Nonclassical Size Dependence of Permeation Defines Bounds for Passive Adsorption of Large Drug Molecules

Article abstract of DOI:10.1021/acs.jmedchem.6b01483

Macrocyclic peptides are considered large enough to inhibit “undruggable” targets, but the design of passively cell-permeable molecules in this space remains a challenge due to the poorly understood role of molecular size on passive membrane permeability. Using split-pool combinatorial synthesis, we constructed a library of cyclic, per-N-methlyated peptides spanning a wide range of calculated lipohilicities (0 < AlogP < 8) and molecular weights (～800 Da < MW < ～1200 Da). Analysis by the parallel artificial membrane permeability assay revealed a steep drop-off in apparent passive permeability with increasing size in stark disagreement with current permeation models. This observation, corroborated by a set of natural products, helps define criteria for achieving permeability in larger molecular size regimes and suggests an operational cutoff, beyond which passive permeability is constrained by a sharply increasing penalty on membrane permeation.

Full text of DOI:10.1021/acs.jmedchem.6b01483

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Article
Non-Classical Size Dependence of Permeation Defines
Bounds for Passive Adsorption of Large Drug Molecules.
Cameron R Pye, William M Hewitt, Joshua A Schwochert, Terra D. Haddad,
Chad E. Townsend, Lyns Etienne, Yongtong Lao, Chris Limberakis, Akihiro
Furukawa, Alan M Mathiowetz, David A. Price, Spiros Liras, and R. Scott Lokey
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01483 • Publication Date (Web): 06 Jan 2017
Downloaded from http://pubs.acs.org on January 8, 2017
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Journal of Medicinal Chemistry
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`Non-Classical Size Dependence of Permeation Defines Bounds for Passive Adsorption of Large
Drug Molecules.
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Cameron R. Pye†, William M. Hewitt†, Joshua Schwochert†, Terra D. Haddad†, Chad E. Town-
send†, Lyns Etienne†, Yongtong Lao†, Chris Limberakis∥, Akihiro FurukawaΔ, Alan M. Mathio-
wetz, David A. Price○, Spiros Liras○, and R. Scott Lokey*†
† Department of Chemistry and Biochemistry, University of California, Santa Cruz, California
95064, United States
∥
World Wide Medicinal Chemistry, Groton Laboratories, Pfizer Inc. Groton, Connecticut 06340,
United States
○ World Wide Medicinal Chemistry, Cambridge Laboratories, Pfizer Inc. Cambridge, Massachu-
setts 02139, United States
Δ Modality Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, To-
kyo 140-8710, Japan
ABSTRACT: Macrocyclic peptides are considered large enough to inhibit “undruggable” targets,
but the design of passively cell-permeable molecules in this space remains a challenge due to the
poorly understood role of molecular size on passive membrane permeability. Using split-pool
combinatorial synthesis, we constructed a library of cyclic, per-N-methlyated peptides spanning a
wide range of calculated lipohilicities (0 < AlogP < 8) and molecular weights (~800Da < MW <
~1200Da). Analysis by the Parallel Artificial Membrane Permeability Assay (PAMPA) revealed a
steep drop-off in apparent passive permeability with increasing size, in stark disagreement with
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Page 2 of 34
current permeation models. This observation, corroborated by a set of natural products, helps
define criteria for achieving permeability in larger molecular size regimes and suggests an opera-
tional cut-off beyond which passive permeability is constrained by a sharply increasing penalty
on membrane permeation.
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INTRODUCTION
Predicting membrane permeability is a key challenge in medicinal chemistry, as diffusion
across membranes is required for orally delivered drugs and for drugs with intracellular targets. In
modern medicinal chemistry, the “drug-likeness” of a compound is often evaluated by comparing
its properties to those of a historical training set of drugs, giving rise to knowledge-based struc-
ture-property relationships to help guide the design of drugs with favorable properties. These
quantitative (and qualitative) models include Lipinski’s Rule of 5 (Ro5), which provides a simple
metric for predicting ADME properties based on molecular weight (MW), number of hydrogen
bond donors and acceptors, and octanol-water partition coefficient (LogP).1 Compounds that do
not follow Lipinski’s rules are more likely to be rejected from screening collections or eliminated
from consideration in medicinal chemistry campaigns, thus reinforcing the criteria and potential-
ly hindering exploration outside this well-defined chemical space. Indeed, there are a number of
notable outliers that challenge the wisdom of a strict adherence to these ADME rules. Particularly
noteworthy are compounds with molecular weights above 1000 Da that show significant passive
membrane permeability and, in some cases, oral bioavailability.2 Of these rule-breaking mole-
cules, some are macrocyclic natural products such as the cyclic peptide cyclosporine A (CSA), a
1202-Da compound that is cell permeable by passive diffusion and has an oral bioavailability of
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28%. Outliers such as CSA suggest that compounds outside the Rule of 5 are able to exhibit fa-
vorable ADME properties and that potentially vast regions of chemical space remain underuti-
lized. Recent work has focused on the classification of these “beyond Rule of 5” (bRo5) com-
pounds using QSPR techniques based on calculable molecular properties that often belie their
physical underpinnings.3,2,4 The question remains to what extent CSA and similar compounds are
true outliers, or whether they point toward untapped chemical potential and the prospect of de-
signing permeable molecules with molecular weights well outside conventional boundaries.
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There is an extensive body of literature on the physics of passive membrane permeability, da-
ting back to the solubility-diffusion theory (1) proposed by Overton in the 19th century.5 This
model treats the membrane as a thin slab of hydrocarbon solvent in which the rate of membrane
permeation (the permeability coefficient Pm) is equal to the hydrocarbon/water partition coeffi-
cient Khc/w times the membrane diffusion coefficient D, divided by the membrane thickness (δ):
ꢆ
ꢃꢄ/ꢅ
(1)
ꢀꢁ = ꢂ
ꢇ
ꢈ ꢊ
ꢉ
(2)
ꢆ =
ꢋꢌꢍꢎ
This model’s only form of size dependence is the diffusion term, which has been approximat-
ed by the Stokes-Einstein equation (2) and is dependent on the radius of the solute (r), the viscos-
ity of the medium (η), and the temperature (T). However, this diffusion term alone does not accu-
rately capture the observed size dependence on permeability, even within Ro5 space. More recent
models have attempted to address this discrepancy by asserting that diffusion through the or-
dered membrane more closely resembles the diffusion of solutes in polymers.6 In polymer-type
diffusion, the solute migrates through the medium by traversing a series of stochastically appear-
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ing holes which are of greater volume than that of the compound.5 Extending this principle,
Xiang and Anderson proposed that there was an additional penalty associated with a lateral pres-
sure that must be overcome during partitioning into the membrane in order to create a cavity of
sufficient volume to accommodate the solute.8,9 Recently these models have been successfully ap-
plied to the prediction of permeabilities for compounds in the “extended Ro5” size range
(500~850 Da) but have yet to successfully predict the permeability of compounds approaching the
weight of CSA.10,11
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Only a handful of natural products and, more recently, synthetic cyclic peptides, have been
described with molecular weights above ~1000 that show bone fide passive membrane permeabil-
ity.12,13 This raises the question whether or to what extent the design rules developed for extended
Ro5 compounds will apply to these significantly larger systems. Herein, we report the application
of a synthetic library approach towards understanding the impact of lipophilicity and molecular
size on the intrinsic permeability of peptidic macrocycles in the size range of CSA and similar
natural products. We designed a library of cyclic peptides with MW > ~800, and assayed their
permeability in order to elucidate the size dependence of permeability in this space. All com-
pounds were permethylated at the backbone to eliminate intramolecular hydrogen bonding
(IMHB) and the resulting conformational effects as potentially complicating factors. After ac-
counting for aqueous solubility and using experimentally determined hydrocarbon-water parti-
tion coefficients, we observed a steep, unprecedented size penalty for increasing molecular size in
the bRo5 space that cannot be accounted for using current physics-based or QSPR models of
permeability.
RESULTS
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Aggregation Imposes a Limit on Apparent Permeability. In order to quantify permeability
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over a wide range of sizes and lipophilicities, we synthesized a split pool library of hydrophobic
octapeptides that spanned a MW range from 784 to 938. All library members contained a Tyr res-
idue for linkage to the resin via the side chain phenol, as well as a D-Pro residue to facilitate cy-
clization. The additional residues were comprised of combinations of Ala, Leu, and Ile. All of the
library members had the same backbone stereochemistry arising from a previous, unpublished
scaffold whose high predicted permeability was compromised by poor solubility. Additionally, in
the final synthetic step before cleavage from solid support all the scaffolds were permethylated.
Complete N-methylation was designed to eliminate the possibility of intramolecular hydrogen
bonding in the backbone, thus providing a controlled model system for evaluating the impact of
size and lipophilicity independent of backbone conformation. A separate library was synthesized
in tandem using Ile in place of Leu to test whether the shape of the alkyl sidechains impact any
observed permeability trends.
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1
0.8
0.6
0.4
0.2
0
b)
# Ala
# Ile/Leu
AlogP
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5
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-0.6 0.7
3.3 4.4 5.9
Figure 1. Octapeptide Split Pool Library
(A) Split-pool synthetic scheme for per-methylated Ala scan library. All library members con-
tained a Tyr and Pro residue and the remaining 6 positions were either Ala, Leu, or Ile. (B)
PAMPA permeability of Ala scan library for each unique composition. Di-Ala substitution was the
dominantly permeable species in Leu and Ile containing library sub-pools.
The parallel artificial membrane permeability assay (PAMPA) is a model of membrane perme-
ability that is widely used as a cell-free system for measuring passive permeability and has been
shown to correlate with in vivo oral bioavailability.14,15 PAMPA allows for the assessment of a com-
pound’s passive membrane permeability without the convoluting factors of paracellular diffusion
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Journal of Medicinal Chemistry
and active efflux and transport mechanisms present in cell-based models. Previously we have
shown that the assay can be multiplexed, using LCMS to enable simultaneous quantification and
deconvolution of complex mixtures.16 Using this methodology, we discovered several novel cyclic
hexapeptides with good to excellent membrane permeability. Both the Leu/Ala and Ile/Ala librar-
ies were assayed using this multiplexed PAMPA methodology in order to evaluate the permeabil-
ity of all library species. Only compounds comprised of two Ala and four Xaa residues (Xaa = Leu
or Ile) showed appreciable permeability (Fig 1b). This result suggested that there is an optimal
lipophilicity window in which significant permeability can be achieved within this system. We
selected individual compounds from the library for synthesis and assayed their permeabilities in
PAMPA as well as a cell-based permeability assay using a low-efflux MDCK cell line in which P-
glycoprotein expression has been reduced (MDCKII-LE).17 While the magnitudes of the permea-
bilities for the individual compounds were higher, the trends observed in the libraries were in
good agreement with the trends observed in the assay of mixtures, demonstrating that the trend
was not an artifact of the multiplexed in vitro assay and that the observed permeabilities correlat-
ed with composition irrespective of the specific sequences. (SI Figure 1).
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nona-
octa-
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MW 912-1123
MW 799-1009
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O
O
HO
N
N
O
N
O
O
X =
Ala
Abu
Nva
Nle
N
N
N
O
N
O
deca-
X4
N
X8
O
X5
N
N
X7
O
X6
O
MW 997-1207
Figure 2. Lipophilicity Scanning Library
Lipophilicity Scanning Library. In order to more thoroughly investigate the combined effect
of lipophilicity and MW on permeability, we designed a combinatorial library of permethylated
octa-, nona-, and decapeptides. As in the previous library, each member contained a Tyr and Pro
residue, and the variable positions were populated with a combination of simple, straight chain
aliphatic residues: Ala, aminobutyric acid (Abu), norvaline (Nva), and norleucine (Nle) (Figure
2). The library was designed to span a wide range of molecular weights (797-1237) and lipophilici-
ty (0 < AlogP < 8), with compositional isomers that sampled lipophilicities in increments of one –
CH2– group, corresponding to 0.4 log units. The octa-,nona-, and decapeptide libraries were ana-
lyzed separately. Permeabilities were determined for all –CH2– (i.e., AlogP) increments, which
eluted separately on a reverse phase (C18) HPLC gradient (H2OꢀACN), and were thus evaluated
as groups of constitutional isomers (Figure 2a). As ring size increased, the overall permeabilities
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Journal of Medicinal Chemistry
decreased, even among compounds in the same AlogP range. This general inverse correlation be-
tween MW and permeability has been observed previously for small sets of low-MW
compounds.18 However, we could not determine whether the magnitude of the MW penalty ob-
served in our system was consistent with current models. In addition, we observed a bilinear rela-
tionship between permeability and lipophilicity. This decrease in apparent permeability beyond
AlogP ~ 4.5 has been reported for other model systems and is consistent with knowledge-based
models such as the Rule of 5 and others which place an upper limit on lipophilicity.19,20,21 However,
there is a lack of consensus as to whether this decrease in permeability at higher AlogP values is
due to poor solubility or sequestration into the PAMPA membrane, and the phenomenon may
depend on the particular system involved.21,22
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In contrast to AlogP, which is a calculated descriptor based on octanol/water partition coeffi-
cients, experimental partition coefficients between water and hydrocarbon solvents such as 1,9-
decadiene have been shown to provide a good correlation with experimental permeabilities in
physics-based permeability models.23 Octanol can form hydrogen bonds with solute and there-
fore provides a poor model for the desolvation of highly polar groups such as hydrogen bond do-
nors.24 However, in a series of homologous compounds that differ only by hydrocarbon chain
lengths, a high correlation is observed between octanol/water and hydrocarbon/water partition
coefficients.24 In order to determine whether the apparent permeabilities observed in our system
are concordant with current physics-based models, we set out to 1) determine their hydrocar-
bon/water partition coefficients experimentally and 2) account for the decrease in solubility at
higher lipophilicities in order to determine their intrinsic permeabilities.
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-6.0
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-8.0
-9.0
-10.0
-3.0
-1.0
1.0
3.0
SFlogKhc/w
b)
3.0
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1.0
0.0
R² = 0.98
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-2.0
-3.0
3.0AlogP
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7.0
c)
-4
-5
-6
-7
-8
R² = 0.71
-8
-7
-6
-5
-4
logPapp MDCK-LE
Figure 3. Variable Ring Size Lipophilicity Scanning Library
All plots include octa- (blue), nona- (orange), and decapeptides (grey). (A) logPapp vs SFlogKhc/w
should be a linear relationship (eq 1), we observe deviation from linearity in each system at
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logKhc/w > 1. (B) Experimental 1,9-decadiene partition coefficients (SFlogKhc/w) vs. AlogP. The Lin-
ear regression (broken red line) was used to extrapolate values outside the detection limits. (C)
logPapp of PAMPA vs MDCK-LE for individual compounds. Linear shape of the data suggests that
the PAMPA assay is correlated with the more biologically relevant cell based assay. The trends of
individual compounds were in agreement with those observed in the mixtures (SI Table 1).
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We selected 1,9-decadiene as the hydrocarbon solvent for shake-flask partition coefficient meas-
urements (SFlogKhc/w) since it has been reported to give the best correlation to experimental
permeabilities.8 We found a very good linear fit over ~5 log units between experimental 1,9-
decadiene/buffer partition coefficients and AlogP (Figure 3b), suggesting that partitioning into
hydrocarbon in this system is dominated by the aliphatic side chains and that conformational ef-
fects (e.g., by way of steric shielding) are either small or similar for all library members. A linear
regression was used to extrapolate logKhc/w values for compounds that were below the detection
limits of the instrument in either of the two phases. The MDCK-LE permeabilities for individual
compounds were measured and compared to their permeabilities in the PAMPA system. The line-
arity of the data suggests that the PAMPA assay is correlated with the more biologically relevant
cell based assay. The lower values in PAMPA vs. MDCK may be related to differences in mem-
brane retention between the two systems. 25
Solubility Adjustment via Filtration
The solvent-diffusion model predicts a linear relationship between log of intrinsic permeabil-
ity (logPo) and membrane partition coefficient (logKmem) with unit slope, and this behavior has
been observed for a variety of compounds, including small linear peptides.26,27 At higher lipo-
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Journal of Medicinal Chemistry
philicities our cyclic peptides deviate substantially from this expected linear relationship,
prompting us to test whether this decrease in apparent permeability could be attributed to a de-
crease in water solubility at the higher logKhc/w values (Figure 3A).
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Apparent permeability coefficients (Papp) are typically calculated based on the assumption
that the concentration of compound added to the donor compartment (in practice usually ~10
µM) represents the solution concentration of the permeable species in the assay. In order to de-
termine intrinsic permeabilities (P0), we used a filtration solubility assay to determine the con-
centration of soluble, monomeric (or small < 200-nm oligomeric) compound for each AlogP (or
SFlogKhc/w) increment. Solutions were prepared using the same concentration and buffer condi-
tions as those used in the permeability assay. The solutions were then filtered through 0.2-µ nylon
filters so that only very small aggregates or monomeric species could pass through, and then
quantitated by LC/MS using samples dissolved in MeOH as standards. The buffer-to-MeOH in-
tensity ratios for each group of isomers thus provided a “solubility decrement” factor for adjusting
Papp values based on true solubilities. Above logKhc/w ~ -1, solubilities fell off precipitously as a
function of logKhc/w (Figure 4a-c).
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Analogous to the adjustment commonly applied to permeants with ionizable groups, we ad-
justed the Papp value by the soluble fraction available (fsol) using eq 3.28 When we applied this sol-
ubility adjustment to Papp, we observed a linear relationship as predicted by models relating in-
trinsic permeability Po to Khc/w as indicated in Figure 4 by the regression lines (orange markers).
While linearity is observed (consistent with a linear free energy relationship between partition
coefficients and membrane transport rates), the slope is notably less than 1 (0.47,0.48,0.53 for the
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octa-,nona-,and deca- libraries respectively). This suggests that the change in Po is not fully ex-
plained by the change in Khc/w
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.
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ꢏ
ꢏ
(3)
=
ꢀ
ꢀ ∗ꢓ
ꢐꢑꢑ
ꢒ
ꢔꢒꢕ
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1
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a)
-4.0
-5.0
-6.0
-7.0
-8.0
-9.0
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0.1
0.0
R² = 0.93
-3.0 -1.0
1.0
3.0
SFlogKhc/w
b)-4.0
1.0
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-8.0
-9.0
R² = 0.98
-10.0
-3.0 -2.0 -1.0 0.0 1.0 2.0 3.0
SFlogKhc/w
c)
-4.0
1.0
0.9
0.8
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-10.0
-3.0 -2.0 -1.0 0.0 1.0 2.0 3.0
SFlogKhc/w
Figure 4. Solubility Adjusted Intrinsic Permeabilities
Filtration solubility (unbroken red lines) and logPapp for the (a) octa-, (b) nona- , (c) decapep-
tides (Figure 2) (blue markers). Solubility-adjusted Po values were obtained by dividing the Papp
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by the relative solubility in MeOH for each point (orange markers). The linearity of Po and Khc/w is
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3
indicated by a linear fit of the solubility-adjusted values (red broken lines).
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Non-Classical Diffusion of bRo5 Compounds. Despite the linear relationship between
logKhc/w and LogP0, the slope was significantly less than 1 and the intercepts decreased with in-
creasing ring size, suggesting that there is an additional permeability penalty not accounted for
with the solubility adjustment. The solubility-diffusion model has served as the basis for more
modern treatments of predicting the role of size in membrane diffusion (e.g., the barrier-domain
model).8,11,29 In order to compare scaffolds of different lipophilicities and sizes, one can solve eq 1
for the apparent membrane diffusion term Dmem to get the following:
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ꢀ ∗ꢇ
ꢒ
(4)
ꢆꢁꢖꢁ =
ꢂ
ꢁꢖꢁ
where Po is our solubility-adjusted Papp, Kmem is the Khc/w value from the 1,9-decadiene partition
experiments, and δ is the thickness of the membrane in cm. Using a reported thickness 0.125 cm,
we plotted diffusions of compounds across the PAMPA membrane.30 It should be noted that solv-
ing for Dmem in the solubility diffusion equation is not necessarily evidence for a diffusion-
mediated mechanism, since the size dependence may arise from a combinations of factors includ-
ing partitioning penalties.8 Plotting the apparent membrane diffusion of small molecules (using
published experimental logKhc/w and logPo values31) versus their McGowan volume32 reveals a
roughly exponential relationship that follows the same form as Einstien-Stokes diffusion (Figure
5, black broken line). However, with our library compounds we find a steep drop-off beginning at
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Journal of Medicinal Chemistry
a molecular volume of ~1000 Å3 (corresponding to a MW of ~1000Da) that deviates from the ex-
ponential approximation (Figure 5 blue points).
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Extension to other scaffolds. To ensure that this trend was not particular to the macrocyclic
scaffolds used in our study, we selected a variety of commercially available or synthetically tracta-
ble cyclic peptide natural products in this size range that we anticipated could be passively per-
meable. This diverse set of compounds allowed us to extend our observations beyond the per-
methylated model system to scaffolds that are potentially more relevant in a drug discovery con-
text. Using the panel of assays described above, we determined their apparent Dmem values (Fig-
ure5, orange markers). These data followed the same sharp downward trend above a volume of
~1000 Å3 (MW ~ 975) that was seen for the octa-, nona-, and decapeptides described above. The
sharp decrease in logDmem observed in this MW range is neither predicted by models with molec-
ular volume dependences in the diffusion term (V-n) nor in the partition term (exp[-Vn]) (nor a
combination of both).29,18,8,
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Generalizing these results, the logKhc/w required to achieve the generally accepted minimum
threshold for permeability of Papp = 1 x 10-6 cm/s can be approximated at a given molecular volume
(Figure 6). As volume increases, the logKhc/w required to overcome the size penalty is so great that
aqueous solubility begins to rapidly decline. In practice, this applies an upper limit on the size of
compounds that are likely to be passively permeable and confines large molecular weight scaf-
folds to existing in a narrow lipophilicity window (green shaded area). These large compounds
must have lipophilicities that are high enough to overcome the steep size dependence observed
but low enough to maintain reasonable aqueous solubility. It should be noted that the relation-
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ship observed in our system between molecular size and intrinsic permeability is in agreement
with the observation by Doak, et al. that very few orally delivered drugs exist beyond MW ~ 1000.2
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Ro5
bRo5
1E-4
1E-5
Vumon
Danoprevir
1E-6
Scytalidamide
1E-7
1NMe3
1E-8
Xifaxan
Stylissamide G
CSALeu
CSA
1E-9
Cordyheptapeptide B
Cylindrocyclin
1E-10
1E-11
0
250
500
750 1000 1250 1500
Volume (Å3)
Figure 5. Membrane diffusion vs molecular Volume. Diffusion was determined by eq 3 using
experimental values of Po and Khc/w. The volume calculated from a 2D structure using a parame-
terized method. Small, Ro5 molecules (green points) roughly follow an exponential trend while
the bRo5, lipophilicity scanning library members (Figure 2) (blue points) are subject to a much
steeper size penalty. A selection of unrelated synthetic and natural products were subjected to the
same analysis to ensure the trends are general (orange points).
DISCUSSION AND CONCLUSIONS
In this work we set out to investigate the relationship between lipophilicity, size, and mem-
brane permeability in a molecular weight range yet to be thoroughly interrogated. By using split-
pool combinatorial syntheses and multiplexed assays we were able to evaluate thousands of com-
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Journal of Medicinal Chemistry
pounds displaying a wide range of sizes and lipophilicities. The use of permethylated amide
backbones and straight chain alkyl sidechains allowed us to probe compositional trends without
convoluting factors arising from intramolecular hydrogen bonding and associated conformation-
al effects. Physical models that describe permeability refer to the intrinsic permeability (Po) as
opposed to the experimentally observed apparent permeability (Papp), which can make compari-
son to experimental values difficult. Notably, the slopes of the relationships between logPo and
logKhc/w were less than unity, indicating that the incremental increase in logKhc/w for each addi-
tional –CH2- group corresponds to a smaller relative increase in logP0. Given that molecular size
also increases with lipophilicity in these plots, the deviation from unity is consistent with a strong
size dependence on permeability. By adjusting our experimental Papp for solubility we were able to
compare P0 values with other published datasets. Solving the general permeability equation (eq 1)
for the size dependent diffusion term we discovered a steep size dependence for compounds with
volumes in the bRo5 molecular weight range. We can only speculate about the mechanistic origin
of this size dependence. It appears that there could be more than one rate limiting step for the
permeation of these large hydrophobic macrocycles. It is clear from Figure 5 that Stokes-Einstein
diffusion cannot account for the size dependence observed, but other more restrictive diffusion
schemes akin to the diffusion of solutes through polymeric matrices might be at play as has been
suggested by others.6 Additionally, increased re-solvation penalties associated with departition-
ing from the membrane may cause membrane exit to become an additional rate limiting step. 33
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The model compounds presented here – permethylated species with only one hydrogen bond
donor, the phenolic hydroxyl – are not necessarily representative of many bRo5 compounds that
contain multiple hydrogen bond donors, where conformational effects may be much more im-
portant. Polar groups in the backbone of therapeutically relevant macrocycles have been shown
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to be important for protein target binding4 and the ability to adopt lipophilic conformations is
required to mitigate the deleterious effect of these polar groups on permeability. The low dielec-
tric conformation that CSA adopts in the membrane substantially decreases the desolvation cost
of partitioning, and might minimize the effective radius, allowing CSA not only to “smuggle” po-
lar backbone groups into the cell, but also to do so in a conformation that minimizes the cavity
size needed to accommodate it.26 These 3D effects of conformation are not captured in the simple
2D molecular volume used in plotting Dmem, which may be why CSA lies to the right of the regres-
sion line in Figure 5. Previous studies into the effect of size on permeability have used the 3D, ra-
dius of gyration descriptor; this descriptor requires the knowledge of the conformational land-
scape which is difficult to determine for large ring systems described in this paper and as such is
not used.3 However, the separate set of diverse compounds (Fig. 5 orange points) containing hy-
drogen bond donors (and conformation-dependent properties) followed roughly the same sharp
decline in membrane diffusion as our model compounds when using the thermodynamic solubil-
ity adjustments and equilibrium logKhc/w values, suggesting that the observed size dependence is
not particular to scaffolds that lack conformational effects. It should be noted that these com-
pounds are also cyclic peptide species (structures and characterization provided in Supplemen-
tary Information), and though their chemical nature is substantially more diverse than the library
described herein, the generality of these trends has yet to be thoroughly investigated with non-
peptidic compounds in this space.
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Predicting precise values of Po ab initio for a diverse set of molecules is a difficult task that re-
quires knowledge of the molecules’ conformational states as well as the composition of the mem-
brane in question. However, the bulk physical properties that contribute to the intrinsic permea-
bility are relatively simple: Kmem (approximated as Khc/w) and size (approximated as ~MW). In
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contrast to intrinsic permeability (P0), apparent permeability (Papp) depends on additional factors
which must be considered, including the aqueous boundary layer, pKa, and aqueous solubility.
Even the most permeable species in this range are well below the aqueous boundary threshold
(around 10-4 cm/s), leaving solubility and pKa as the main factors to consider aside from size and
lipophilicity when assessing the potential apparent permeability of bRo5 molecules. To achieve
Kmem values that are high enough to offset the intrinsic size penalty in bRo5 space, any ionizable
groups are likely to be prohibitively deleterious. The fraction of available compound depends on
both solubility and pKa, and if a large compound is not fully soluble at the required lipophilicity
to permeate, the addition of an ionizable group, while it may substantially increase the soluble
fraction, will likely decrease Kmem prohibitively. However, in principle a balance could be achieved
with the incorporation of solubilizing groups (e.g., morpholine or other tertiary amines) whose
pKa values are close to neutral. It has been shown in previous studies that conformational flexi-
bility can help improve solubility of bRo5 macrocyclic compounds in some cases, though the ex-
act mechanism by which the “chameleonicity” gives rise to these desirable properties is still poor-
ly understood.34,35,36 Poor aqueous solubility can also be alleviated (to a point) by modern phar-
maceutical formulation chemistry. The use of nm-scale dispersions and other solubility-
enhancing delivery systems such as emulsifiers and liposomes can greatly improve the oral bioa-
vailability of highly lipophilic drugs.37
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Figure 6. Permeable Space
A crude approximation of the minimum logKhc/w required to maintain a permbeability of 10-
6cm/s (black line), using Dmem values from the Einstein-Stokes for Ro5 compounds (Vol < 850 Å3)
and a 3rd order polynomial fit of the relationship between LogKhc/w and molecular volume for
bRo5 compounds (Vol > 850 Å3). High logKhc/w will likely result in poor aqueous solubility limit-
ing the Papp. This in turn applies an upper limit to the size of a molecule for passive membrane
permeability (green shaded region) since the necessary logKhc/w required to overcome the size
penalty would render the compound insoluble.
Cyclic peptides and peptide-containing macrocycles have received a lot of attention due to
their synthetic tractability and a renewed enthusiasm for their potential to exhibit drug-like
pharmacokinetic properties.38 While a variety of combinatorial strategies (DNA-encoded, mRNA
display, and phage display libraries etc.) have been developed that can yield macrocycles with po-
tencies that rival those of antibodies, they tend to produce molecules with multiple polar side
chains. While these residues can be critical for high affinity, they can virtually eliminate the pos-
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Journal of Medicinal Chemistry
sibility for passive cell permeability.39,40 There are other modalities that can be employed to effect
cell permeability which rely on various non-passive or pseudo-passive mechanisms (e.g., mi-
cropintocytosis, membrane disruption, possibly other uncharacterized transport pathways).41–43
For example, charge-mediated transport mechanisms employed by incorporating a series of cati-
onic residues have been demonstrated to overcome the passive permeability problems by bypass-
ing the mechanism altogether.44 Yet, although these strategies have been effective in cell culture
and even in animals43, there are still no compounds in this class that have progressed through
clinical trials, perhaps due to the complexities associated with cation-mediated transport in the
varied cell types found in biological tissues. Despite the severe constraints placed on molecular
volume by conventional, passive membrane diffusion, the permeable chemical space between
MW 500 and 1000 remains virtually untapped. Nature has provided us with a diverse set of exam-
ples that manage to walk the line between permeability and solubility. In addition to CSA, scaf-
folds with very different structures such as cylindrocyclin and patellamide C have drug-like pas-
sive permeabilities.28 Therefore, while the challenge of achieving both permeability and solubility
increases sharply beyond the Rule of 5, so does the number of synthetically accessible scaffolds.
Mining this chemical space for compounds of therapeutic value will be the challenge of this field
for decades to come.
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EXPERIMENTAL
General
All chemicals were commercially available and used without further purification. For reverse-
phase column chromatography, a SNAP C18 cartridge (30 g, Biotage) was employed. NMR spectra
were recorded in CDCl3 , Pyridien d5, or DMSO d6 (depending on solubility) on a Varian 500
MHz NMR instrument with Unity Plus console and 5 mm broad-band probe at 25 °C. Chemical
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shifts were referenced to residual solvent proton signals. The purities of individual compounds
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were tested by HPLC (Waters 1525) with an attached mass spectrometer (Micromass ZQ, waters)
and PDA detector (Waters 2998) through a 3.5 μm C18 column (XBridge BEH C18 4.6 × 50 mm).
The mixture of water (0.1% formic acid) and ACN (0.1% formic acid) was used as an eluent, of
which ACN percentage was increased stepwise (0–2 min, 20%; 2–10 min, linear gradient from 20
to 100%; 10–12 min, 100%) with a flow rate of 1.2 mL/min. Pure, diverse compounds were analyzed
on the same instrument and solvent system using a Thermo Accucore C18 column (50x4.6mm)
with 2.6μm packing material. The ACN was again increased stepwise (0–1 min, 20%; 2–10 min,
linear gradient from 20 to 100%; 2–4 min, 100%) with a flow rate of 1.9 mL/min. The absorbance
at 220 nm wavelength was used to calculate the purity and all individual compounds were at least
95% pure. LC-MS analyses for PAMPA were performed on a UHPLC (UltiMate 3000, Dionex) with
attached mass spectrometer (Orbitrap Velos Pro, Thermo Scientific) through a 1.9 μm C18 column
(Hypersil GOLD 30 × 2.1 mm, Thermo Scientific). The mixture of water (0.1% formic acid) and
ACN (0.1% formic acid) was used as an eluent, using a short trap and elute method ACN was held
at an initial value of 10% for 1min followed by a rapid linear gradient (30sec) to 95% which was
held for 1.75min before returning to starting column conditions with a flow rate of 1 mL/min.
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Cyclic Peptide Synthesis
Permethylated peptides were synthesized on via Fmoc-Tyrosine-OAllyl 2-chlorotrityl resin (0.3–
0.5 mmol/g) loaded through the sidechain phenol. Couplings were performed using standard
Fmoc coupling conditions (Fmoc amino acid/HATU/DIPEA in DMF, 1.5 h). The linear peptides
were deprotected (N-terminal Fmoc and C-terminal Allyl) using a solution of Pd(Ph3P)4 and pi-
peridine. Cyclization was performed on-resin using PyBOP/HOAt/DIPEA for 3 h in DMF. The
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resulting cyclic species were then permethylated by deprotonating using 3 eq potassium tert-
butoxide in DMSO/THF followed by immediate treatment with MeI (10 eq). The library com-
pounds were cleaved using 5% TFA in DCM, evaporated and used as crude mixtures. Individual
compounds were purified as described above. Pure compounds from the diverse compound set
were synthesized using a variety of standard Fmoc based solid phase synthesis techniques includ-
ing HATU and HBTU couplings and incorporation of pre N-methylated monomers. The synthetic
schemes and characterization of each unique compound is detailed in the supplemental infor-
mation.
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Shake Flask logKhc/w Assay
Reagent grade 1,9-decadiene was saturated with H2O prior to use. 1 μL of ~10 mM DMSO stock
was transferred to 1 mL of 1,9-decadiene/H20 (1:1). After thorough agitation, the system was al-
lowed to reach equilibrium overnight. 100 μL aliquots of each phase were taken and evaporated
under a stream of air. Each aliquot was then suspended in MeOH and quantified via LC-MS.
Filtration solubility
Filtration solubility samples were prepared by transferring 10 μL of ~10 mM DMSO stock solution
to 990 µL of PBS (pH 7.4). The solution was vortexed and allowed to reach the thermodynamic
solubility product overnight. The solution was then centrifuged at 16k g for 5 min and the super-
natant was then filtered through a 0.2 µm syringe driven filter. The first half of the filtrate (~0.5
mL) was discarded and the second half was quantified via LCMS. A reference solution was pre-
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pared by diluting 10 μL of the same DMSO stock into MeOH and quantified immediately after
preparation via LCMS.
Page 26 of 34
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PAMPA
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The PAMPA assay was run using a 96-well filter plate with 0.45 μm hydrophobic Immobilon-P
membrane supports (Millipore). 5 μL of 1% lecithin in n-dodecane was applied to the filter to
serve as the artificial membrane. Library mixtures or individual compounds were prepared from
DMSO stocks diluted in PBS to a final concentration of 5% DMSO and ~10 μM. The filter donor
plate was then placed into the acceptor plate which contained 300 μL of 5% DMSO PBS and were
allowed to incubate for 14 h at 25 °C in triplicate. After the incubation the donor and acceptor
plates were separated and the concentration of each compound in the donor and acceptor wells
was quantified by LC-MS to calculate Papp.
ALogP Calculation
ALogP values were calculated by using the method described by Ghose et. al.45
Supporting Information Available:
Detailed experimental procedures, additional data for compound studied herein, as well as
characterization of pure compounds.
AUTHOR INFORMATION
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Corresponding Author
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* E-mail: slokey@ucsc.edu
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* phone : 831-459-1307
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Author Contributions
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The manuscript was written through contributions of all authors
ABBREVIATIONS
ACN,
acetonitrile;
COMU,
(1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-
morpholino-carbenium hexafluorophosphate; DBU, 1,8- Diazabicyclo[5.4.0]undec-7-ene; DCM,
dichloromethane; DIAD, diisopropyl azodicarboxylate; DMF, N,N-dimethylformamide; DIC,
diisopropylcarbodiimide; DIPEA, diisopropylethylamine, DMSO, dimethylsulfoxide; DVB, divi-
nylbenzene; EtOH, ethanol; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, N,N,N',N'-Tetramethyl-
O-(1H-benzotriazol-1-yl)-uronium hexafluoro-phosphate; HOAt, 1-hydroxy-7-azabenzotriazole;
IR, infrared; LCMS, liquid chromatography mass spectrometry; MeOH, methanol; N2, nitrogen;
NMP, 1-methyl-2-pyrrolidinone; PAMPA, Parallel Artificial Membrane Assay; PTFE, Polytetraflu-
oroethylene; PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; RP,
reverse phase; SPPS, solid phase peptide synthesis; t-BuOH, tert-butoxide; TFA, trifluoroacetic
acid; THF, tetrahydrofuran
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