Some of these compounds may also be substrates for poorly understood active transport mechanisms of absorption. As a result of these problems, advancing projects with bRo5 chemical matter frequently requires empirically driven PK campaigns with a high cost in terms of project timelines, compound synthesis, and animal screening. An emerging area of focus for research on bRo5 drug design is the exploration of macrocycles,7−9 catalyzed by the observation that several orally bioavailable macrocyclic drugs such as CsA are significantly bRo5 and demonstrate chameleon-like conformational flexibility that may mask polar groups through intramolecular hydrogen bond formation. While considerable effort is being devoted to the prospective design of orally bioavailable macrocyclic peptide drugs,10−14 less attention has been paid to the design principles for acyclic bRo5 compounds. In this respect, our present retrospective analysis of AbbVie’s bRo5 projects may provide valuable insights into acyclic bRo5 compounds due to the larger Received: May 15, 2017 Published: September 19, 2017 A

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Table 1. New FDA Approvals (2014 to Present)a of Oral bRo5 Drugs

a

drug

year approved

therapeutic area

MW

cLogP

HBD

N+O

velpatasvir venetoclax elbasvir grazoprevir cobimetinib daclatasvir edoxaban ombitasvir paritaprevir netupitant ledipasvir ceritinib

2016 2016 2016 2016 2015 2015 2015 2014 2014 2014 2014 2014

HCV oncology HCV HCV oncology HCV cardiovascular HCV HCV nausea from chemotherapy HCV oncology

883.02 868.44 882.0 766.90 531.31 738.88 548.06 894.13 765.89 578.59 889.00 558.14

2.5 10.4 2.6 −2.0 5.2 1.3 −0.9 1.3 1.1 6.8 0.9 6.5

4 3 4 3 3 4 3 4 3 0 4 3

16 14 16 15 5 14 11 15 14 5 14 8

Source: www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugInnovation/.

determine if lessons could be gleaned from an analysis of the physicochemical properties together with their preclinical PK data. As a first step, we analyzed the property space of all compounds in the AbbVie preclinical DMPK database, which is a large repository of DMPK data generated in vitro and in vivo for AbbVie’s compound collection. For the present analysis, we focused primarily on the large collection of rat oral bioavailability (F) data (n = 8647 at the time of this analysis) which was accumulated during the course of numerous drug discovery programs where oral low dose screening of new compounds was utilized to identify promising compounds. While the data were curated to remove studies in which PK boosters were utilized which would clearly skew the oral bioavailability, no attempt was made to account or correct for the effect of formulations on oral bioavailability, as suitable formulations are typically identified early in projects and modified as needed throughout the course of the project. We then calculated the physicochemical properties for the compounds in the database, including number of rotatable bonds (NRB), number of aromatic rings (NAR), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), molecular weight (MW), topological polar surface area (TPSA), number of nitrogens and oxygens (N+O), cLogP, and cLogD, among others. Using this data, it was then possible to identify the compounds that violate Lipinski’s Ro5 (n = 1116) by virtue of failing >1 rule. The compound collection encompassed a wide range of molecular properties, such as MW = 500−1410, cLogP = −5.8 to 13.9, HBD = 0−7, HBA = 2−15, N+O = 3−20, and NRB = 3−26, providing excellent representation of bRo5 chemical matter from 85 projects and 165 chemical series. As part of our preliminary analysis of the data, we examined the properties of the compounds that demonstrated oral F ≥ 5% in order to estimate the “possible to be orally absorbed” space. Similar to the observations made by Kihlberg et al.,2 in their analysis of a set of launched and clinical bRo5 compounds, very high MW (≤1132) and TPSA (≤229) along with a wide range of cLogP values (−5.5 to 13.3) were tolerated. However, we also observed that there were no compounds with HBD > 6, HBA > 14, or NRB > 19 that were orally bioavailable, also in close agreement with Kihlberg’s observations. In order to investigate correlations between molecular properties and oral F, the bRo5 compound set was divided (binned) into quartiles based on their observed F, with the upper quartile (Q4) defined as acceptable bioavailability (oral F

proportion of acyclic bRo5 compounds present in AbbVie’s compound collection. By ascertaining the key learnings from past successes and failures bRo5, it is hoped that more rapid progress can be made on the emerging opportunities for medicinal chemists to have an impact in the treatment of diseases with large unmet medical need. While several excellent retrospective studies have been published on the analysis of marketed and clinical bRo5 compound collections,2−6 they provide only a partial picture of potential success in the vast bRo5 chemical space and little information about bRo5 failures. Although no single organization’s compounds may be fully representative of the vast bRo5 chemical space, we reasoned that conducting an analysis of both our clinical and preclinical compound collections with available preclinical PK data may provide a better picture of potential success bRo5 by widening the representation of bRo5 chemical space while adding information about bRo5 failures. Herein, we present the result of a retrospective analysis conducted on AbbVie’s preclinical DMPK database in which we correlated the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) results with in silico physicochemical properties associated with the compounds. We present a simple multiparametric scoring function (AB-MPS) which correlated the preclinical PK results with physicochemical properties. We also present case studies for projects with bRo5 compounds that advanced to the clinic, including HCV NS5A inhibitors, HCV NS3/4A protease inhibitors, and Bcl-2 inhibitors.

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PHYSICOCHEMICAL PROPERTY ANALYSIS OF PRECLINICAL DMPK DATABASE Since the appearance of Lipinski’s rules, a large number of additional pass/fail criteria as well as multiparametric scoring functions based on physicochemical properties have emerged and a recent review by Meanwell provides an excellent overview of the strengths and weaknesses of these approaches.15 In addition, several useful retrospective analyses of the druglikeness of compounds prepared by medicinal chemists over the past decades have recently appeared.16−19 Naturally, the compound data sets employed for older retrospective analyses are influenced by the more druggable types of target proteins that medicinal chemists have investigated for which compounds were prepared, in contrast to the current focus on less druggable targets. In order to more fully understand the effect of physicochemical properties on compounds bRo5, we sought to focus exclusively on the analysis of bRo5 compounds to B

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Figure 1. Effect of multiple physicochemical properties on oral bioavailability F: box plot of binned oral bioavailability (red = F ≤ 2.5, blue = 2.5 < F ≤ 27.28, green = F > 27.28) versus average values for physicochemical properties. Comparison circles are shown for α level = 0.05. White line = median value.

> 27.3), the lower quartile (Q1) defined as poor bioavailability (oral F ≤ 2.5) and the interquartile range (IQR, Q2 and Q3) (2.5 < oral F ≤ 27.3) defined as moderate bioavailability. Box plots of the binned oral F versus the averages of key physicochemical properties known to effect oral absorption including average TPSA, NRB, MW, cLogD, and NAR appear in Figure 1. From this qualitative analysis, we observed that compounds with acceptable oral bioavailability (Q4, F > 27.3) demonstrated lower values for these key physicochemical properties, indicative of overall better druglike properties than those with moderate and poor oral F. Clearly, bRo5 compounds with acceptable oral bioavailability had superior physicochemical properties than compounds with poor bioavailability. One notable exception to trends observed in Figure 1 was the relationship between oral F and cLogD, one of the most influential physicochemical properties that broadly affects the DMPK properties of drugs. Previous studies have illustrated the importance of maintaining lipophiliciy within a narrow range in order to achieve cell permeability and solubility.5,7 If one considers the propensity of a compound to have good oral bioavailability, good permeability is a key prerequisite, with LogD a key determinant of good passive permeability. It is wellknown that both high hydrophilicity (low LogD < 1) and lipophilicity (high LogD > 5) have negative effects on permeability. As an illustration of this, we analyzed 82 000 compounds with available parallel artificial membrane permeability assay (PAMPA) data and binned by poor (Papp ≤ 2 × 10−6 cm/s) and good (Papp > 10 × 10−6 cm/s) permeability, with moderate permeability between these two values. The distributions of these three bins are shown in Figure 2, with the

Figure 2. Effect of cLogD on PAMPA permeability. Red = PAMPA ≤ 2. Yellow = 2 < PAMPA ≤ 10. Green = PAMPA > 10.

compounds with highest permeability (>10 × 10−6 cm/s) showing an almost normal distribution with a peak at around cLogD = 3. As shown in Figure 2, the shape of the cLogD distribution observed for compounds with good permeability was narrower than the distributions observed for the compounds with moderate and poor permeability. In contrast to the shape of the distribution observed for compounds with good permeability, the compounds with poor permeability exhibited two maxima, one centered at cLogD = 1 and one centered at cLogD = 4. As the curve for compounds with low C

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Table 2. Physicochemical Property Averages by Rat Oral Bioavailability Quartile quartile

AB-MPS

NRB

ΔLogD

NAR

HBD

bRo5 Set (n = 1116)a 1.9 3.6 2.0 3.0 4.2 2.4 3.4 4.4 2.9 2.8 4.1 2.4 −0.33 −0.26 −0.23 Tier 7 Set (n = 2643)b 1.5 2.9 1.7 2.1 3.3 1.8 2.6 3.6 2.3 2.1 3.3 1.9 −0.25 −0.18 −0.12

4 2, 3 1 all (averages) r with F

>27.3 >2.5 and ≤27.3 ≤2.5 18.8

14.0 18.8 19.8 17.9 −0.41

8.6 11.5 12.1 10.9 −0.35

4 2, 3 1 all (averages) r with F

>34.8 >3.8 and ≤34.8 ≤3.8 23.1

11.0 13.8 15.3 13.4 −0.28

6.6 8.3 9.1 8.0 −0.24

12.5 16.4 17.5 15.7 −0.35

Molecular Weight of >500 Set (n = 1662)c 7.7 1.6 3.2 1.8 10.2 2.4 3.8 2.1 10.7 2.9 4.0 2.6 9.7 2.32 3.7 2.2 −0.30 −0.28 −0.26 −0.22

4 2, 3 1 all (averages) r with F a

F range (rat)

>31.2 >2.9 and ≤31.2 ≤2.9 21.2

MW

TPSA

N+O

cLogP

cLogD

655.1 754.6 723.2 721.9 −0.22

123.2 137.2 148.3 136.5 −0.19

9.9 11.2 11.5 10.9 −0.18

5.3 6.9 5.2 6.1 −0.09

4.2 5.5 4.1 4.8 −0.08

521.1 583.6 594.8 571.0 −0.17

92.8 99.1 115.8 101.7 −0.13

7.4 8.1 9.0 8.2 −0.13

4.8 5.7 4.8 5.4 −0.09

3.8 4.7 3.9 4.3 −0.10

600.6 687.1 669.4 661.7 −0.22

112.2 124.3 137.0 124.5 −0.19

9.0 10.0 10.6 9.9 −0.18

4.4 5.7 4.6 5.2 −0.12

3.9 4.8 3.8 4.3 −0.08

Fail >1 Lipinski rule. bFail ≥1 Lipinski rule. cFail Lipinski rule MW > 500.

shown in Figure 3. We observed that compounds with acceptable oral bioavailability (F > 27.3%) demonstrated a

permeability demonstrates, having cLogD in the range from 2 to 4 does not ensure good permeability and other factors must account for their poor permeability. Given the fact that both high and low cLogD have a negative effect on permeability, we reasoned that deviation from this value of cLogD would have a negative effect on oral absorption and therefore oral bioavailability. As a result we considered cLogD = 3 to be the sweet spot for optimal permeability, and the expression ΔLogD = Abs(cLogD-3) was used as a descriptor for weighting the effect of cLogD on permeability. As an example, for a compound with cLogD = 3, there is a zero negative effect on absorption and therefore oral bioavailability. As observed in Figure 1, ΔLogD demonstrated a clear trend and compounds with acceptable oral F showed lower values than those with moderate or low oral F. A more quantitative analysis of the average physicochemical properties for compounds in each quartile set appears in Table 2. In a similar analysis to that conducted by Veber,20 we also reported the correlation coefficients r between oral F and the calculated properties. For the bRo5 set, weak negative correlations were observed between oral F and a number of properties including NRB (r = −0.35), ΔLogD (r = −0.33), NAR (r = −0.26), and HBD (r = −0.23). While weaker negative correlations were also observed for MW (r = −0.22), TPSA (r = −0.19), and N+O (r = −0.18), there was no correlation with cLogP or cLogD. On the basis of the observed correlations in Table 2, we sought to evaluate simple multiparametric functions that might provide a higher correlation with oral F and provide the potential to be used prospectively to evaluate bRo5 compounds. Toward this goal, we selected the three properties with the highest correlation coefficients with oral F and combined them as shown below in eq 1 to arrive at a simple scoring function, which we called AbbVie multiparametric score (AB-MPS). We found that AB-MPS provided a higher negative correlation coefficient with oral F (r = −0.41) (Table 2), wherein lower AB-MPS values were correlated with higher oral F. The distributions of AB-MPS as a function of binned oral F rat were also visualized using a box plot for the bRo5 set, as

Figure 3. Box plot of binned oral F versus AB-MPS value. Red = F ≤ 2.5, blue = 2.5 < F ≤ 27.28, green = F > 27.28. Comparison circles are shown for α level = 0.05. White solid line = median, white dashed line = average.

lower average AB-MPS value of 14 with a much narrower distribution (IQR = 5.4) than compounds with poor (IQR = 9.4) and moderate (IQR = 10.2) oral bioavailability. The narrower IQR for AB-MPS for compounds with acceptable oral F resulted from correspondingly narrower IQR values for NRB and ΔlogD, while the IQR for NAR was similar across the oral F bins. We also observed that an individual compound that deviates from druglikeness in one property (ΔLogD, NAR, or NRB) can achieve a low AB-MPS value by having more druglike values for the remaining properties, a principle also captured in other multiparametric scoring functions such as the simple property forecast index (PFI)21,22 and the more sophisticated quantitative estimate of druglikeness (QED).23 However, we found that PFI provided a poorer correlation (r = D

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−0.15) with the oral F data set than AB-MPS, while QED provided a similar correlation (r = 0.34) with the oral F data set to AB-MPS. By virtue of its simple calculation, AB-MPS may offer a simple mnemonic for medicinal chemists to evaluate bRo5 chemical matter, with AB-MPS values of ≤14 predicting a higher probability of success. Unlike pass/fail metrics, AB-MPS also provides for a continuum of scores for medicinal chemists to evaluate druglikeness rather than relying on cutoff values. AB‐MPS = Abs(cLogD‐3) + NAR + NRB

(1)

We next conducted a similar analysis of compounds regarded to be in poor druglike space by virtue of failing at least one of Lipiniski’s rules, which coincides with tier 7 physicochemical property space as defined by Cox et al.24 The tier 7 set (n = 2643) provided additional chemical diversity with compounds from 184 projects and 382 chemical series. As shown in Table 2 for the tier 7 set, we observed weak negative correlations between oral F and a number of properties including NRB (r = −0.24), ΔLogD (r = −0.25), and NAR (r = −0.18), similar to the results observed with the bRo5 set. In this case as well, ABMPS provided the highest correlation (r = −0.28). As many bRo5 projects target large molecules to achieve adequate potency, we also analyzed the set of compounds with MW > 500 (n = 1662). Like the previous bRo5 and tier 7 sets, we observed weak negative correlations between oral F and ABMPS (r = −0.35), NRB (r = −0.30), ΔLogD (r = −0.28), and NAR (r = −0.26). Interestingly, we observed higher negative correlation between AB-MPS and oral F as the MW increased (r = −0.41 for MW > 600 and r = −0.46 for MW > 700), indicating an increased usefulness of the metric in less-desirable compound space. Oral bioavailability is defined by the equation F = FaFgFh, where Fa = fraction absorbed, Fg = fraction escaping intestinal metabolism, and Fh = fraction escaping hepatic metabolism. Given that oral bioavailability can be influenced by many factors such as formulation, dose, solubility, intestinal metabolism, and hepatic metabolism, in addition to permeability, we also looked at absorption in the context of FaFg, which is a measure of intestinal absorption without the complexity of considering hepatic metabolism.25,26 Assuming primarily hepatic clearance for large bRo5 drugs, we used the equation FaFg = F/(1 − Clp/ Qh), where Clp is the total clearance and Qh is hepatic blood flow. Again, the thresholds for binning acceptable, moderate, and poor FaFg were defined by the overall distribution, with acceptable FaFg > 0.39 (Q3) and poor FaFg ≤ 0.06 (Q1). When we examined the distribution of AB-MPS values relative to binned FaFg (Figure 4), we again observed a lower average ABMPS value (13.5) with a narrower distribution (IQR = 4.7) for compounds with acceptable FaFg compared to compounds with moderate and low FaFg. Permeability is an important factor that contributes to the likelihood of good oral absorption. While this is not the only factor influencing oral absorption, high intrinsic permeability will significantly improve the odds of achieving good oral absorption and therefore bioavailability. With this in mind, we revisited the PAMPA permeability data that were available for the bRo5 set (n = 7345) and analyzed the relationship between AB-MPS and PAMPA permeability. As shown in the box plot of AB-MPS versus binned PAMPA (Figure 5), we observed that compounds with good PAMPA permeability (Papp > 10 × 10−6 cm/s) demonstrated lower AB-MPS values (average = 11) with a narrower distribution (IQR = 2.6) compared to compounds with moderate or low PAMPA permeability. In

Figure 4. Box plot of binned oral FaFg versus AB-MPS value. Red = FaFg ≤ 0.06, blue = 0.06 < FaFg ≤ 0.39, green = FaFg > 0.39. Comparison circles are shown for α level = 0.05. White solid line = median, white dashed line = average.

Figure 5. Box plot of binned PAMPA versus AB-MPS value for bRo5 compounds. Red = PAMPA ≤ 2, blue = 2 < PAMPA ≤ 10, green = PAMPA > 10. Comparison circles are shown for α level = 0.05. White solid line = median, white dashed line = average.

addition, we observed a weak negative correlation (r = −0.28) between PAMPA and AB-MPS for this bRo5 compound set. While the trend between good PAMPA and superior druglike properties is clear, one caveat with the interpretation is that we observed a higher percentage of invalid data (14%) for the bRo5 set compared to the Ro5 compliant set (8%). Thus, the poor physicochemical properties of bRo5 compounds such as low solubility may result in poor recovery and a higher invalid data rate, confounding the prediction of in vivo oral bioavailability based on in vitro PAMPA. Lokey et al. recently reported on the challenges of interpreting PAMPA permeability for a series of macrocyclic peptides, noting that low Papp values in PAMPA may result from higher membrane retention and observing a steep drop-off in permeability for compounds with MW > 1000.7 While the PAMPA assay provides a measure of passive permeability without the influence of transporters, one must consider the potential role of active transport in the efflux and E

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uptake of bRo5 compounds.2,6 In our data set, we found that the median efflux ratio for bRo5 compounds (MDCK MDR1 efflux ratio = 17.2) to be significantly higher (p < 0.001 by oneway ANOVA) than that for Ro5 compliant (MDCK MDR1 efflux ratio = 2.4). While the effect of efflux transport on oral absorption and tissue distribution has been widely studied, the effect of uptake transporters is significantly less well understood. Although there are many suggestions for the contribution of uptake transporters in intestinal absorption of drugs, information on the potential transporter molecules responsible for the intestinal absorptive process is limited, with peptide transporter PEPT1 and organic anion transporting polypeptide (OATP) transporters such as OATP1A2 and OATP2B1 among the most studied.27 While the scope PEPT1 substrate specificity may be too narrow to accommodate bRo5 drugs, OATP1A2 and OATP2B1 have been proposed to play a role in the intestinal absorption of several bRo5 drugs, such as montelucast, clarithromycin, and HIV protease inhibitors, with broader substrate selectivity.28 The coexpression of OATPs in the liver leading to potential increased hepatic clearance may confound understanding the role of intestinal uptake by these transporters on the disposition of oral drugs. As molecular structures get progressively larger, the probability of having an opportunity to undergo a form of self-organization, be it internal hydrogen bonding, salt bridge formation, π-stacking, or hydrophobic collapse, increases and eventually goes to unity. Such self-organization processes may lead to chameleon-like behavior where molecules may achieve both an active and a permeable conformation by masking both lipophilic and polar residues as required for binding to a protein target and crossing a lipid bilayer.6,11,29−31 This means that two-dimensional proxies for physicochemical properties such as TPSA, cLogD, and molecular volume calculated from 2D structures, which cannot consider self-organization, will come to overestimate these parameters progressively as those molecules get larger. Several measures to quantify the degree of three-dimensionality of molecules have been proposed including fraction of sp3 carbons (Fsp3), normalized principal moments of inertia ratio (NPR), and plane of best fit (PBF).32 While similar average Fsp3 (0.35) and NPR1 + NPR2 (1.07) values were observed for the bRo5 and Ro5 compound sets, the PBF observed for the bRo5 set (1.38) was significantly higher (p < 0.001 by one-way ANOVA) than for the Ro5 set (0.83), consistent with a high degree of three-dimensionality for the bRo5 set. A recent study of the effects of 3D shape on permeability for bRo5 compounds employed radius of gyration (Rgyr) and 3D-PSA as surrogates for hydrodynamic radius, although such methods require approximations and assumptions to be made about the molecular conformations of a molecule including selecting a single low energy conformation.33 With the large structural diversity and molecular complexity found in the current bRo5 data set, we reasoned that selecting a single conformation was an unreasonable simplification due to the myriad of potential conformations and focused our analysis on 2D molecular descriptors, despite the limitations. Given the relationship between low AB-MPS and acceptable oral absorption for bRo5 compounds, we analyzed a set of drugs delivered orally in humans (n = 852) which was culled from a collection of 1211 marketed drugs. As originally noted by Veber,20 one must use caution in the analysis of drug databases due to “a need to assume that all orally administered drugs are intended to be absorbed”. Therefore, the route of

administration for marketed compounds was confirmed, removing compounds that are administered orally but display no or subtherapeutic systemic exposures, act locally in the GI tract or oral cavity, or are prodrugs that are metabolized before absorption of the parent molecule. Prodrugs that are largely absorbed prior to metabolism to the parent were included in set. After calculating the physicochemical properties, we identified 138 bRo5 drugs in the collection, including 69 orally administered and 69 parenterally administered drugs. As shown in Figure 6, the average AB-MPS value for the oral bRo5 set

Figure 6. Box plot of binned route of administration versus AB-MPS value for bRo5 compounds. Comparison circles are shown for α level = 0.05. White solid line = median, white dashed line = average.

was 14.9, while the parenterally administered drugs demonstrated a higher average AB-MPS value. The average AB-MPS value for the oral bRo5 set (15) was comparable to the average AB-MPS value (14) for the internal bRo5 compounds with acceptable oral bioavailability. This suggests that from a prospective design perspective, bRo5 compounds targeted to occupy property space defined by AB-MPS ≤ 15 will have enhanced odds of achieving reasonable levels of oral absorption. Interestingly, analysis of the Ro5 compliant orally administered marketed drugs in the set (n = 768) revealed a low average AB-MPS value of 8.9 with an IQR of 5.2, suggesting that AB-MPS value, as a key indicator of acceptable oral absorption of compounds, transcends compounds both within and outside the Ro5.

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CASE STUDIES It is now becoming recognized that compounds bRo5 appear to be particularly well-suited for drugging important but less druggable targets in oncology, virology, and infectious diseases, and a number of excellent reviews on the topic have recently appeared.3 In the case studies included here, we have selected some of our recent drug discovery research programs in HCV and oncology that highlight some of the benefits and challenges of conducting medicinal chemistry with bRo5 chemical matter. In each case study, we’ve highlighted the obstacles to successful drug discovery for the particular targets while focusing on the properties of both successful and failed compounds. Discovery HCV NS5A Inhibitors. Today, highly potent HCV NS5A inhibitors, together with HCV NS5B polymerase or HCV NS3 protease inhibitors, form the backbone of the F

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Table 3. Early HCV NS5A Inhibitors

Table 4. Structures, Replicon Potencies, Oral Bioavailability, and Properties of NS5A Inhibitors

a

EC50 determined using methods previously reported.41

challenges. NS5A has no known enzymatic activity, and while it interacts with many viral and host proteins as well as RNA, its precise biological functions have yet to be elucidated. As a result, discovery of the early classes of NS5A inhibitors resulted from investigation of compounds that inhibited HCV replication in a replicon assay, with concomitant development

highly efficacious and well-tolerated direct-acting antiviral (DAA) combination therapies used clinically to cure HCV. In fact, 5 of 12 new bRo5 drugs approved by the FDA in the past 2 years are HCV NS5A inhibitors including ledipasvir, ombitasvir, daclatasvir, elbasvir, and velpatasvir. However, the discovery and development of NS5A inhibitors faced significant G

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of resistant variants that mapped to NS5A. A recent review34 highlighted many of the early NS5A inhibitors, and although diverse in structure (Table 3), a common feature of the inhibitors included the selection of resistance-associated variants at Y93 of genotype 1b (GT1b), particularly Y93H. A key challenge for the early compounds was modest potency against GT1b and significantly weaker potency against GT1a compared to GT1b. It is important to note that worldwide HCV GT1 is the most prevalent form and in the U.S. approximately 55% of GT1 infections are GT1a. In addition, the chemical series in Table 3 suffered from poor druglike properties including high cLogP and high molecular weights. The combination of modest potency and poor properties highlights a key challenge for the early series in achieving the very high exposures in vivo that would be required to adequately suppress viral replication. AbbVie investigated a series of naphthyridine- and pyrrolopyrimidine-based compounds, represented by compound 6 (Table 3), that demonstrated nanomolar potency against GT1b but were >20-fold weaker against GT1a.35 Compound 6 demonstrated a robust viral load decline in a GT1b HCV-infected chimpanzee and selected resistance-associated variants within NS5A, thus providing in vivo proof of concept for the antiviral efficacy of the series. Mostly bRo5, the compounds in the series generally showed good passive permeability (PAMPA Papp > 2 × 10−6 cm/s) while suffering for poor metabolic stability. Ultimately, the series lacked adequate tractable SAR, potency against GT1a and oral PK to enable further development. Although counterintuitive, moving toward less druglike chemical matter resulted in the discovery of successful NS5A inhibitors. X-ray crystallographic studies of NS5A domain I protein fragments from both GT1a and 1b have revealed the formation of symmetrical dimers.36,37 While the biological relevance of these dimeric forms is unknown, it is believed that inhibitors may function by binding to these structures, providing stability to the dimeric form and thereby impacting several aspects of HCV expression and regulation.38,39 This mode of action is supported by the discovery of highly potent NS5A inhibitors with symmetric dimer or dimer-like structures, which presumably provide improved interactions with a dimeric form of the NS5A protein. The discovery of symmetry-based NS5A inhibitors resulted in compounds with dramatically improved potencies, particularly against GT1b. At the same time, the dimeric structural motif resulted in significantly higher molecular weight compounds with poor physicochemical properties and additional challenges for series progression and development. Daclatasvir, a symmetric compound discovered as part of a lead optimization campaign starting from dimers derived from chemically unstable compound 3, was the first HCV NS5A inhibitor to demonstrate proof of concept in human clinical trials.40 A symmetry-based medicinal chemistry approach in our laboratories identified several novel series compounds that were potent inhibitors of both GT1a and 1b replicons, and two series are shown in Table 4. Dimerization of the scaffold found in compound 6 resulted in series A compounds such as 7 and 8 which showed subnanomolar potencies against GT1b. The GT1a potency, however, was highly dependent on the linking strategy, with the N-phenylbenzyl linkage in 8 providing single-digit nanomolar GT1a potency, while the ethylenediamine linked amide 7 was inactive. The compounds in series A generally had low solubility (phosphate pH 7.2 = 0.5−7.4 μM) and negligible permeability in the PAMPA assay. Attempts to reduce the high

lipophilicity and NAR resulted in compounds with similarly poor properties but markedly weaker potencies, and none of the nine compounds tested in rat PK has measurable plasma drug levels. Another symmetry-based medicinal chemistry approach identified N-phenyl-core compounds (series B in Table 4) that were potent inhibitors of both GT1a and 1b replicons, with lower lipophilicity and NAR.41 Like compound 8, compound 9 demonstrated good potency at GT1a and 1b, although it was 72-fold more potent against GT1b than GT1a. In contrast to 8, compound 9 showed moderate oral bioavailability in rat (28%). Compound 10, with its conformationally constrained pyrrolidine core, demonstrated dramatically higher replicon potency, particularly for GT1a, compared to the acyclic analog 10. Addition of a tert-butyl group to the central phenyl ring resulted in the discovery of 11 (ABT-267, ombitasvir), which demonstrated 14 pM and 5 pM potency against GT1a and GT1b, respectively.41 The S,S-trans stereochemistry was found to be the optimal substitution pattern for the central pyrrolidine ring, with the R,R stereoisomer being significantly less active against GT1a. It is well-known from the development of HIV PIs that plasma protein binding (PPB) can markedly decrease the clinical efficacy of antiviral compounds.42 Like many bRo5 drugs, the NS5A inhibitors in Table 4 were >99 bound to human plasma proteins, and accurate determination of their free fractions presented a considerable challenge without access to radiolabeled materials due to the very low drug concentrations. In order to assess the magnitude of the effect of PPB on potency by a practical method, we evaluated the replicon assay potency in the presence of 40% human plasma (HP). The GT1a potencies of the highly lipophilic compounds 7 and 8 shifted by 15- to 30-fold in the presence of 40% HP. Compound 9 showed a smaller shift and the EC50 values increased by 7- to 10-fold when the assay was performed in the presence of 40% HP. Compound 11 retained high potency when the assay was performed in the presence of HP, with 186 pM and 56 pM potencies against GT1a and GT1b, respectively. Retrospective application of the AB-MPS function to the symmetry-based NS5A series was informative. As shown in Figure 7, there was generally good correlation between the

Figure 7. AB-MPS values for symmetry-based NS5A inhibitor versus observed FaFg in rat PK. Red = FaFg ≤ 0.06, blue = 0.06 < FaFg ≤ 0.39, green = FaFg > 0.39. Comparison circles are shown for α level = 0.05. White solid line = median, white dashed line = average. H

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Table 5. In Vitro Activity of HCV NS3/4A PIs in the Replicon Assaya

stable replicon EC50 (nM) 1a

a

transient replicon EC50 (nM) (fold resistance)

1b

3a

1a variant

1b variant

3a

compd

0% HPb

40% HP

0% HP

40% HP

0% HP

R155K

D168V

R155K

D168V

WT

12 13 14 15 16 17 18 19

1.4 1.1 0.5 0.8 0.7 0.7 0.2 0.5

30 11 12 7 9 6 7 7

0.4 0.8 0.4 0.5 0.5 0.5 0.1 2

9 7 10 7 11 8 3 12

NTc NTc NTc 64 NTc 7 0.8 8

3 (6) 2 (7) 0.4 (4) 0.3 (3) 0.3 (3) 0.7 (18) 2 (65) 0.6 (0.8)

94 (177) 41 (146) 33 (290) 14 (149) 14 (127) 8 (168) 3 (97) 0.4 (2)

2 (2) 1.6 (2) 0.7 (2) 0.5 (1) 0.4 (1) 0.4 (4) 0.03 (0.5) 8 (33)

5 (2) 3 (4) 2 (6) 1.5 (5) 1.3 (4) 0.9 (9) 0.1 (2) 3 (11)

NTc NTc NTc 83 124 4 140 are generally associated with poor permeability and oral bioavailability.20 It has recently been hypothesized that NS5A inhibitors may, through formation of intramolecular hydrogen bonds (IHB) in nonpolar environments such as lipid bilayers, effectively mask their high polarity.43,44 NMR methods and more recently chromatographic techniques such as EPSA45−47 have been shown to be useful for detection of IHB. We retrospectively measured the EPSA values and found that the EPSA for 10 was 83 and was indeed lower than the TPSA value and in the range of EPSA values associated with I

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Table 6. Pharmacokinetic Properties of HCV NS3/4A PIsa rat iv compd 12 13 14 15 16 17 18 19

t1/2 2.4 NAb 1.2 2.1 4.1 3.2 5 1.7

dog po

Clp 4.7 NAb 0.21 2.3 1.6 2.5 1.1 3.1

t1/2 b

NA NAb 1.8 1.9 4.7 3.5 4.6 1.5

iv

po

AUC/D

F

t1/2

Clp

t1/2

AUC/D

Fc

FaFg

0.008 NAb 4.75 0.14 0.19 0.16 0.4 0.09

3.9 NAb 99.5 28 27 40 43 27

1.4 2 1 3.5 2.9 2.3 3.2 2.2

0.47 0.07 0.34 0.04 0.02 0.02 0.01 0.07

nf 2 1.5 2.2 2.9 2 4 2.1

0.3 18 6.76 18 46.8 85 138 11.6

13 123 200 74 88 140 81 76

0.2 NAb 1.5 0.8 0.9 1.4 0.8 0.8

Units: t1/2 (h); Cl (L h−1 kg−1); Cmax (μg/mL); AUC0−24h (μg·h/mL); F (%); dose (mg/kg). bNot available. cCalculated based on iv dosed at 1 mg/kg and po dosed at 3 mg/kg.

a

GT1 stable replicons, while compound 16 demonstrated 1.5fold weaker potency compared to 15 against GT3a transient replicons. An extensive SAR evaluation identified the phenanthridinebased inhibitors such as 17 as a promising series to achieve broad genotypic antiviral activity as well as desirable PK properties. Through intensive evaluation of the SAR, the substitution of the phenanthridine group and the capping group of the P3-amine were identified as two areas that significantly affected the virology and PK. Compound 17, with an 8fluorophenanthridine group and compound 18, with a 3trifuoromethoxy-8-fluorophenanthridine group, provided equal or better potency to 15 against GT1 replicons in the presence of 40% human plasma (HP) (Table 5). Both compounds also demonstrated comparable EC50 values and fold-resistance against GT1 variants to 15. In contrast to the weak GT3a potency of 15, compound 17 exhibited single-digit nanomolar potency against GT3a replicon, although it was approximately 10-fold weaker against GT3 compared to GT1 (Table 5). Compound 18 yielded potency against GT3a that was within 10-fold of the GT1 EC50 values. By extending the ligand− enzyme interaction into the P4 site, compound 19, bearing a capped tert-butyl alanine P4 group, showed a higher folddifference between GT3a and GT1 replicons in comparison to 18. However, compound 18 provided a different resistance profile against GT1 variants in that it exhibited no loss in potency against GT1aR155K variant and only 2-fold loss in potency against GT1aD168V replicon, a marked difference to the capped-P3 analogs. The compounds in the series generally exhibited large shifts (6- to 35-fold) in the replicon EC50 values obtained in the presence of 40% HP. For example, compound 18 exhibited more than a 30-fold shift in potency against GT1 with the addition of 40% HP, likely a result of the high plasma protein binding. Fluorination of the phenanthridine moiety impacted the PK properties as observed in the quinoxaline series (Table 6). Compound 17 with a fluorinated phenanthridine produced similar half-life and plasma concentration following oral dosing in rat when compared to compound 16. Similar clearance and nearly 2-fold higher plasma concentrations were achieved by 17 dosed in dog in comparison to 16. Compound 18, having an additional C3-trifluoromethoxy group on the phenanthridine, exhibited long plasma half-life and more than 2-fold higher plasma level following oral dosing in both rat and dog than 16. On the other hand, compound 19, containing the extended P4 functional groups, demonstrated

(WT) GT1 as well as resistance-associated variants GT1a R155K, GT1b R155K, and GT1b D168V, within a single-digit fold of the WT EC50 value. However, the GT1a D168V variant still conferred significant fold resistance to 12. In addition, the PK of 12 was characterized by high iv clearance, poor plasma concentration following oral dosing, and poor bioavailability in both rat and dog (Table 6). We conducted SAR to improve both the resistance profile and PK for the series. For example, fluorination of the quinoxaline resulted in compound 13 which was equipotent to 12 against WT GT1 and resistanceassociated variants with similar fold resistance. Incorporation of a trifluoromethyl group provided incremental improvement to the antiviral activity. Compounds 14 and 15 were equipotent to 12 against WT GT1 while exhibiting improved activity against resistance variants of approximately 3- to 10-fold (Table 5). The impact of fluorination and trifluoromethylation of the quinoxaline group was particularly significant to the PK properties of the analogs (Table 6). Compound 13, with the introduction of the C7-F to the quinoxaline, exhibited significantly lower clearance (0.07 L h−1 kg−1) and higher plasma level AUC/dose (18 μg·h/mL, per mg/kg) following oral dosing in dog in comparison to 12. Compound 14, bearing the C3-CF3 also showed lower clearance, higher oral plasma concentration, and bioavailability in rat while demonstrating higher plasma concentrations after oral dosing in dog. Incorporating both substitutions to the quinoxaline, compound 15 showed 2-fold lower clearance, 18-fold higher plasma concentration following oral dosing in rat, and 7-fold improved bioavailability in comparison to 12. Similarly, it demonstrated low clearance and high plasma concentration following oral dosing in dog, consistent with the trend observed in compounds 13 and 14. Examining the replacement of the cyclopropyl group of the acylsulfonamide revealed that the addition of a methyl group to the cyclopropyl ring had a positive effect on PK while the antiviral activity profiles remained equivalent to the corresponding unsubstituted analogs. Compound 16 was equipotent to 15 in the WT and resistant replicons (Table 5). The PK of 16 was superior to 15 with a long half-life, improved clearance, and plasma concentration after oral dosing in rat or dog (Table 6). Despite achieving good antiviral potency in the GT1 replicons and PK properties, the quinoxaline series did not offer improvement to potency against broad HCV genotypes benchmarked by potency in the G3a replicon. Compound 15 showed >80-fold weaker activity against GT3a compared to J

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superior resistance profile, pan-genotypic potency, and PK, and it is currently being investigated in combination with methyl {(2S,3R)-1-[(2S)-2-{5-[(2R,5R)-1-{3,5-difluoro-4-[4-(4fluorophenyl)piperidin-1-yl]phenyl}-5-(6-fluoro-2-{(2S)-1-[N(methoxycarbonyl)-O-methyl-L-threonyl]pyrrolidin-2-yl}-1Hbenzimidazol-5-yl)pyrrolidin-2-yl]-6-fluoro-1H-benzimidazol-2yl}pyrrolidin-1-yl]-3-methoxy-1-oxobutan-2-yl}carbamate (ABT-530, pibrentasvir) in clinical trials.53 Discovery of Bcl-2 Family Inhibitors. Nearly 2 decades ago, Abbott began a project aimed at developing inhibitors of the antiapoptotic proteins Bcl-2 and Bcl-xL for use in oncology indications. Bcl-2 and Bcl-xL are involved in a signal transduction pathway controlling the intrinsic apoptosis program and in so doing undergo protein−protein interactions involving large and hydrophobic protein surfaces. We thus anticipated challenges in producing compounds that would have properties necessary for oral administration. In fact, compounds resulting from this effort were characterized by two large groups that interacted with two deep hydrophobic pockets situated several angstroms apart, referred to as P2 and P4 (Figure 8). A notable feature of the series was the discovery

limited plasma half-life and concentrations in both animal species following oral dosing. Selected physicochemical properties for the compounds appear in Table 7. Retrospective determination of AB-MPS Table 7. Physicochemical Properties of HCV NS3/4A PIs compd

AB-MPS

cLogD

TPSA

EPSA

solubilitya

permeability

12 13 14 15 16 17 18 19

12.05 11.91 11.92 11.79 11.35 11.22 14.51 13.65

0.95 1.09 2.08 2.21 2.65 3.22 4.51 3.65

202.8 202.8 202.8 202.8 202.8 189.9 190.9 184.2

112 108 104 99 89 116 114 104

>500 >500 150 155 20.6 1.9 0.65 2.2

3.5b 2.8c 5.4c 5.1b 12.1b 0.19b 0.06b 0.28b

a b

Critical aggregation concentration (CAC) buffer (pH 7.2) (μM). PAMPA Papp 10−6 cm/s. cCaco-2 Papp 10−6 cm/s.

revealed a narrow range (from 12 to 14.5) of values indicating that the compounds would be predicted to have moderate or high oral availability, consistent with the observations in Table 6. The AB-MPS values for the marketed NS3/4A are as follows: grazoprevir (10.5) and paritaprevir (12.2). By virtue of their macrocyclic structures, the compounds have druglike NRB (