Effect of heterocycle content on metal binding isostere coordination

Article abstract of DOI:10.1039/d0sc02717k

Bioisostere replacement is a core concept in modern medicinal chemistry and has proven an invaluable strategy to address pharmacodynamic and pharmacokinetic limitations of therapeutics. The success of bioisostere replacement is often dependent on the scaffold that is being modified (i.e., "context dependence"). The application of bioisostere replacement to a picolinic acid fragment was recently demonstrated as a means to expand a library of metal-binding pharmacophores (MBPs) to modulate their physicochemical properties, while retaining their metal binding and metalloenzyme inhibitory activity. Here, metal binding isosteres (MBIs) with different nitrogen-containing heteroarenes is explored. This resulted in a number of new MBIs that were evaluated for their physicochemical properties and metal binding features. It was observed that the coordination behavior of an MBI is dependent on the identity and arrangement of the heteroatoms within each heteroarene. To further understand the observed coordination chemistry trends, density functional theory (DFT) calculations were performed. Theory indicates that preferences in coordination geometry are largely determined by the electronic character of the heteroarene scaffold. These results provide important insights into the development of novel MBI scaffolds that can serve to broaden the scope of scaffolds for metalloenzyme inhibitor development.

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Effect of heterocycle content on metal binding
isostere coordination†
Cite this: DOI: 10.1039/d0sc02717k
*
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Benjamin L. Dick,
Ashay Patel and Seth M. Cohen
Bioisostere replacement is a core concept in modern medicinal chemistry and has proven an invaluable
strategy to address pharmacodynamic and pharmacokinetic limitations of therapeutics. The success of
bioisostere replacement is often dependent on the scaffold that is being modified (i.e., “context
dependence”). The application of bioisostere replacement to a picolinic acid fragment was recently
demonstrated as a means to expand a library of metal-binding pharmacophores (MBPs) to modulate
their physicochemical properties, while retaining their metal binding and metalloenzyme inhibitory
activity. Here, metal binding isosteres (MBIs) with different nitrogen-containing heteroarenes is explored.
This resulted in a number of new MBIs that were evaluated for their physicochemical properties and
metal binding features. It was observed that the coordination behavior of an MBI is dependent on the
identity and arrangement of the heteroatoms within each heteroarene. To further understand the
observed coordination chemistry trends, density functional theory (DFT) calculations were performed.
Theory indicates that preferences in coordination geometry are largely determined by the electronic
character of the heteroarene scaffold. These results provide important insights into the development of
novel MBI scaffolds that can serve to broaden the scope of scaffolds for metalloenzyme inhibitor
development.
Received 12th May 2020
Accepted 16th June 2020
DOI: 10.1039/d0sc02717k
rsc.li/chemical-science
tetrazoles that possess some similarities (i.e., pKa), but differ in
other aspects (i.e., lipophilicity).10,11 In an effort to increase the
Introduction
chemical diversity and to improve the drug-likeness of MBPs,
carboxylic acid bioisostere replacement was applied to the
pyridine-2-carboxylic acid (picolinic acid) MBP, generating
a novel set of metal binding isosteres (MBIs).12 This exercise in
bioisostere replacement was successful in generating a set of
viable metal coordinating motifs varying in their physico-
chemical properties (i.e., pKa, log P, log D7.4) without compro-
mising their metalloenzyme inhibitory activity.
Herein, we report a diverse series of nitrogen heteroarene
scaffolds containing a carboxylic acid that were explored for
bioisostere replacement. MBIs were synthesized and their
metal binding ability assessed via examining bioinorganic
model complexes. Certain MBIs had their physicochemical
properties determined and coordination chemistry behav-
iour analysed via computational methods. This expanded
study examines a wide variety of heteroarene scaffolds
allowing for examination of the effects of bioisostere
replacement on the coordination abilities of the resulting
MBIs, and shows that the effect of bioisostere replacement is
context dependent.13 The ndings presented here demon-
strate that while the application of bioisostere replacement
to MBPs may be a straightforward concept, the coordination
behaviour and physiochemical properties are dependent on
the parent scaffold and require consideration when
employed in metalloenzyme drug discovery.
Metalloenzymes are an undervalued class of therapeutic targets
relative to the entirety of clinical targets.1 Clinically approved
metalloenzyme inhibitors are limited in scope, specically with
respect to the metal binding motif, which are oen dominated
by a select few metal binding functional groups.1,2 This over-
reliance on a limited set of metal binding functional groups
(a.k.a., metal-binding pharmacophores, MBPs) capable of
binding active site metal ions is a barrier to developing next-
generation metalloenzyme inhibitors. Efforts to apply
fragment-based drug discovery (FBDD) to metalloenzymes has
produced new libraries of potential MBPs.3–5 However, some
new MBPs may possess physiochemical properties that limit
their viability for use in drug candidates.6,7
In medicinal chemistry, functional groups that adversely
modulate the pharmacological properties of a drug candidate or
introduce clear pharmacological liabilities are oen replaced
with alternate functional groups with broadly similar properties
referred to as bioisosteres.8,9 For example, carboxylic acids are
oen replaced with other acidic functional groups, such as
Department of Chemistry and Biochemistry, University of California San Diego, 9500
Gilman Drive, La Jolla, CA 92093-0358, USA. E-mail: scohen@ucsd.edu
† Electronic supplementary information (ESI) available: Experimental procedures,
synthetic procedures, crystal data tables, and supplementary gures and tables.
CCDC 1999816–1999840. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/d0sc02717k
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starting from 5 (synthetic details can be found in the ESI†). The
tetrazoles were synthesized from the corresponding nitriles via
a 3 + 2 cycloaddition with sodium azide yielding the indazole-3-
Results and discussion
Scaffold selection and MBI synthesis
tetrazole
(1c),
benzimidazole-2-tetrazole
(2c),
1,2-
Carboxy-substituted heteroarene MBPs were examined as
possible candidates for MBI development. The candidates for
study included indazole-3-carboxylic acid (1), benzimidazole-2-
carboxylic acid (2), 1,2-benzisothiazole-3-carboxylic acid (3),
benzothiazole-2-carboxylic acid (4), and 1,2-benzisoxazole-3-
carboxylic acid (5) (Fig. 1). Benzoxazole-2-carboxylic acid
would be a logical addition to this series, but was not
included as the scaffold is known to readily decarboxylate.14
Compounds 1, 2, 4, and 5 were commercially available, while
benzisothiazole-3-tetrazole (3c), benzothiazole-2-tetrazole (4c),
and 1,2-benzisoxazole-3-tetrazole (5c). To obtain the oxadiazo-
lone derivatives, the N-hydroxy amidine was rst synthesized for
each scaffold from the respective nitrile using hydroxylamine,
which was isolated and then cyclized with ethyl chloroformate
yielding the indazole-3-oxadiazolone (1d), benzimidazole-2-
oxadiazolone (2d), 1,2-benzisothiazole-3-oxadiazolone (3d),
benzothiazole-2-oxadiazolone (4d), and 1,2-benzisoxazole-3-
oxadiazolone (5d).
3 was synthesized according to a published procedure.15
A
series of both acyclic (A and B series) and cyclic (C and D
series) bioisosteres were selected for synthesis. For the acyclic
MBIs the starting material was generally the corresponding
carboxylic acid (1, 2, 3, 4, and 5). The corresponding O-methyl
indazole-3-hydroxamic acid (1a) and O-methyl benzimidazole-
2-hydroxamic acid (2a) were generated from the carboxylic
acid using peptide coupling conditions. O-Methyl 1,2-
benzisothiazole-3-hydroxamic acid (3a) and O-methyl
benzothiazole-2-hydroxamic acid (4a) were synthesized from
the carboxylic acid using acid chloride coupling conditions. The
indazole-3-hydroxamic acid (1b) and benzimidazole-2-
hydroxamic acid (2b) were generated from the carboxylic acid
using peptide coupling conditions. 1,2-Benzisothiazole-3-
hydroxamic acid (3b) was synthesized from the carboxylic acid
using acid chloride coupling. Benzothiazole-2-hydroxamic acid
(4b) was synthesized from ethyl benzothiazole-2-carboxylate
and hydroxylamine under basic conditions. The O-methyl 1,2-
benzisoxazole-3-hydroxamic acid (5a) and 1,2-benzisoxazole-3-
hydroxamic acid (5b) were both synthesized from 5 by gener-
ating the acid chloride and combining it with the correspond-
ing hydroxyl amine.
Synthesis and characterization of model complexes
To determine the metal binding ability of each MBI in a bio-
inorganic context, TpPh,MeZn(MBI) (TpPh,Me ¼ hydrotris(5,3-
methylphenylpyrazolyl)borate) complexes were prepared.
These model complexes are mimetic of a tris(histidine) Zn2+
metalloenzyme active site and are useful for characterizing MBI
coordination chemistry.12,19 TpPh,MeZn(MBP) complexes of 1, 2,
3, 4, and 5 were prepared and crystallized. It was found that all
of these MBPs coordinate in a bidentate fashion via the nitrogen
atom of the heteroarene and the carboxylate oxygen (Fig. 2). The
only exception was for TpPh,MeZn(5), which coordinated to the
Zn2+ ion in a monodentate fashion only through the carboxylate
donor (Fig. 2), indicating the 1,2-benzisoxazole scaffold has
signicantly decreased coordination ability. Interestingly, the
Zn–heteroarene nitrogen bond distances for the N,S hetero-
arenes (3 and 4) were slightly longer than the
corresponding N,N heteroarenes (1 and 2), by about 0.10 to 0.15
˚
A, while the Zn–carboxylate bond distances exhibited a smaller
˚
deviation (ꢀ0.05 A) (Table S6†). This difference in bond lengths
suggest an overall decrease in the donor ability of the N,S
heterocycle scaffolds relative to the N,N scaffolds.
The cyclic MBIs (C and D series, Fig. 1) of 1, 2,16 3,17 and 418
were all synthesized starting from the commercially available or
published synthesis of the corresponding nitrile derivatives.
The nitrile derivative of 5 was prepared in a similar fashion
Next, the coordination ability of the O-methyl hydroxamate
MBIs (A series) was analysed (Fig. 3). The X-ray structures of
TpPh,MeZn(MBI) with 2a, 3a, and 4a exhibit the same bidentate
coordination (through the heterocycle and the carbonyl oxygen
of the O-methyl hydroxamate) that was observed for the pyridine
scaffold.12 Interestingly, the structures of the TpPh,MeZn(MBI)
complexes of 1a and 5a show bidentate coordination only
through the oxygen donor atoms of the O-methyl hydroxamate.
The bidentate coordination through the O-methyl hydroxamate
has only rarely been observed crystallographically and not in the
presence of alternative coordination motifs.20,21 This bidentate
Fig. 2 Crystal structure of TpPh,MeZn(MBP) complexes of 1, 2, 3, 4, and
Fig. 1 Heterocyclic MBPs (top row, compounds 1–5) that were 5. Phenyl groups of TpPh,Me have been removed for clarity.
chosen for MBI development (all other rows).
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Fig. 6 Crystal structure of TpPh,MeZn(MBI) complexes of 1d, 2d, 3d, 4d,
and 5d. Phenyl groups of TpPh,Me have been removed for clarity.
Fig. 3 Crystal structure of TpPh,MeZn(MBI) complexes of 1a, 2a, 3a, 4a,
and 5a. Phenyl groups of TpPh,Me have been removed for clarity.
crystallographically (Fig. 5 and 6). Interestingly, the coordina-
tion mode of 1c was almost monodentate in character with
˚
a Zn–heterocycle bond distance of 2.61 A. The coordination
mode of 2c was clearly bidentate in character, with a Zn–
˚
heterocycle bond distance of 2.28 A, as had been observed
before with the pyridine-2-tetrazole complex.12 The complex
with 3c again showed monodentate binding with the Zn–
Fig. 4 Crystal structure of TpPh,MeZn(MBI) complexes of 1b, 2b, 3b, 4b,
and 5b. Phenyl groups of TpPh,Me have been removed for clarity.
˚
heterocycle distance ꢀ3.05 A, while the complex with 4c
exhibited bidentate binding with an elongated Zn–heterocycle
distance of 2.48 A. The TpPh,MeZn(MBI) complex with 5c again
˚
coordination through an ether-like coordination motif (1a and
5a), when there is a suitable nitrogen heterocycle donor avail-
able, suggests that the coordination ability of the nitrogen
atoms are compromised due to electronic effects. Additionally,
it is unlikely that sterics play a signicant role in the observed
coordination chemistry, as in the case of comparing the
difference in coordination between 1a to 3a, the only difference
in both structures is a single atom substitution (N versus S). To
further probe the effect of bioisostere replacement on each
scaffold, the hydroxamate series of bioisosteres was analysed
crystallographically, as the hydroxamate is a stronger ligand
than the respective O-methyl hydroxamate.
presented monodentate coordination with a Zn–heterocycle
˚
distance of 3.17 A. To evaluate whether these coordination
trends were due to a steric effect, a more sterically bulky
heterocycle bioisostere, oxadiazolone, was investigated. The
oxadiazolone TpPh,MeZn(MBI) complexes (Fig. 6) exhibited the
same coordination motifs and bond distances as the tetrazole
complexes, suggesting an important electronic contribution to
the observed coordination trends and no signicant steric
impediments.
Based on the TpPh,MeZn(MBI) structures of both the acyclic
and cyclic MBI complexes, it is evident that the heterocyclic
scaffold greatly inuences MBI binding. The heteroarenes pre-
senting a 1,3-heteroatom arrangement (series 2 and 4) appear to
retain bidentate coordination and short metal–ligand bond
distances. In contrast, the heteroarenes bearing a 1,2 arrange-
ment of heteroatoms vary signicantly in their preferred coor-
dination mode and bond distances. For example, the series
based on the 1,2-benzisoxazole scaffold (5a–d) exhibited either
monodentate coordination or coordination through the bio-
isostere replacement alone (5a and 5b). These results suggest
that retention of the coordination mode upon carboxylate
replacement is dependent on the coordination character/ability
of the parent scaffold, which appears to be affected by hetero-
atom identity and pattern within the heteroarene scaffold.
Taken together, the crystallographic data suggest the
TpPh,MeZn(MBI) model complex is a useful evaluative tool for
metal binding, and may be useful to recapitulate coordination
behaviour of similar substitutions in metalloenzyme inhibi-
tors.22 To further understand the electronic effect of each bio-
isostere replacement, the physicochemical properties of the
MBIs were measured.
The TpPh,MeZn(MBI) complexes with the hydroxamate series
(1b, 2b, 3b, 4b, and 5b) exhibited similar trends as was observed
with the O-methyl hydroxamate series, with one exception.
Compounds 1b and 5b were observed to coordinate through the
hydroxamate only and 2b and 4b were bound through the
heterocycle and the hydroxamate (Fig. 4), which are all consis-
tent with the O-methyl hydroxamate series. In contrast, 3b did
not coordinate in the same manner as 3a, but rather was
coordinated to the Zn2+ center through the hydroxamic acid in
a bidentate fashion (like 1b and 5b). The combined results of
the O-methyl hydroxamate and hydroxamate series (A and B
series) suggests signicant electronic differences not only
depend on heteroarene heteroatom content, but also the
arrangement of the heteroatoms in the ring system.
To gain further insight into the coordinative ability of these
MBIs, heterocyclic MBIs (series C and D) were analysed
Physicochemical analysis
Upon determining that the MBIs of the different series have
different coordination abilities to the TpPh,MeZn(MBI) bio-
inorganic model system, an evaluation of physicochemical
properties was performed. The acidity (pKa) of the bioisostere
Fig. 5 Crystal structure of TpPh,MeZn(MBI) complexes of 1c, 2c, 3c, 4c,
and 5c. Phenyl groups of TpPh,Me have been removed for clarity.
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Table 1 Physicochemical values from all O-methyl hydroxamate (1a– Table 2 Calculated bond lengths for select TpMeZn(MBI) coordination
5a) and oxadiazolone (1d–5d) MBIs
complexes using uB97x-D/def2-TZVPP
log Pa
log D7.4
Zn–carboxylate
(A)
Zn–N
(heteroarene, A)
a
Compound
pKa
˚
˚
MBP
1a
2a
3a
4a
5a
1d
2d
3d
4d
5d
8.43
7.21
7.44
6.34
6.35
4.73
4.04
4.50
2.87
3.74
1.37
1.28
1.83
1.91
1.53
2.51
1.55
2.86
3.05
2.26
1.33
0.88
1.55
0.81
0.44
ꢁ0.16
ꢁ1.75
0.11
1
2
3
4
1.953
2.021
1.959
1.994
1.955
1.996
2.255
2.161
2.261
2.193
2.319
2.199
5
Benzoxazole-2-
carboxylic acid
ꢁ0.43
Zn–tetrazole
˚
(A)
Zn–N
(heteroarene, A)
ꢁ0.24
˚
MBI
a
All pKa and log P experiments yielded standard deviations of <0.05.
1c
2c
3c
4c
1.948
2.033
1.951
2.004
1.953
1.985
2.784
2.283
2.706
2.348
2.959
2.496
groups of the O-methyl hydroxamic ester (1b–5b) and oxadia-
zolone MBIs (1d–5d) were determined as representative sets.
Acidity was measured using a potentiometric method with or
without methanol as a cosolvent (Table 1).23 The compounds
were also evaluated for their lipophilicity (log P) via a potentio-
metric method, which was combined with the measured pKa MBI
value to determine log D7.4 (Table 1).
The physicochemical values between MBIs vary signicantly,
even in cases where the only difference between compounds is
a single atom replacement or arrangement in the heteroarene.
Comparing isomeric MBIs such as, 1a and 2a, yields an interesting
trend with the 1,3-heteroatom arrangement (2a) MBIs possessing
a substantially lower pKa value than the corresponding 1,2-isomer
(1a). Interestingly, single atom substitutions had a strong effect on
the log P values, causing a signicant shi in some cases (2d versus
4d). These observed differences were also manifest in the log D7.4
values, as they are derived from both measurements. Overall, the
pKa values are signicantly affected by the heteroatom positioning
and identity, yielding a set of substituted heteroarene MBIs that
span a range of two pKa units, suggesting the heteroarene scaffold
strongly affects the electronics of the bioisostere replacement and
vice versa. To better understand how electronic effects are inu-
enced by the positioning and identity of the heteroatoms within
these heteroarene rings, computational analysis of the coordina-
tion complexes was performed.
5c
Benzoxazole-2-tetrazole
Zn–oxadiazolone
(A)
Zn–N
(heteroarene, A)
˚
˚
1d
2d
3d
4d
1.942
2.004
1.949
1.976
1.940
1.957
2.776
2.391
2.740
2.491
2.910
2.738
5d
Benzoxazole-2-
oxadiazolone
regarding the computational methodology can be found in the
Experimental section below.
In agreement with the bond lengths determined from X-ray
crystallography (Table S6†), computations show that the
carboxylate/isostere–metal bonds of TpMeZn(MBP) complexes
are generally shorter than the heteroarene–metal (i.e., N–Zn)
bonds (Table 2). The greatest deviation between the experi-
mentally and computationally determined bond lengths is
observed when the ligating heteroarene engages in long range
interactions with the Zn (3c, 3d, 5c, and 5d). However, the
computational results recapitulated the experimental results
with respect to the observed trends in Zn–MBI bond distances,
Computational analysis
To understand the electronics of the MBIs and energetics of the with 1,3-heteroatom arrangement MBIs (e.g. 2, 2c, 4, 4c)
resulting TpPh,MeZn(MBI) model complexes, an evaluation of producing shorter Zn–heteroarene bond distances than their
each complex via density functional theory (DFT) was per- 1,2-isomers (e.g. 1, 1c, 3, 3c).
formed. All computations were performed using uB97x-D/def2-
Computed free energies (1 atm at 298 K) determined at the
TZVPP as it generally recapitulates the geometries observed uB97x-D/def2-TZVPP level of theory indicate that bidentate
experimentally.24 An added benet of performing these calcu- coordination through both the heteroarene and carboxylate
lations is to exclude the effect of noncovalent interactions (N,O) in the TpMeZn(1) complex is 4.7 kcal molꢁ1 lower in
present in the crystal structures that may inuence the observed electronic energy than isomeric bidentate coordination through
modes of coordination.
A
phenyl truncation of the only the carboxylate (O,O), whereas the analogous coordination
TpPh,MeZn(MBI) models was used to reduce computational cost, modes of the isomeric TpMeZn(5) complexes are closer to being
with the phenyl substituents present on the TpPh,Me ligand isoenergetic (DG ¼ 1.2 kcal molꢁ1) (Fig. 7). These differences in
being replaced with a hydrogen atom (i.e., TpMe). Additionally, relative stabilities can be attributed to differences in electronic
the benzoxazole scaffold (Fig. S26†) was investigated even structure of the heterocyclic ring systems. The 1,2-benzisoxazole
though it could not be accessed synthetically.14 Further details (5) contains a more electronegative oxygen atom adjacent to the
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Fig.
8
uB97x-D/def2-TZVPP-optimized geometries of: isomeric
TpMeZn(2) and TpMeZn(1) (top and bottom left, respectively), isomeric
TpMeZn(benzoxazole-2-carboxylic acid) and TpMeZn(5) (top and
bottom middle, respectively), and isomeric TpMeZn(4) and TpMeZn(5)
(top and bottom right, respectively). Energies (in kcal molꢁ1) are free
energies determined at the uB97x-D/def2-TZVPP level of theory.
TpPh,MeZn(2c) are more stable than the those that featuring an
indazole ligand (e.g., TpPh,MeZn(1c)) because through greater
electron delocalization 2c more effectively shis electron
density toward the coordinating nitrogen atom of the hetero-
arene than its isomer 1c. A smaller difference in the stability of
TpPh,MeZn(4c) and TpPh,MeZn(3c) pair of complexes is due to
both the reduced aromaticity of the benzothiazole (4) moiety
and the reduced electronegativity of sulfur.25,26 These compu-
tational data provide a rationalization for the tighter coordi-
nation, and likely better donor ability, observed for the
benzimidazole and benzothiazole MBPs/MBIs (2, 4, 2c, 4c, 2d,
4d). Furthermore, ligand binding interactions were analysed by
using computational data to determine the favourability of
isodesmic reactions (Fig. S29, ESI†). These calculations gener-
ally show that for the 1,3-heterocyclic MBPs/MBIs coordinate
more strongly than their 1,2-isomers. Interestingly, this pref-
erence does not hold when this analysis is performed on the
isomeric 1,2-benzisothiazole/1,3-benzothiazole TpMeZn(MBI)
complexes.
Fig. 7 A comparison of the structures and relative Gibbs free energies
(kcal molꢁ1) for carboxylate only (O,O) and isomeric heteroarene/
carboxylate (N,O) coordination of (A) TpMeZn(1) and (B) TpMeZn(5)
using uB97x-D/def2-TZVPP level of theory.
ligating nitrogen atom than the indazole (1), making it a poorer
electron donor than 1. Furthermore, the negative charge of the
carboxylate is more effectively withdrawn in 5 due to the greater
electron-withdrawing character of the 1,2-benzisoxazole.25
Differences in the electronic character of heteroarene ligands 1–
5, 1a–5a, 1b–5b, 1c–5c, and 1d–5d, not only modulate ligand–
metal bond lengths, but ultimately inuence the actual modes
by which these ligands coordinate metal ions.
Computational analysis of the TpPh,MeZn(MBI) complexes
with ligands 1a–5a and 1b–5b is complicated by the possibility
of multiple coordination modes (e.g., N,O or N,N coordination),
various coordination geometries, and conformational exibility
of the hydroxamate and hydroxamic methyl ester ligands. As
shown in Fig. 9, computational modelling of the phenyl trun-
cated complex TpMeZn(1a), reveals the crystallographically
observed mode of coordination is the lowest energy mode of
coordination predicted computationally. Because there were
many potential modes of coordination with TpPh,MeZn(MBI)
complexes with ligands 1a–5a and 1b–5b, geometry optimiza-
tions were performed on the complete, phenyl-substituted
TpPh,MeZn(1a) complex. The inclusion of the phenyl substitu-
ents also correctly predicts that the preferred coordination
geometry involving heteroarene coordination via the nitrogen
atom and the oxygen atom of 1a. Interestingly, these calcula-
tions show that the phenyl substituents reduce the energetic
differences between the various possible modes of coordination
Theory shows that regardless of the nature of the hetero-
arene substituent, heteroarenes with
a
1,3-heteroatom
arrangement (2, 4, and benzoxazole-2-carboxylic acid) coordi-
nate with shorter bond lengths to the Zn2+ center than do their
1,2-isomers (1, 3, and 5). In examining the isomeric pairs (1 vs.
2) the computed energy difference is largest when comparing
1,2-benzisoxazole-containing
complexes,
TpMeZn(5),
TpMeZn(5c), and TpMeZn(5d), to their benzoxazole isomers
(Fig. 8, S27 and S28†). In contrast, the corresponding energy
differences between isomeric benzothiazole- and 1,2-
benzisothiazole-ligated TpPh,MeZn(MBI) complexes (e.g.,
TpPh,MeZn(4) and TpPh,MeZn(3)) are much smaller, ranging in
energy from ꢀ4 to ꢀ9 kcal molꢁ1. These energetic preferences
can be attributed to differences in the aromaticity of the het-
eroarenes. For instance, benzimidazole-ligated complexes, like
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resulting mode of coordination to a bioinorganic model
complex. This likely dependence of the success of bioisostere
replacement on the electronic character of each scaffold
suggests a “context dependence” in isostere replacement for
MBPs. These results and analyses provided here allow for an
understanding and prediction of future MBI success. In addi-
tion, the results described here can be applied to MBP devel-
opment, particularly to other heteroarene MBP scaffolds. This
work demonstrates that MBIs are useful scaffolds for FBDD of
metalloenzymes, but the effect of bioisosteres on metal coor-
dination must be understood to successfully implement the use
of isosteres for metalloenzyme inhibition.
Experimental
General information
Starting materials and solvents were purchased and used
without further purication from commercial suppliers (Sigma-
Aldrich, Alfa Aesar, EMD, TCI, etc.). Detailed synthetic routes for
each MBI are provided in the ESI.† [(TpPh,Me)ZnOH] (TpPh,Me
hydrotris(5,3-methylphenylpyrazolyl)borate) was synthesized as
reported using [KTpPh,Me], which was prepared as previously
reported.27
¼
General synthesis and crystallization of TpPh,MeZn(MBI)
model complexes
[(TpPh,Me)ZnOH] (60 mg, 0.11 mmol) was dissolved in 15 mL of
CH2Cl2 in a 50 mL round-bottom ask. The MBP/MBI
(0.11 mmol, 1 equiv.) in 10 mL of MeOH was added, and the
reaction mixture was stirred overnight under a nitrogen atmo-
sphere (note: in a select few cases, to conserve material, the
amounts of [(TpPh,Me)ZnOH] and MBI were halved while
keeping solvent volumes constant). The resulting mixture was
evaporated to dryness via rotary evaporation and subsequently
a minimal amount (ꢀ5 mL) of benzene was added, if that was
insufficient to dissolve the residue a minimal amount of MeOH
was added (ꢀ1–3 mL). The solution was ltered to remove any
undissolved solids. The resulting complex in benzene or
benzene/MeOH was recrystallized using vapor diffusion with
pentane; crystals typically formed within one week.
Fig. 9 A comparison of various coordination modes of phenyl-trun-
cated TpMeZn(1a) (left) and phenyl substituted TpPh,MeZn(1a) (right)
complexes. Relative Gibbs free energies, in kcal molꢁ1, determined
using uB97x-D/def2-TZVPP level of theory are reported below each
structure.
by ꢀ1 kcal molꢁ1, relative to the TpMeZn(1a) results. Overall,
these computational analyses suggest that while the phenyl
groups contribute slightly to the observed modes of coordina-
tion, the major driving force behind MBI coordination is ligand
electronics.
Physicochemical properties analysis
Physicochemical properties were determined using a Sirius T3
instrument. All titrations, both pKa and log P, were performed
in 0.15 M KCl with 0.5 M HCl and KOH. The pKa of a compound
was determined by analysing each MBI sample in triplicate
Conclusions
The development of a series of MBIs based on a diverse range of using potentiometric titrations.23 Experiments were typically
heteroarene scaffolds was shown to produce an interesting performed over a pH range of 2.0–12.0. Standard deviations
range of expected, as well as unexpected, coordination modes. were derived from tting all three replicate experiments. For
DFT calculations were able to reproduce the experimental water insoluble compounds methanol was added and the ob-
results from X-ray crystallography. From these data it was tained apparent pKa (psKa) was extrapolated to the aqueous pKa
determined that the content and arrangement of the hetero- by the Yasuda–Shedlovsky procedure.28,29 log P was determined
arene signicantly modies the coordination ability of the via potentiometric titrations in the presence of varying ratios of
resulting MBP or MBI. Specically, the heteroarene aromaticity octanol and water.30 The presence of octanol shis the pKa of
and subsequent donation ability of the heteroarene donor ionizable species, and based on the shi, a log P can be deter-
atom(s) was determined to be essential to dictating the mined. Measurements for log P determination were typically
Chem. Sci.
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performed over a pH range of 2.0–12.0. Three experiments with
varying ratios of water : octanol were performed, allowing for
a standard deviation to be determined from the tting of all
measurements. MBI sample sizes were #0.5 mg for both pKa
and log P measurements.
2 Y. Yang, X. Q. Hu, Q. S. Li, X. X. Zhang, B. F. Ruan, J. Xu and
C. Liao, Curr. Top. Med. Chem., 2016, 16, 384–396.
3 J. A. Jacobsen, J. L. Fullagar, M. T. Miller and S. M. Cohen, J.
Med. Chem., 2011, 54, 591–602.
4 C. V. Credille, Y. Chen and S. M. Cohen, J. Med. Chem., 2017,
60, 9912.
5 C. V. Credille, C. N. Morrison, R. W. Stokes, B. L. Dick,
Y. Feng, J. Sun, Y. Chen and S. M. Cohen, J. Med. Chem.,
2019, 62, 9438–9449.
6 A. Y. Chen, P. W. Thomas, A. C. Stewart, A. Bergstrom,
Z. Cheng, C. Miller, C. R. Bethel, S. H. Marshall,
C. V. Credille, C. L. Riley, R. C. Page, R. A. Bonomo,
M. W. Crowder, D. L. Tierney, W. Fast and S. M. Cohen, J.
Med. Chem., 2017, 60, 7267–7283.
7 C. Perez, J. Li, F. Parlati, M. Rouffet, Y. Ma, A. L. Mackinnon,
T. F. Chou, R. J. Deshaies and S. M. Cohen, J. Med. Chem.,
2017, 60, 1343–1361.
8 G. A. Patani and E. J. LaVoie, Chem. Rev., 1996, 96, 3147–
3176.
Computational methodology
All DFT computations were performed using Gaussian 09.31 All
geometry optimizations were performed at the uB97x-D/def2-
TZVPP level of theory.24,32,33 Frequency calculations were per-
formed to conrm that each structure was in fact a local
minimum (stationary point) on the potential energy surface. All
computed free energies were calculated assuming standard
state conditions of 1 atm in the gas phase at 298 K. Initial
coordinates for computation were prepared using Gaussview
5.0.9 and Avogadro 1.2.0.34–36 Pymol 2.3 was used to render
images of the DFT-optimized structures presented herein.37
9 H. L. Friedman, Natl. Acad. Sci., 1951, 206, 295–362.
10 T. Lassila, J. Hokkanen, S. M. Aatsinki, S. Mattila,
M. Turpeinen and A. Tolonen, Chem. Res. Toxicol., 2015,
28, 2292–2303.
11 H. Pajouhesh and G. R. Lenz, NeuroRx, 2005, 2, 541–553.
12 B. L. Dick and S. M. Cohen, Inorg. Chem., 2018, 57, 9538–
9543.
13 C. Ballatore, D. M. Huryn and A. B. Smith 3rd,
ChemMedChem, 2013, 8, 385–395.
14 I. I. F. Boogaerts and S. P. Nolan, J. Am. Chem. Soc., 2010, 132,
8858–8859.
15 N. J. Hrib, J. G. Jurcak, K. L. Burgher, P. G. Conway,
H. B. Hartman, L. L. Kerman, J. E. Roehr and A. T. Woods,
J. Med. Chem., 1994, 37, 2308–2314.
16 D. Cundy and G. Simpson, Aust. J. Chem., 1996, 49, 199–203.
17 O. Barba, G. J. Dawson, T. M. Krulle, R. J. Rowley, D. Smyth
Conflicts of interest
The authors declare the following competing nancial inter-
est(s): S. M. C. is a co-founder, has an equity interest, and
receives income as member of the Scientic Advisory Board for
Cleave Therapeutics and is a co-founder, has an equity interest,
and a member of the Scientic Advisory Board for Forge Ther-
apeutics. Both companies may potentially benet from the
research results of certain projects in the laboratory of S. M. C.
The terms of this arrangement have been reviewed and
approved by the University of California, San Diego in accor-
dance with its conict of interest policies.
Acknowledgements
Support was provided by the National Institutes of Health (R01
GM098435; R01 AI149444). B. L. D. was supported by the
National Institute of Health Molecular Biophysics Training
Grant (T32GM008326-26). Computational studies by A. P. were
supported by the National Science Foundation (MCB-1020765),
National Institute of Health (R01 GM31749 and R01
GM095970), Howard Hughes Medical Institute, National
Biomedical Computation Resource (NBCR), and NSF super-
computer centers through Prof. J. Andrew McCammon (U.C.
San Diego); we also thank Prof. McCammon for helpful
discussions. A. P. thanks M. D. Burkart for nancial support
through the National Institutes of Health (NIH GM095970). We
thank Prof. Arnold Rheingold, Dr Milan Gembicky, and Dr
Curtis Moore (U.C. San Diego) for assistance with crystallo-
graphic data collection and structure determination. We also
thank Dr Yongxuan Su for mass spectrometry sample analysis at
The Molecular Mass Spectrometry Facility at U.C. San Diego.
and
G.
H.
Thomas,
Prosidion
Limited,
UK
WO2006085118A2, 2006.
18 A. L. Gryshuk and J. Perkins, Lawrence Livermore National
Security, LLC, USA, US20110213124A1, 2011.
19 D. T. Puerta and S. M. Cohen, Inorg. Chem., 2003, 42, 3423–
3430.
20 S. M. George, J. H. Nam, G. Y. Lee, J. H. Han, B. K. Park,
C. G. Kim, D. J. Jeon and T.-M. Chung, Eur. J. Inorg. Chem.,
2016, 2016, 5539–5546.
21 J. H. Lee, E. A. Jung, G. Y. Lee, S. H. Han, B. K. Park, S. W. Lee,
S. U. Son, C. G. Kim and T.-M. Chung, ChemistrySelect, 2018,
3, 6691–6695.
22 T. Heinrich, H. P. Buchstaller, B. Cezanne, F. Rohdich,
J. Bomke, M. Friese-Hamim, M. Krier, T. Knochel,
D. Musil, B. Leuthner and F. Zenke, Bioorg. Med. Chem.
Lett., 2017, 27, 551–556.
23 D. Schonherr, U. Wollatz, D. Haznar-Garbacz, U. Hanke,
K. J. Box, R. Taylor, R. Ruiz, S. Beato, D. Becker and
W. Weitschies, Eur. J. Pharm. Biopharm., 2015, 92, 155–170.
References
1 A. Y. Chen, R. N. Adamek, B. L. Dick, C. V. Credille, 24 J. D. Chai and M. Head-Gordon, Phys. Chem. Chem. Phys.,
C. N. Morrison and S. M. Cohen, Chem. Rev., 2019, 119,
1323–1455.
2008, 10, 6615–6620.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci.

View Article Online
Chemical Science
Edge Article
25 A. R. Katritzky, K. Jug and D. C. Oniciu, Chem. Rev., 2001,
101, 1421–1450.
26 A. R. Katritzky, V. Feygelman, G. Musumarra, P. Barczynski
and M. Szafran, J. Prakt. Chem., 1990, 332, 870–884.
27 D. T. Puerta and S. M. Cohen, Inorg. Chim. Acta, 2002, 337,
459–462.
S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam,
M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev,
A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski,
R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth,
P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels,
¨
28 M. Yasuda, Bull. Chem. Soc. Jpn., 1959, 32, 429–432.
29 B. Pesce, Electrolytes: Proceedings of an International
Symposium Held in Trieste June, 1959, Pergamon, 1962.
O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and
D. J. Fox, Gaussian 09 Revision D.01, Gaussian Inc,
Pittsburg PA, 2009.
30 B. Slater, A. Mccormack, A. Avdeef and J. E. A. Comer, J. 32 F. Weigend, Phys. Chem. Chem. Phys., 2006, 8, 1057–1065.
Pharm. Sci., 1994, 83, 1280–1283.
31 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
33 F. Weigend and R. Ahlrichs, Phys. Chem. Chem. Phys., 2005,
7, 3297–3305.
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, 34 Avogadro: an open-source molecular builder and visualization
B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, tool. Version 1.2.0, http://avogadro.cc.
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, 35 R. Dennington, T. A. Keith and J. M. Millam, GaussView
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, Version 5, 2016.
R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, 36 M. D. Hanwell, D. E. Curtis, D. C. Lonie, T. Vandermeersch,
O. Kitao, H. Nakai, T. Vreven, J. J. A. Montgomery, E. Zurek and G. R. Hutchison, J. Cheminf., 2012, 4, 17.
J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, 37 PyMOL, The PyMOL Molecular Graphics System, Version 2.2
¨
Schrodinger, LLC.
E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi,
J. Normand, K. Raghavachari, A. Rendell, J. C. Burant,
Chem. Sci.
This journal is © The Royal Society of Chemistry 2020