Systematic study of effects of structural modifications on the aqueous solubility of drug-like molecules

Article abstract of DOI:10.1021/acsmedchemlett.6b00451

Aqueous solubilities and activities have been measured for 17 members of the quinolinyltriazole series of inhibitors of human macrophage migration inhibitory factor (MIF). Systematic variation of a solvent-exposed substituent provided increases in solubil

Full text of DOI:10.1021/acsmedchemlett.6b00451

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Letter
Systematic Study of Effects of Structural Modifications
on the Aqueous Solubility of Drug-like Molecules
Jose A. Cisneros, Michael J Robertson, Brandon Q. Mercado, and William L Jorgensen
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00451 • Publication Date (Web): 01 Dec 2016
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Systematic Study of Effects of Structural Modifications on the
Aqueous Solubility of Drug-like Molecules
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José A. Cisneros, Michael J. Robertson, Brandon Q. Mercado, and William L. Jorgensen*
Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107
S Supporting Information
KEYWORDS: Aqueous solubility, MIF inhibitors, crystallography
ABSTRACT: Aqueous solubilities and activities have been measured for 17 members of the quinolinyltriazole series of in-
hibitors of human macrophage migration inhibitory factor (MIF). Systematic variation of a solvent-exposed substituent
provided increases in solubility from 2 µg/mL for the parent compound 3a up to 867 µg/mL. The low solubility of 3a re-
sults from its near-planar structure and an intermolecular hydrogen bond, as revealed in a small-molecule X-ray structure.
Removal of the hydrogen bond yields a 3-fold increase in solubility, but a 7-fold drop in activity. 5b emerges as the most
potent MIF inhibitor with a Ki of 14 nM and good solubility, 47 µg/mL, while 4e has both high potency and solubility.
Aqueous solubility is well known as a critical property in
the development of drugs.1-5 Poor solubility is associated
with difficulties in the reliable performance of assays, in
obtaining oral formulations for in vivo administration, and
in bioavailability. On the other hand, high solubility may
lead to poor cell permeability and rapid excretion. The
optimal range for the aqueous solubility S of drugs intend-
ed for oral delivery is ca. 10 µM to 10 mM or, equivalently,
4 – 4000 µg/mL for a compound with a molecular weight
of 400.2 Oral drugs with solubilities below 1 µM are very
rare.
Poor solubility is a common issue in drug discovery
efforts since hydrophobic compounds bind well to target
proteins for the same reasons that proteins fold to shield
their hydrophobic groups from the aqueous environment.
The problems may arise early from poorly soluble com-
pounds that are obtained as hits in high-throughput
screening. It often becomes aggravated during lead optimi-
zation since the most reliable way to increase potency is to
add hydrophobic substituents that fill hydrophobic regions
in the target’s binding site. Consistently, target proteins
with highly hydrophobic binding sites are particularly
prone to inhibitor designs with poor solubility. A classic
example is non-nucleoside inhibitors of HIV-1 reverse
transcriptase, NNRTIs.6 The binding site in this case fea-
tures a cluster of residues with aliphatic and aromatic side
chains, and poor solubility has characterized many classes
of NNRTIs. This is especially true for diaminopyrimidines
including the FDA-approved drugs etravirine and rilpi-
virine (1). In comparison to 1, we were able to increase the
solubility ca. 700-fold to 14 µg/mL for the triazine ana-
logue with a morpholinylpropoxy substituent (2) while
retaining excellent potency in infected T-cell assays.7 This
illustrates the basic principle for improving solubility
without loss of potency: add conformationally flexible sub-
stituents with polar groups to a site in the inhibitor that is
solvent-exposed in the complex with the protein. The ben-
efit of flexibility includes the entropic gain from populating
more conformers in solution than in the crystal. A corol-
lary is to reduce planarity for inhibitors with multiple ar-
omatic rings, which also leads to less tight packing in the
crystalline state.8 However, the addition of polar groups
comes with uncertainties since they may also form stabiliz-
ing hydrogen bonds in the crystal.
More recently, solubility has become an issue in our
development of inhibitors of the tautomerase activity of
human macrophage migration inhibitory factor (MIF).9
Specifically, surprisingly low solubility of 2.2 µg/mL was
found for the parent quinolinyltriazole 3a (R = H), which
arose from de novo design. However, modeling indicated
that substituents at the 6- and 7-positions of the quinoline
ring should be solvent exposed. This expectation was con-
firmed by obtaining X-ray crystal structures for 3a and the
R = methoxyethoxy (MOEO) analogue 3d (Figure 1). The
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Figure 1. Rendering from the 1.8-Å crystal structure of 3d bound
to MIF (PDB ID: 4WRB).9 Carbon atoms of 3d are colored yel-
low. Some residues in front of the ligand have been removed for
clarity. Coordination of Lys32A is highlighted with dashed lines;
the methoxyethoxy group on C6 of the quinoline is solvent-
exposed.
Figure 3. Close-up of the intermolecular hydrogen bonding in the
crystal structure of 3a.
addition of a substituent at C3 of the quinoline is not viable
as it is accompanied by large decreases in potency in view
of the slot-like binding site (Figure 1) and coordination of
Lys32.9 In all, aqueous solubilities and inhibition constants
Ki were determined for 17 analogues with 11 different sub-
stituents, as reported in Table 1. The aqueous solubilities
were measured with a standard shake-flask procedure.6,9,12
Saturated solutions are obtained by stirring for two days in
Britton-Robinson buffer (pH 6.5), followed by filtration
(Acrodisc syringe, 0.2 µm pore) and UV-vis analysis (Ag-
ilent 8453). Piroxicam has been used as a control more
than 10 times yielding S = 6.5 ±1.7 µg/mL, which is con-
sistent with a reference value of 6.36 ±0.04 µg/mL.12 The
inhibition constants were also determined as before using
4-hydroxyphenylpyruvic acid (HPP) as the substrate.9,13,14
Inhibitory activity is monitored by measuring formation of
the borate complex of the enol product at 305 nm using a
Tecan Infinite F500 plate reader. 9 of the 17 inhibitors
have been reported previously;9,13 the synthetic and spec-
troscopic details for the new compounds (3b, 3c, 3e, 3f, 3i,
4c, 4e, 6) are provided in the Supporting Information.
As reflected in Table 1, to improve solubility we favour
substituents that contain alkyleneoxy linkages with hy-
droxy, amino, and carboxylic acid termini. Amide and ester
components are viewed as less desirable for drug pro-
spects owing to their potential decomposition by proteo-
lytic enzymes. All compounds showed solubility gains over
the parent 3a. Among the smallest substituents, the bene-
fits for the hydroxyethoxy and aminoethoxy analogues 3b
and 3c were modest, while the carboxymethoxy analogue
binding site is again seen to include multiple residues with
aromatic and aliphatic side chains. The poor solubility of
3a in spite of its four nitrogen atoms and hydroxyl group
can be attributed to its expected near planarity,10 which
has now been confirmed by a small molecule crystal struc-
ture.11 As illustrated in Figure 2, the nearly planar mono-
mers are well-stacked in the crystal structure and there
are also intermolecular hydrogen bonds between the
quinoline nitrogen atoms and phenolic hydroxyl groups
(2.73 Å) in adjacent molecules (Figure 3). As expected, 3a
in isolation or in the crystal adopts a conformation with
the quinoline nitrogen atom anti to N3 of the triazole ring
to minimize lone-pair repulsion, while the conformation is
syn in the complex with MIF to provide optimal coordina-
tion of the ammonium group of Lys32A (Figure 1).
Under the circumstances, it was decided to explore the
solubility range that could be obtained by systematic varia-
tion of the substituent R in the 6-position of 3 and 4. Two
analogues of 59 were also considered along with the
deshydroxy compound 6. Disruption of planarity by, e.g.,
Figure 2. Illustration of the packing in the 0.84-Å crystal struc-
ture of 3a. The space group is Pca21. The thermal ellipsoids are
depicted at 50% probability level. The CCDC ID is 1514977.
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In Table 1, computed octanol/water partition coeffi-
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Table 1. Computed Octanol/Water log Ps, Experimental
Aqueous Solubility at pH 6.5 (S in µg/mL), and Ki (µM)
cients, ClogP and QPlogP, have been included as deter-
mined by ChemDraw and QikProp.18,19 These are of inter-
est as a measure of hydrophobicity and since log P and log
S values are known to be correlated. The solubility equa-
Cp
3a
3b
3cb
3d
3e
R
H
ClogP
3.80
3.22
3.29
3.98
3.85
4.89
3.36
4.24
3.04
3.35
4.24
3.38
4.06
3.63
5.70
5.64
2.48
QPa
3.32
2.88
2.12
3.49
3.81
4.07
2.43
2.82
2.64
2.77
3.16
2.98
3.90
3.30
5.54
4.70
3.32
S
2.2
2.6
3.7
3.6
2.4
3.4
13.9
48.5
365
9.1
27.2
37.0
19.2
867
6.1
Ki
0.23
0.53
0.26
0.20
0.147
0.27
0.36
0.161
0.20
0.144
0.074
0.048
0.039
0.037
0.024
0.014
1.37
HOCH2CH2O
H2NCH2CH2O
H3COCH2CH2O
H3CO(CH2CH2O)2
2-THP-CH2O
H2N(CH2CH2O)2
4-Mr(CH2CH2O)2
HOOCCH2O
H2N(CH2CH2O)2
4-Mr(CH2CH2O)2
HOOCCH2O
HOOC(CH2)3O
HOOCCH2OCH2CH2O
H3COCH2CH2O
HOOC
tion of Yalkowsky estimates log S = 0.5 - log P - 0.01(tm
-
25) where tm is the melting point,2,5,20 and a simpler rela-
tionship is log S = - log P - 0.2 with an rms error of 1.0 log
unit.9 Thus, log S does decrease linearly with increasing
hydrophobicity as represented by log P, but much quanti-
tative uncertainty is associated with the crystalline state as
reflected in tm and other terms that have been introduced
to represent it in predictive methods.2 The log P predic-
tions with the present methods are in generally good ac-
cord with an average difference of 0.62, though the dis-
crepancies for 3c, 3h, and 4b are greater than 1 log unit.
Presently, log P is not found to be a good predictor of log S.
For 3a – 3e, the log P values would suggest solubilities in
the 10-3 to 10-4 M range, while the observed values are
closer to 10-5 M owing presumably to the π-stacking in the
crystals (Figure 2). Differences in log P are also not a good
gauge of differences in log S for the present compounds.
For example, 5a might be expected to be 100-fold less sol-
uble than 3d based on log P, but it is more soluble; similar-
ly, the enhanced solubility of 4e is not reflected in its log P.
Concerning the Yalkowsky equation, routine measure-
ment of melting points is not common today in drug dis-
covery settings. However, we did measure the melting
points of several of the compounds. They are 200 – 205 °C
for 3d, 268 – 270 °C for 4d, and 230 – 235 °C for 4e. Appli-
cation of the Yalkowsky equation using the average of the
two log P values in Table 1, then yields predicted log S val-
ues of -5.0, -5.9, and -5.0 for the three compounds, respec-
tively, which translate to 3.5, 0.5, and 3.9 µg/mL. Thus,
compared to the experimental solubilities in Table 1, the
prediction for 3d is accurate, but the enhanced solubilities
for 4d and 4e are completely missed. Even differences in
solubility are difficult to predict, and, if solubility is an im-
portant issue, there is no alternative but to measure it.
Finally, for the Ki results, the tautomerase assay was
repeated for all of the listed compounds in this report in
order to limit variations from the protein preparation, in-
cubation times, and spectrometer.13 For 3a – 3i, the activi-
ties are mostly in a narrow range of 0.2 – 0.4 µM, which is
expected since the structural variations are only for the
solvent-exposed substituent; additional modulation may
come from interactions with residues on the surface of
MIF. For example, the greater potency for 3e may result
from the CH3O(CH2CH2O)2 appendage curling back and
making hydrophobic contacts with the rings of Pro33 and
Pro34, which are in the grey area above Lys32 in Figure 1.
The present Ki results for 3a, 3g, and 3h also agree well
with Kd values that we reported recently for these com-
pounds from a fluorescence polarization assay.14 As found
previously,9,14 addition of the fluorine in going to 3 to 4,
e.g., 3g to 4a or 3h to 4b, enhances the activities ca. 3-fold
owing to contact with Met101 and enhancement of the
hydrogen bond between the phenolic hydroxyl group and
Asn97. Modeling also suggested introduction of the phe-
noxy group at C5 of the quinoline fragment to pick up con-
8
9
3f
3gb
3hc
3i
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4ab
4b
4c
4d
4e
5a
5b
6d
47.2
1046
HOOCCH2O
7e
27.3
a
b
c
d
QPlogP. TFA salt. Mr = morpholinyl. Des-hydroxy
analogue of 3i. e (R)-ISO-1.
3i provided a striking solubility boost to 365 µg/mL.
Though it worked well in the present cases, in general, the
impact of addition of substituents with a carboxylic acid is
uncertain since carboxylic acids often form hydrogen-
bonded dimers in their crystals.4,15 The purely ether con-
taining substituents in 3d, 3e, and 3f also just gave modest
improvements for the solubility, and it is notable that addi-
tion of a second ethyleneoxy unit in going from 3d to 3e
was not helpful. However, significant gains were found
with amino-containing substituents in 3g and 3h.16 It is
well known that introduction of morpholine and related
heterocycles is normally successful in improving aqueous
solubility.5,7 The groups are expected to be protonated at
physiological pH, which benefits hydration, and their satu-
rated, non-planar character eschews tight crystal packing.
Turning to 4a – 4e, addition of the fluorine atom adja-
cent to the phenolic hydroxyl group reduces solubility by
factors of ca. 2 - 10 for the matched pairs 4a and 3g, 4b
and 3h, 4c and 3i. This is the typical direction in view of
the increase in hydrophobicity except in special cases
where the fluorine is 2 or 3 carbon atoms removed from an
oxygen.5,17 Lengthening the linker by two methylene units
in going from 4c to 4d reduced the solubility from 37 to 19
µg/mL; however, addition of an ethyleneoxy group in pro-
gressing to 4e was very beneficial yielding a 23-fold boost
to 867 µg/mL. For 5a and 5b, the improvements with a
carboxylic acid group are again apparent. Then, with 6,
removal of the intermolecular hydrogen bond in the crys-
tal was tested; the result of 1046 µg/mL reflects a 3-fold
increase over the solubility of 3i.
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tacts with Tyr36,9,14 which has yielded the most potent
compounds, 5a and 5b. In addition, the importance of the
hydrogen bond between the phenolic OH and Asn97 was
confirmed by the increase in Ki to 1.37 µM for 6 from 0.20
µM for 3i. As an additional control, the reference MIF in-
hibitor (R)-ISO-1 (7)21 was also assayed; the observed Ki of
27.3 µM is similar to the prior average value of 24 µM from
multiple measurements.14
(4) Zhang, L.; Zhu, H.; Mathiowetz, A.; Gao, H. Deep understand-
ing of structure-solubility relationship for a diverse set of organic
compounds using matched molecular pairs. Bioorg. Med. Chem.
2011, 19, 5763-5770.
(5) Walker, M. A. Improving Solubility by Structural Modifica-
tion. Top. Med. Chem. 2015, 9, 69-106.
(6) Jorgensen, W. L. Computer-aided discovery of anti-HIV
agents. Bioorg. Med. Chem. 2016, 24, 4768-4778.
(7) Bollini, M.; Cisneros, J. A.; Spasov, K. A.; Anderson, K. S.;
Jorgensen, W. L. Optimization of diarylazines as anti-HIV agents
with dramatically enhanced solubility. Bioorg. Med. Chem. Lett.
2013, 23, 5213-5216.
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(8) Ishikawa, M.; Hashimoto, Y. Improvement in Aqueous Solu-
bility in Small Molecule Drug Discovery Programs by Disruption
of Molecular Planarity and Symmetry. J. Med. Chem. 2011, 54,
1539
(9) Dziedzic, P.; Cisneros, J. A.; Robertson, M. J.; Hare, A. A.;
Danford, N. E.; Baxter, R. H.; Jorgensen, W. L. Design, Synthesis,
and Protein Crystallography of Biaryltriazoles as Potent Tau-
tomerase Inhibitors of Macrophage Migration Inhibitory Factor. J.
Am. Chem. Soc. 2015, 137, 2996-3003.
(10) Dahlgren, M. K.; Schyman, P.; Tirado-Rives, J.; Jorgensen,
W. L. Characterization of Biaryl Torsional Energetics and its
Treatment in OPLS All-Atom Force Fields. J. Chem. Info. Model.
2013, 53, 1191-1199.
The principal purpose of this study was to explore sys-
tematically the effects of variations of a solvent-exposed
substituent on aqueous solubility in a drug-like series. The
related work in the literature is largely scattered and less
extensive, though the matched pair study of Zhang et al.4
and the study of polyether and alcohol substituents by Zhu
et al.22 are particularly notable. The sequence of substitu-
ents in Table 1 may be of use to others who are faced with
a similar challenge, though the quantitative outcomes will
undoubtedly be different for other molecular series. How-
ever, the present and earlier results5,7 do point to the utili-
ty of the addition of polyether chains terminated with a
morpholine ring or surrogates for improving aqueous sol-
ubility. In the present case, addition of polyether chains
with a carboxylic acid were also highly effective.
(11) Crystals of 3a were grown via vapor diffusion with DMSO
and water. A resulting crystal was diffracted under a cold nitro-
gen stream on a Rigaku MicroMax 007HF+ equipped with a Sat-
urn 944+ CCD detector with Cu Kα. The structure was solved with
direct methods. Heavy atoms were refined anisotropically, and
hydrogen atoms were refined using a riding model. The hydrogen
atom isotropic displacement parameters were fixed to the U value
of the atoms to which they are linked multiplied by 1.2. The mole-
cules pack well with a pseudo-slipped stacked morphology. The
median plane formed by the triazole ring creates angles of 8.0(3)°
and 23.9(4)° with the quinoline and phenol groups, respectively.
(12) Baka, E.; Comer, J. E. A; Takács-Novák, K. Study of equilib-
rium solubility measurement by saturation shake-flask method using
hydrochlorothiazide as model compound. J. Pharm. Biomed. Anal.
2008, 46, 335-341.
(13) Cisneros, J. A.; Robertson, M. J.; Valhondo, M.; Jorgensen,
W. L. Irregularities in enzyme assays: The case of macrophage migra-
tion inhibitory factor. Bioorg. Med. Chem. Lett. 2016, 26, 2764-2767.
(14) Cisneros, J. A.; Robertson, M. J.; Valhondo, M.; Jorgensen,
W. L. A Fluorescence Polarization Assay for Binding to Macrophage
Migration Inhibitory Factor and Crystal Structures for Two Potent
Inhibitors. J. Am. Chem. Soc. 2016, 138, 8630-8638.
(15) Etter, M. C. Encoding and decoding hydrogen-bond patterns
of organic compounds. Acc. Chem. Res. 1990, 23, 120-126.
(16) The primary amines 3c, 3g, and 4a were prepared as their TFA
salts. This resulted from use of trityl protecting groups that are re-
moved with TFA yielding the salts. Attempted isolation of the free
amines by treatment with NaHCO3 and extraction with DCM or ethyl
acetate gave poor yields owing to low solubility of the amines.
(17) Lee, W.-G.; Chan, A. H.; Spasov, K. A.; Anderson, K. S.;
Jorgensen, W. L. Design, Conformation, and Crystallography of 2-
Naphthyl Phenyl Ethers as Potent Anti-HIV Agents. ACS Med. Chem.
Lett. 2016, 7, 0000-0000. doi: 10.1021/acsmedchemlett.6b00390
(18) ChemBioDraw 12.0.2, CambridgeSoft Inc., Cambridge, MA,
2014.
(19) QikProp 4.0, Schrödinger Inc., New York, NY, 2014.
(20) Ran, Y.; He, Y.; Yang, G,; Johnson, J. L. H.; Laskowski, S. H.
Estimation of aqueous solubility of organic compounds using the
general solubility equation. Chemosphere 2002, 48, 487-509.
(21) Lubetsky, J. B.; Dios, A.; Han, J.; Aljabari, B.; Ruzsicska, B.;
Mitchell, R.; Lolis, E.; Al-Abed, Y. The tautomerase active site of
macrophage migration inhibitory factor is a potential target for
ASSOCIATED CONTENT
Supporting Information. Synthetic procedures, NMR and
HRMS spectral data for all new compounds, and crystallo-
graphic details for 3a. This information is available free of
charge via the Internet at http://pubs.acs.org .
AUTHOR INFORMATION
Corresponding Author
* william.jorgensen@yale.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Gratitude is expressed to the National Institutes of Health
(GM32136) for research support and to the National Science
Foundation for a fellowship for MJR (DGE-1122492).
ABBREVIATIONS
DCM, dichloromethane; THP, tetrahydropyran; TFA, trifluoro-
acetic acid.
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H. When poor solubility becomes an issue: From early stage to
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ACS Paragon Plus Environment