Structure-Permeability Relationship of Semipeptidic Macrocycles - Understanding and Optimizing Passive Permeability and Efflux Ratio

Article abstract of DOI:10.1021/acs.jmedchem.0c00013

We herein report the first thorough analysis of the structure-permeability relationship of semipeptidic macrocycles. In total, 47 macrocycles were synthesized using a hybrid solid-phase/solution strategy, and then their passive and cellular permeability was assessed using the parallel artificial membrane permeability assay (PAMPA) and Caco-2 assay, respectively. The results indicate that semipeptidic macrocycles generally possess high passive permeability based on the PAMPA, yet their cellular permeability is governed by efflux, as reported in the Caco-2 assay. Structural variations led to tractable structure-permeability and structure-efflux relationships, wherein the linker length, stereoinversion, N-methylation, and peptoids site-specifically impact the permeability and efflux. Extensive nuclear magnetic resonance, molecular dynamics, and ensemble-based three-dimensional polar surface area (3D-PSA) studies showed that ensemble-based 3D-PSA is a good predictor of passive permeability.

Full text of DOI:10.1021/acs.jmedchem.0c00013

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Article
Structure-Permeability Relationship of Semi-Peptidic Macrocycles –
Understanding and Optimizing Passive Permeability and Efflux Ratio
Antoine Le Roux, Emilie Blaise, Pierre-Luc Boudreault, Christian Comeau, Annie Doucet, Marilena Giarrusso,
Marie-Pierre Collin, Thomas Neubauer, Florian Koelling, Andreas H. Göller, Lea Seep, Dieudonné T
Tshitenge, Matthias Wittwer, Maximilian Kullmann, Alexander Hillisch, Joachim Mittendorf, and Éric Marsault
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00013 • Publication Date (Web): 26 May 2020
Downloaded from pubs.acs.org on May 26, 2020
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Structure-Permeability Relationship of Semi-
Peptidic Macrocycles – Understanding and
Optimizing Passive Permeability and Efflux Ratio
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Antoine Le Roux,†,‡ Émilie Blaise,†,‡ Pierre-Luc Boudreault,†,‡ Christian Comeau,† Annie Doucet,†
Marilena Giarrusso,† Marie-Pierre Collin,$ Thomas Neubauer,$ Florian Koelling,$ Andreas H.
Goeller,$ Lea Seep,$ Dieudonné T. Tshitenge,$ Matthias Wittwer,$ Maximilian Kullmann,$
Alexander Hillisch,$ Joachim Mittendorf, $ and Eric Marsault†,*
† Department of Pharmacology-Physiology and Institut de Pharmacologie de Sherbrooke; 3001,
12e av nord ; Sherbrooke (Québec), J1H 5N4.
$ Bayer AG; Drug Discovery, Pharmaceuticals; D-42096 Wuppertal, Germany.
AUTHOR CONTRIBUTIONS
‡ALR, EB and PLB contributed equally to this manuscript.
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KEYWORDS.
Macrocycles, semi-peptidic, cellular permeability, structure-permeability relationship, efflux.
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ABSTRACT
We herein report the first thorough analysis of the structure-permeability relationship of semi-
peptidic macrocycles. In total, 47 macrocycles were synthesized using a hybrid solid
phase/solution strategy, then their passive and cellular permeability was assessed using the
PAMPA and Caco-2 assay, respectively. Results indicate that semi-peptidic macrocycles generally
possess high passive permeability based on the PAMPA assay, yet their cellular permeability is
governed by efflux as reported in the Caco-2 assay. Structural variations led to a tractable structure-
permeability and structure-efflux relationship, whereby linker length, stereoinversion, N-
methylation and peptoids site-specifically impact permeability and efflux. Extensive NMR,
molecular dynamics and ensemble-based 3D-PSA studies showed that ensemble-based 3D-PSA is
a good predictor of passive permeability.
INTRODUCTION
In recent years, macrocycles have generated a lot of interest as scaffolds for drug discovery.1-5 As
a class, they possess the ability to bridge the properties of small molecules, with physicochemical
profiles conducive of permeability and oral bioavailability, with those of biologics, characterized
by large interacting surfaces suitable for difficult targets such as protein-protein interactions, yet
devoid of passive permeability.6 Marketed macrocyclic drugs (ca 70 in total) largely belong to
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natural products and peptides.3 While natural products, often structurally complex, possess some
level of bioavailability to fulfill their natural function, macrocyclic peptides are often poorly
permeable and mostly administered parenterally, with the exception of cyclosporine.3 Owing to
their easier synthetic accessibility, potential for diversity and compatibility with chemo-biological
methods, the latter are under intense scrutiny. To unlock this class, a major challenge is to guide
permeability optimization.4, 7-13
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Among clinical candidates, designer semi-peptidic macrocycles are a growing subset3 that can
leverage (1) the large diversity of commercially available amino acids, (2) reduced peptidic
character and (3) distinct chemical space compared to their fully peptidic congeners.4, 14, 15 While
the structure-permeability of cylic hexa-/heptapeptides and a collection of synthetic macrocycles
has been studied,7, 10, 16-24 semi-peptidic macrocycles have not been systematically studied.
To elucidate the impact of structural features on permeability, we generated 47 derivatives of
scaffold A (Fig. 1), composed of a tripeptide cyclized head-to-tail with an alkyl linker. The Leu-
Ala-Phe peptide was chosen because in NMR the corresponding signals are easily distinguished,
making the analysis simpler. Also, the cLogD7.5 of the corresponding variations on this scaffold
varies between ca 1.5 and 3.5. The impact of discrete structural changes on passive and cellular
permeability was assessed using the PAMPA and Caco-2 assays, respectively. Contextually, most
permeability studies of cyclic hexapeptides relied on the low efflux MDCK assay, a low efflux
cellular passive permeability assay.7-12 In contrast, this study takes into account both passive and
cellular permeability, including efflux as a key component of permeability.
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sequence
stereochemistry
N-alkylation
peptoid
R1 = Leu
O
R2 = Ala
O
O
N
H
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NH
HN
N
H
linker
R3 = Phe
length
A
Figure 1. Representative structure of macrocycles in this publication (C6 alkyl linker and (L)
stereochemistry shown as examples
RESULTS AND DISCUSSION
Impact of ring size (Table 1) - Analogs were synthesized using a hybrid approach wherein the
linear, alkylated tripeptide was assembled on solid phase then macrocyclized in solution (see
Experimental Section, Supplementary Scheme S1 and Supplementary Information Procedure 2 ).14,
15, 25
Since size is expected to influence lipophilicity and flexibility,8, 19, 26, 27 linker length was varied
from 2 to 10 atoms (ring sizes 12-20, 1-9). In this series, passive permeability steeply increases
with ring size from 12- to 14-membered rings 1-3, with PAMPA -LogPe ranging from5.6 to 4.7 (-
LogPe < 6 is considered good permeability, -LogPe > 7 impermeable, see Supplementary Section
4c for details). A plateau around -LogPe 4.6 was observed for 14- to 20-membered rings 3-9. In
the Caco-2 assay, small macrocycles displayed high efflux and low cellular permeability. The latter
increases with ring size and decreasing efflux, with maximal permeability and minimal efflux for
19-membered ring 8.
Table 1. Influence of ring size on permeability (sequence: Leu-Ala-Phe)
Caco-2 Papp (Efflux)[a]
Nr
Linker
Ring size
12
PAMPA -LogPe
5.6
1
-(CH2)2
4.7 (>20)
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-(CH2)3
-(CH2)4
-(CH2)5
-(CH2)6
-(CH2)7
-(CH2)8
-(CH2)9
-(CH2)10
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7.7 (>20)
6.4 (>20)
4.9
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4.6
4.6
4.6
4.5
4.5
19.4 (>20)
21.5 (19.0)
69.3 (5.2)
60.7 (3.2)
82.4 (3.3)
45.1 (6.1)[b]
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[a] Papp given in nm/s. Efflux calculated as Papp (B-A)/ Papp (A-B). [b] 63% recovered.
These first results suggest that passive permeability is high, although cellular permeability
appears driven by high efflux. The involvement of P-gp in efflux was confirmed by running the
Caco-2 assay in the presence of P-gp inhibitor ivermectin.28 In these conditions (Suppl. Table T1),
2 and 5 possess Papp of 95 and 211 nm/s, with efflux ratios of 2.5 and 1.7, respectively. The impact
of ring size, on the other hand, suggests a role for either lipophilicity or flexibility in the interaction
with P-gp. Furthermore, flexibility may be a key component to modulate permeability (aka
chameleonic properties).29
To better understand passive permeability on a molecular level, we conducted a series of
descriptor-based property calculations and molecular dynamics (MD) simulations on this first
subset. Comparison of 3D-PSA of multiple conformers of 1 (-LogPe 5.6) in chloroform with those
of the more flexible compound 5 (-LogPe 4.6) suggests that the higher rigidity of 1 leads to more
exposed polar amide groups not involved in IMBHs. The higher flexibility of 5 enables polar NH-
groups to be buried in the macrocycle and a higher degree of shielding of carbonyl groups by the
amino acid side chains. Thus, carbonyl groups of 1 are solvent-exposed, increasing 3D-PSA and
lowering permeability. In the more flexible 5, the amide NH groups can form intramolecular H-
bonds (IMHBs), lowering 3D-PSA and increasing permeability. The difference in 3D-PSA of 1 vs
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5 is about 20Å2 based on conformational sampling and MD simulations in chloroform. Thus, the
permeability cliff between 1 and 5 may be attributed to the higher rigidity of 1 due to its smaller
size, leading to higher 3D-PSA.
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Figure 2. Comparison of different conformations with the corresponding impact on 3D-PSA in
CHCl3 for 1 and 5. a) Intramolecular hydrogen bonds (IHMBs) across 50k MD-snapshots; b) 5
kcal/mol conformer ensemble. c) Representative conformer from b) in good agreement with the
mean IHMBs and 3D-PSA compared to a). d) Low Dielectric Conformations (LDCs)
demonstrating the tendency to maximize IHMBs.
Impact of peptide sequence (Table 2) - Using 5 as a reference scaffold, the next question was
to identify the structural determinants of permeability and efflux. Firstly, sequence permutations,
which do not alter calculated 2D properties, marginally influence passive permeability and efflux,
which both remain high (PAMPA -LogPe 4.2 to 4.6 and efflux >18), while cellular permeability
remains low to moderate (Papp 6 to 21). Notably, Ala in position 3 (i.e., 11, 12) leads to poor cellular
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permeability, presumably due to the lower ability of the small Ala residue to shield the H-bond
donor ability of the adjacent amine.21
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Table 2. Influence of sequence order on permeability
Nr
5
Sequence
Caco-2 Papp (Efflux)[a]
21.5 (18.9)
15.1 (>20)
PAMPA -LogPe
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Leu-Ala-Phe-C6
Ala-Leu-Phe-C6
Leu-Phe-Ala-C6
Phe-Leu-Ala-C6
Ala-Phe-Leu-C6
Phe-Ala-Leu-C6
4.6
4.5
4.2
4.5
4.5
4.5
10
11
12
13
14
6.1 (>20)
5.7 (>20)
17.1 (18.6)
12.0 (>20)
[a] Papp given in nm/s. Efflux calculated as Papp (B-A)/ Papp (A-B).
Impact of stereochemistry (Table 3) - Stereochemistry may impact free energy of desolvation
and permeability,9 depending on side-chain orientation and polarity shielding.30 Thus, the 8
diastereomers of 5 were synthesized and their permeability measured. Whereas stereoinversion
has little impact on passive permeability (PAMPA -LogPe 4.4 to 4.9), inversion in positions 2
[(D)Ala, 16] and 3 [(D)Phe, 17] increases efflux (>20) and decreases cellular permeability (Caco-
2 Papp 1.4 - 2.1). Conversely, inversion in position 1 [(D)Leu, 15] reduces efflux while doubling
Caco-2 Papp (15 vs 5, efflux 7.6 vs. 18.9, Papp 53.5 vs 21.5). In the presence of ivermectin, the Papp
of 15 further increases to 420 while its efflux decreases to 0.9 (Suppl. Table T1), confirming that
passive cellular permeability remains high. Interestingly also, cellular permeability and efflux
mirror in pairs of enantiomers (see 5, 15, 16, 17 vs 21, 20, 19, 18 respectively). This contrasts
with results on polar cyclic hexapeptides, which suggested transporter-mediated uptake based on
enantiomeric differences.31 These observations suggest conformational changes and different
orientations of the isobutyl side chain of Leu, possibly masking polar backbone atoms.
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Table 3. Influence of stereochemistry on permeability
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Nr
5
Sequence
Caco-2 Papp (Efflux)[a]
21.5 (18.9)
53.5 (7.6)
PAMPA -LogPe
Leu-Ala-Phe-C6
4.6
4.5
4.7
4.4
4.9
4.8
4.8
4.9
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(D)Leu-Ala-Phe-C6
Leu-(D)Ala-Phe-C6
Leu-Ala-(D)Phe-C6
(D)Leu-(D)Ala-Phe-C6
(D)Leu-Ala-(D)Phe-C6
Leu-(D)Ala-(D)Phe-C6
(D)Leu-(D)Ala-(D)Phe-C6
2.1 (>20)
1.4 (>20)
1.3 (>20)
1.0 (>20)
56.8 (7.8)
13.3 (>20)
[a] Papp given in nm/s. Efflux calculated as Papp (B-A)/ Papp (A-B).
The impact on efflux is also noteworthy since inversion in position 1 reduces efflux in both
enantiomeric series, suggesting a P-gp molecular recognition based on structural pattern rather
than individual pharmacophoric elements, congruent with broad specificity.32
Impact of N-methylation and acetylation (Table 4) - N-methylation is beneficial for
permeability in cyclic hexa/ heptapeptides when applied site-specifically to solvent-exposed amide
bonds.9,
Guidance from NMR analysis was used to rationally optimize the oral
10, 20, 33
bioavailability of a cyclic hexapeptide20 and an integrin antagonist.34 Thus, site-selective N-
methylation and peptoid replacement were implemented on each NH bond (synthesized as per
Suppl. Schemes S1, S2).35
Table 4. Influence of N-methylation and peptoids on permeability
Nr
5
Sequence
Caco-2 Papp (Efflux)[a]
PAMPA LogPe
Leu-Ala-Phe-C6
21.5 (18.9)
4.6
4.6
4.3
22
23
Leu(NMe)-Ala-Phe-C6
Gly(NiBu)-Ala-Phe-C6
166.9 (2.8)
102.0 (3.4)
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Leu-Ala(NMe)-Phe-C6
14.6 (>20)
2.3 (>20)
41.6 (7.9)
74.1 (4.9)
43.4 (9.4)
11.8 (>20)
4.7
4.7
5.2
4.4
4.3
5.1
Leu-Sar-Phe-C6
Leu-Ala-Phe(NMe)-C6
Leu-Ala-Gly(NBn)-C6
Leu-Ala-Phe-C6(NMe)
Leu-Ala-Phe(NAc)-C6
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[a] Papp given in nm/s. Efflux calculated as Papp (B-A)/ Papp (A-B).
N-methylation or N-acetylation of the secondary amine negatively impact passive permeability
(26, 29, -LogPe > 5). Compared to 5, N-methylation of Leu greatly reduces efflux (18.9 to 2.8) and
increases cellular permeability (22 vs 5, Papp 166.9 vs 21.5 nm/s), while keeping passive
permeability unaffected. In the presence of ivermectin, the Papp of 22 further increases to 557 with
an efflux of 0.67 (Suppl. Table T1). Replacement of Leu by its peptoid (23) has a similar effect
(efflux 3.4, Papp 102.0 nm/s), with marginal increase in passive permeability (23 vs 5, -LogPe 4.3
vs 4.6). In contrast, the same replacements on Ala (24, 25) increases efflux (>20) and reduces
cellular permeability (Papp 14.6 and 2.3 nm/s), with no impact on passive permeability (-LogPe 4.7).
N-methylation or peptoid in position 3 or linker N-methylation are beneficial for cellular
permeability and efflux (26-28), yet to a lower extent. Together, inversion of stereochemistry and
N-methylation/peptoid both influence efflux and cellular permeability site-specifically, with little
impact on passive permeability. In agreement with values for PAMPA permeability, a narrow
distribution of 3D-PSA values could also be observed (Suppl. Table T2).
A strong tendency to maximize IMHBs in aprotic solvents such as CHCl3 (a surrogate of
membrane environment with similar dielectric) is described for larger macrocyclic systems.19, 36
To understand whether this class behaves similarly, NMR and MD studies were conducted in
different solvents to assess the presence of IMHBs and their impact on passive permeability.
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IMHBs are expected to lower PSA and grant higher passive permeability.[7d, 7e, 16] Given that
stereoinversion or N-methylation in position 1 greatly increases permeability, we performed NMR
experiments on 5, 15 and 22 in CDCl3 (hydrophobic) and DMSO (polar), and determined the amide
acidity coefficient using experimental shifts in both solvents (aka NMR A value which gives a
‘quantitative’ assessment of IMHB, Fig. 3 and Supplementary Figure F1).37 Amide groups with A
˂ 0.05 are involved in IMHB, while those with A ˃ 0.16 are not.37 Donor-acceptor pairs for IMHB
were further identified by 2D heteronuclear NOE (2D-HOESY) NMR. The results are summarized
in Fig. 4. In both experiments, amides D1 and D3 in 22 displayed IMHB with A2, which was the
strongest in this series. Stereoinversion in position 1, i.e. 15 vs 5, did not increase IMHBs as
observed by NMR.
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Empirical difficulties in monitoring 2D-NMR cross-peaks and some variabilities in the spectra
suggest a higher flexibility for these molecules in comparison with other published macrocycles
such as hexa/heptapeptides.
Figure 3. NMR A values of the amide groups for macrocycles 5, 15 and 22, determined in
DMSO-d6 and CDCl3, displayed with the potential donor and acceptor positions.
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Figure 4. IMHB patterns for macrocycles 5, 15 and 22 from NMR (top) and MD simulations
(bottom), determined in DMSO-d6 and CDCl3. NMR IMHB patterns are given in classes weak,
medium, strong and are quantitative for individual molecules only, whereas MD IMHB patterns
are comparable between molecules and given in percentages of snapshots displaying respective
IMHB. For compound 22, D2 was discarded due to N-methylation (grey column).
Extensive molecular dynamics (MD) simulations for seven selected compounds (1, 5, 15, 24,
25, 26, 28) in different charge states and solvents were performed using the OPLS3.1 force field.38
A detailed analysis for 5, 15 and 22 is shown in Fig. 4 (bottom) to elucidate preferred
conformational states and common IMHB patterns. Per molecule, five 100 ns MD simulations
were performed at 300 K, starting from five diverse conformers each, both in explicit CHCl3 and
DMSO, producing 50,000 snapshots each that were analyzed for IMHB patterns (Figure 4).
Generally, probabilities of forming IMHB in DMSO are lower (and even lower for H2O, data not
shown), but there is only weak coincidence with NMR data. Based on MD, D1-A2 for all
molecules and D2-A3 for 5 and 15 are the most important IMHB, with occurrences in at least 20%
of the snapshots. We assume incomplete or imbalanced sampling or sub-optimal state analysis39 to
account for these differences. Together, both NMR and MD analyses confirm that structural
modifications impact IMHB patterns, but with different conclusions. Yet, how these impacts
permeability is unclear. These conclusions differ significantly from larger, fully peptidic
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9
macrocycles in terms of IMHB.19 One possible explanation is that the alkyl linker cannot
accommodate strong transannular IMHBs observed in cyclic hexapeptides (which display amide
bonds all around the ring), while imparting higher flexibility, which together contribute to prevent
a conserved IMHB pattern.
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Combining beneficial structural elements (Table 5) - The next question is whether the
observed structure-permeability relationship is synergistic. As testified by compounds 30-39, this
is generally the case for both cellular permeability and efflux, whereas passive permeability
remained invariable. Indeed, analogs 30, 32, 34 and 39, which combine best features at positions
1, 3 and linker, possess very high Caco-2 permeabilities (Papp 346.0, 189.3, 270.3 and 266.0 nm/s)
and no efflux. Interestingly, N-methylation or stereochemical inversion in position 2 reduces
permeability, even combined with positive features on other positions (e.g., 31 vs 22, 33 vs 28),
although this is overweighed by positive features in position 1 (see for example, 37 vs 38).
Ivermectin has marginal effect on the permeability and efflux of compound 34, as expected (Suppl.
Table T1).
Table 5. Combination of structural modifications
Nr
5
Sequence
Caco-2 Papp (Efflux)[a]
21.5 (18.9)
346.0 (0.7)
38.5 (11.1)
189.3 (2.0)
7.7 (>20)
PAMPA -LogPe
Leu-Ala-Phe-C6
4.6
4.6
4.6
4.7
4.6
4.4
4.6
4.5
4.5
30
31
32
33
34
35
36
(D)Leu(NMe)-Ala-Phe-C6
Leu(NMe)-(D)Ala-Phe-C6
(D)Leu-Ala-Phe-C6(NMe)
Leu-(D)Ala-Phe-C6(NMe)
(D)Leu(NMe)-Ala-Phe-C6(NMe)
Leu(NMe)-(D)Ala(NMe)-Phe-C6
Leu(NMe)-(D)Ala-Phe-C6(NMe)
270.3 (0.9)
207.0 (1.1)
63.4 (6.4)
37 Leu(NMe)-(D)Ala(NMe)-Phe-C6(NMe)
200.6 (1.3)
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39
Leu-(D)Ala(NMe)-Phe-C6(NMe)
Gly(NiBu)-Ala-Phe-C6(NMe)
40.0 (9.9)
266.4(1.6)
4.6
4.5
[a] Papp given in nm/s. Efflux calculated as Papp (A-B)/ Papp (BA).
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Impact of expanding side chain diversity (Table 6) - In order to increase the range of passive
permeabilities and explore potential correlations with calculated descriptors, we expanded the
dataset via more polar side chains such as Ser or Tyr (compounds 40-47). As expected, the passive
permeability of alcohol-containing side chains dropped compared to 5. In order to understand
structure-permeability relationship throughout the entire series, we conducted a series of property
calculations.
Table 6. Broader side chain variations
Nr
5
Sequence
PAMPA -LogPe
Leu-Ala-Phe-C6
4.6
>7.8
6.7
40
41
42
43
44
45
46
47
Val-(D)Ser-Gly-C6
Leu-Tyr-Ala-C6
Tyr-Pro-Gly-C6
>7.8
>8.2
6.6
Leu-(D)Tyr-Gly-C6
Leu-(D)Tyr-(D)Ala-C6
Val-(D)Ser-(D)Ala-C6
Ser-(D)Leu-(D)Ala-C6
Tyr-Pro-(D)Ala-C6
6.3
5.5
> 7.8
Understanding passive permeability on a molecular level: analysis of the entire macrocycle
dataset (Figure 5) - Many 3D-permeability prediction models postulate the existence of a low
dielectric conformation (LDC) in hydrophobic environment. Key characteristics of the LDC,
largely derived from cyclic hexapeptides, are the tendency to maximize IMHBs in combination
with a collapsed fold, which shields polarity.19 This led to the notion of chameleonic properties,
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i.e. the ability to modulate conformation and polar surface area as a function of the environment,
necessary for beyond-rule of 5 compounds to reach acceptable permeabilities.29, 40 In contrast to
NMR or MD studies, permeability descriptors can be calculated rapidly to enable in silico library
profiling to support the design of collections with desired physicochemical properties. Several
descriptors were used in the past to predict permeability on larger macrocyclic peptides19, 36 other
subclasses of macrocycles,41 and to predict passive membrane permeability of non-macrocyclic
systems. 8, 42, 43
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Accordingly, a selection of descriptors frequently used for permeability predictions was
calculated for the broader dataset (i.e. compounds 1-47) and correlated against PAMPA data
(Caco-2 permeability was not considered due to the confounding efflux parameter). It is important
to point out that the current permeability data do not show a well-balanced and continuous
distribution: a majority of compounds show good PAMPA permeability, whereas low permeability
is observed only for seven data points (including four left censored measurements with a -LogPe
> 7, which were discarded from regression analysis). Of importance, the dataset covers four -LogPe
units. Table 7 gives a short overview of the selected methods. More descriptors being used for
permeability prediction, methods and information on dataset preparation can be found in the
supporting information (Table T5), as well as a summary of all calculated properties (Table T6).
Table 7. Descriptors used for correlation with PAMPA permeabilities
Nr
Descriptor Name
Conformation resp. coordinates used r2 (r2)[c] Ref
44, 45
4a
cLogD 7.5
2D-Structure
0.41
(0.61)
0.51
19
46
4b
4c
[b]ΔGinsert
LDC
LDC
[a] 3D-PolarSASA
0.55
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4d
[a] 3D-PolarSASA
3D-Conformers in 5 kcal/mol E- 0.64
Window (0.76)
-
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[a] Pipeline Pilot Implementation (Version 16.5.0.143 ; http://www.3dsbiovia.com); [b] Maestro
(Version 2018-1; www.schrodinger.com); [c] left censored data included for regression in
brackets;
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Since the PAMPA assay mimics an artificial lipophilic membrane, a good correlation with
descriptors encoding lipophilicity could be expected. Surprisingly, cLogD7.5 displayed only an r2
of 0.41 (Fig. 5a). The ΔGinsert descriptor claims the existence of a low dielectric conformation
(LDC) with a tendency to maximize IMHBs showed only a medium r2-value of 0.51 (Fig. 5b). As
indicated by NMR and MD studies in the underlying scaffold class, no isolated LDC or highly
conserved IMHB pattern was identified, possibly explaining these r2-values. The 3D-PSA
calculated on the LDC indicates a slight improvement of 0.55 (Fig. 5c). The use of single LDCs
could be problematic, since they tend to form compact conformations with very low 3D-PSA of ~
40Å2, which might not reflect reality. To overcome this, 2D structures were converted to 3D using
Corina,47, 48 then submitted for conformational sampling in chloroform with a maximum of 1,000
conformers48, 49 and a mean 3D-PSA was calculated. Instead of averaging the 3D-PSA across the
entire ensemble,50 only low energy conformers within a 5 kcal/mol energy window using the
OPLS3.1 force field38 were considered. While working on an ensemble-based 3D-PSA, values
increased about 20 Å2 leading to an improved r2-value of 0.64 (Fig 5d). In contrast to single and
static LDC, ensemble-based 3D-PSA recapitulates a hyper-topology that integrates different
IMHB patterns and flexibility via a representative number of conformers within 5 kcal/mol.
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Figure 5 Correlation plots between PAMPA-permeability and selected descriptors. Blue dots
represent left censored data points which were discarded from regression analysis.
The impact of such conformational ensemble analysis becomes obvious when comparing the
rigid 12-membered ring 1 (-LogPe 5.6) with the more flexible 5 (-LogPe 4.6, see Fig. 2). The higher
rigidity of 1 prevents transannular orientation of H-bond donors, yet does not necessarily reduce
the number of H-bonds. Moreover, carbonyl groups of 1 are more solvent exposed, leading to
higher 3D-PSA and lower permeability. Since 5 is more flexible, its 3D-PSA is lower and its
permeability higher. Flexibility is also represented by the size of the 5 kcal/mol ensembles which
is much smaller for 1 (29 conformers) compared to 5 (100 conformers). The mean 3D-PSA of the
5 kcal/mol ensembles matches the mean 3D-PSA across the 50,000 MD-snapshots very closely.
More details can be found in the Suppl. Figures F3, F4 and Suppl. Table T3 and T4). Results of
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an extended descriptor set used for PAMPA permeability predictions can be found in (Suppl.
Figure F2 and Suppl. Table T5). Finally, there was no correlation between measured solubility and
permeability in the PAMPA assay (Suppl. Figure F5).
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CONCLUSION
This first study on the structure-permeability of semi-peptidic macrocycles indicates that this
class possesses distinct behaviour compared to macrocyclic hexa- and heptapeptides. In contrast
to the latter, neither NMR nor MD experiments showed the prevalence of an LDC-conformation
maximizing IMHBs, possibly due to their semi-peptidic nature and the presence of a hydrocarbon
linker. Indeed, the opportunity for strong IMHBs with an alkyl linker is greatly reduced compared
to hexapeptides which feature amide bonds along the whole ring. Moreover, higher rigidity for
smaller rings appears detrimental for passive permeability.
The passive permeability of this class is generally high with a narrow distribution. Its cellular
permeability ranges from low to very high and is efflux-driven, with significant contribution from
P-glycoprotein. Most importantly, while sequence order has little influence on efflux, site-specific
stereoinversion, N-methylation and peptoids are all highly positive in position 1, detrimental in
position 2, and have positive or smaller negative effects in position 3 and the linker. Importantly
also, beneficial features are synergistic, offering a tractable structure-permeability relationship.
In terms of molecular descriptors, 3D-PSA demonstrated the best correlation with passive
permeability for this scaffold. An ensemble-based 3D-PSA is advantageous in terms of eliminating
conformations with very low 3D-PSA, leading to higher r2-values. In agreement with Whitty and
co-workers, we find that a 3D-PSA below 140 Å2 emerges as a reasonable threshold for PAMPA
permeability in this scaffold class 29 and might be a reasonable descriptor for other datasets as well.
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Moreover, the ensemble-based 3D-PSA matches the mean 3D-PSA across 50,000 MD-snapshots
very closely and might be a good alternative for analyzing relevant 3D-conformations in different
solvents, while investing far less computational resources.
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Finally, the lack of impact of IMHB on permeability is consistent with efflux-driven
permeability, suggesting that conformational modulation is an important component of structure-
permeability. Overall, this study is the first to provide a detailed analysis of the structure-
permeability relationship of semi-peptidic macrocycles, a growing class among development
candidates.3, 51
EXPERIMENTAL SECTION
Materials. Protected amino acids were purchased from Chem-Impex International (USA). 2-
chlorotrityl chloride polystyrene resin was purchased from Matrix Innovation (Canada). All other
reagents were purchased from Sigma-Aldrich (Canada) or Fisher Scientific (USA), of the highest
commercially available purity, and were used as received.
Peptide synthesis was performed on Silicycle MiniBlocks. Crude peptides were purified by
reverse-phase chromatography using a Waters Preparative LC (Sample Manager 2767 (Fraction
collector), Binary gradient module 2545, with two 515 HPLC pump and a System Fluidics
Organizer SFO, Photodiode Array Detector 2998: column X Select CSH Prep C18 5 µm OBD 19
x 250 mm2 column, buffer: A: 0.1% HCOOH in H2O, B: 0.1% HCOOH in ACN, Flow 20 mL/min.
Analytical HPLC chromatograms were recorded on a Waters UPLC H-Class with UV detection
PDA equipped with an Acquity UPLC CSH C18 1.7µm 2.1 x 50 mm2 column. MS spectra were
recorded on a Waters SQ Detector 2 (electrospray) instrument, with the following gradient
conditions: 0 → 0.2 min, 5% acetonitrile; 0.2 → 1.5 min, 5 → 95%; 1.5 → 2 min, 95%. MS spectra
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were recorded on a Waters SQ Detector 2 (electrospray) instrument. NMR spectra were recorded
on a Bruker Avance III HD 400 MHz and Neo 500 MHz and are reported relative to deuterated
solvent signals. Data for 1H NMR are reported as follow: chemical shift (δ ppm), multiplicity (s:
singlet, bs: broad singlet, d: doublet, t: triplet, q: quartet, quint: quintet, hex: hexuplet), integration
and coupling constant (Hz). 13C NMR spectra were recorded on a Bruker spectrometer at 101 MHz
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13
and 126 MHz. Data for C NMR are reported in terms of chemical shift (δ ppm). Exact mass
measurements were recorded on an electrospray quadrupole time-of-flight Maxis 3G from Bruker
using positive mode.
Solid phase synthesis.
NB: number of equivalents are given with respect to nominal resin loading. Typically, 200 mg of
resin was used per well, using 24-well Bohdan miniblocks shaken at 600 rpm. Washing times
included shaking for 5 min followed by draining. Volumes are given per well.
Procedure 1 - General synthesis scheme for macrocycles with secondary amine on AA3 (Scheme
S1, macrocycles 1-25, 28-47)
-
AA1 attachment on 2-chlorotrityl chloride resin
A solution of Fmoc-AA1-OH (2 eq) and DIPEA (4 eq) in a mixture of DMF/DCM 1/4 (1 mL per
100 mg resin) was added to the resin, and the mixture was shaken for 1h at room temperature.
After filtration, the resin was washed five times with DMF (1.25 mL).
-
AA2 and AA3 coupling
A solution of Fmoc-AA-OH (3 eq) and HATU (3 eq) in DMF (1 mL for 100 mg resin) was added
to the resin, followed by DIPEA (4 eq), and the mixture was shaken for 2h at room temperature.
After filtration, the resin was washed five times with DMF (1.25 mL).
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Fmoc cleavage with piperidine (AA1 ≠ N-Alkyl amino acids)
5
6
7
8
A solution of 20% piperidine in DMF (1 mL) was added to the resin, and the mixture was shaken
for 10 min at room temperature. After filtration, a fresh solution of 20% piperidine in DMF (1 mL)
was added, and the mixture was shaken for 10 min at room temperature. After filtration, the
mixture was washed five times with DMF (1.25 mL) in the case of the deprotection of AA1, and
twice with DMF, once with iPrOH, once with DCM, once with iPrOH and twice with DCM (1.25
mL each) in the case of AA3.
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Fmoc removal with DBU (AA1 = N-Alkyl amino acids)
A solution of 2% DBU in DMF (1 mL) was added to the resin, and the mixture was shaken for 3
min at room temperature. After filtration, a fresh solution of 2% DBU in DMF (1 mL) was added,
and the mixture was shaken for 3 min at room temperature. After filtration, the mixture was washed
five times with DMF (1.25 mL).
-
AA3 nosylation
A solution of NsCl (4 eq) and 2,4,6-collidine (10 eq) in NMP (1 mL) was added, and the mixture
was shaken for 15 min at room temperature. After filtration, the mixture was washed twice with
DMF, once with iPrOH, once with DCM, once with iPrOH and twice with DCM (each with 1.25
mL).
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Fukuyama-Mitsunobu linker alkylation
A solution of Fmoc-protected alcohol (3 eq) and triphenylphosphine (3 eq) in THF (1 mL) was
added to the resin, followed by DIAD (3 eq). The mixture was shaken overnight at room
temperature. After filtration, the resin was washed twice with DMF, once with iPrOH, once with
DCM, once with iPrOH and twice with DCM (1.25 mL each).
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Simultaneous Fmoc/nosyl removal
5
6
7
8
A solution of n-propylamine (10 eq), piperidine (10 eq) and 2,2’-(ethylenedioxy)diethanethiol (10
eq) in DMF (1 mL) was added to the resin, and the mixture was shaken for 1h at room temperature.
After filtration, a second identical cycle was repeated. After filtration, the resin was washed twice
with DMF, once with iPrOH, once with DCM, once with iPrOH and twice with DCM (1.25 mL
each).
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Resin cleavage
A solution of 30% HFIP in DCM (1 ml for 100 mg resin) was added to the resin, and the mixture
was shaken for 1h at room temperature. After filtration, the resin was washed with DCM (1.25
mL) and shaken for 3 min, then filtered. The combined DCM phases were concentrated in vacuo
then dried.
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Macrocyclization
The above crude linear compound was dissolved in DMF (final concentration: 0.05M, based on
nominal resin loading), then DEPBT (1.1 eq) and DIPEA (3 eq) were added, and the mixture was
stirred overnight at room temperature. The mixture was then filtered over a pad of silica-carbonate
scavenger (10 eq) and washed with 4 x 2 mL of DMF. The combined DMF phases were
concentrated in vacuo and dried, then the crude macrocycles were purified by preparative HPLC-
MS.
All tested compounds were purified to >95% purity (UPLC-UV-MS) and further characterized by
1H, 13C NMR and HRMS.
SUPPORTING INFORMATION
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Supplementary Schemes S1-2, Figures F1-4, Tables T1-7, Smiles structures, Summary of all the
calculated properties for macrocycles 1-47. Experimental details (synthetic, biological and
computational methods) and full characterization of synthetic intermediates and final compounds
can be found in the Supporting Information section of this manuscript.
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AUTHOR INFORMATION
Corresponding Author
*Pr Eric Marsault; Institut de Pharmacologie de Sherbrooke; 3001, 12e av nord; Sherbrooke
(Qc), Canada J1H 5N4; eric.marsault@usherbrooke.ca
Present Addresses
†If an author’s address is different than the one given in the affiliation line, this information may
be included here.
AUTHOR CONTRIBUTIONS
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. ‡These authors contributed equally.
ACKNOWLEDGMENT
This study was funded by Bayer AG, the Natural Sciences and Engineering Research Council
of Canada (RGPIN 2017-05513) and the Canadian Foundation for Innovation.
ABBREVIATIONS
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IMHB: IntraMolecular Hydrogen Bonds. LDC: Low Dielectric Constant. MD: Molecular
Dynamics. PAMPA: Parallel Artificial membrane Permeability Assay. PSA: Polar Surface Area
7
8
9
REFERENCES
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Drug Discovery--an Underexploited Structural Class. Nat Rev Drug Discov 2008, 7, 608-624.
2. Marsault, E.; Peterson, M. L. Macrocycles are Great Cycles: Applications, Opportunities
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3. Giordanetto, F.; Kihlberg, J. Macrocyclic Drugs and Clinical Candidates: What can
Medicinal Chemists Learn from their Properties? J Med Chem 2014, 57, 278-295.
4. Marsault, E.; Peterson, M. L. Practical Medicinal Chemistry with Macrocycles: Design,
Synthesis and Case Studies. Wiley (Hoboken) 2017.
Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. The Exploration of Macrocycles for
5.
6.
Levin, J. Macrocycles in Drug Discovery. Royal Society of Chemistry (London) 2014.
Cardote, T. A.; Ciulli, A. Cyclic and Macrocyclic Peptides as Chemical Tools to
Recognize Protein Surfaces and Probe Protein-Protein Interactions. ChemMedChem 2016, 11,
787-794.
7. Beck, J. G.; Chatterjee, J.; Laufer, B.; Kiran, M. U.; Frank, A. O.; Neubauer, S.; Ovadia,
O.; Greenberg, S.; Gilon, C.; Hoffman, A.; Kessler, H. Intestinal Permeability of Cyclic
Peptides: Common Key Backbone Motifs Identified. J Am Chem Soc 2012, 134, 12125-12133.
8.
Guimaraes, C. R.; Mathiowetz, A. M.; Shalaeva, M.; Goetz, G.; Liras, S. Use of 3D
Properties to Characterize Beyond Rule-of-5 Property Space for Passive Permeation. J Chem Inf
Model 2012, 52, 882-890.
9.
White, T. R.; Renzelman, C. M.; Rand, A. C.; Rezai, T.; McEwen, C. M.; Gelev, V. M.;
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