Carborane-Containing Matrix Metalloprotease (MMP) Ligands as Candidates for Boron Neutron-Capture Therapy (BNCT)

Article abstract of DOI:10.1002/cmdc.202000470

Based on the previously reported potent and selective sulfone hydroxamate inhibitors SC-76276, SC-78080 (SD-2590), and SC-77964, potent MMP inhibitors have been designed and synthesized to append a boron-rich carborane cluster by employing click chemistry

Full text of DOI:10.1002/cmdc.202000470

Accepted Article
Title: Carborane-containing Matrix Metalloprotease (MMP) Ligands as
Candidates for Boron Neutron Capture Therapy (BNCT)
Authors: Daniel P. Becker, Marlon R. Lutz, Sebastian Flieger, Andre
Colorina, John Wozny, and Narayan S. Hosmane
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000470
Link to VoR: https://doi.org/10.1002/cmdc.202000470
01/2020

10.1002/cmdc.202000470
ChemMedChem
FULL PAPER
Carborane-containing Matrix Metalloprotease (MMP) Ligands as
Candidates for Boron Neutron Capture Therapy (BNCT)
Marlon R. Lutz, Jr.[a], Sebastian Flieger[b], Andre Colorina[c], John Wozny[d] , Narayan S. Hosmane[d],
and Daniel P. Becker[b]
[a] Dr. M. Lutz
Biosynthetic Technologies
6320 Intech Way, Indianapolis, IN 46278, (USA)
[b] S. Flieger, Prof. Dr. D Becker
Department of Chemistry and Biochemistry
Loyola University Chicago, Chicago, IL 60660, (USA)
E-mail: dbecke3@luc.edu
[c] A. Colorina
Regis Technologies, Inc.
8210 Austin Ave., Morton Grove, Illinois 60053, (USA)
[d] J. Wozny, Prof. Dr. N. Hosmane
Department of Chemistry and Biochemistry
Northern Illinois University, DeKalb, IL 60115, (USA)
Supporting information for this article is given via a link at the end of the document.
Abstract: Based on previously reported potent and selective sulfone
hydroxamate inhibitors SC-76276, SC-78080 (SD-2590), and SC-
77964, potent MMP inhibitors have been designed and synthesized
to append a boron-rich carborane cluster employing click chemistry
for targeting tumor cells that are known to upregulate gelatinases.
Docking against MMP-2 suggests binding involving the hydroxamate
zinc-binding group, key H-bonds by the sulfone moiety with the
peptide backbone residues Leu 82 and Leu 83, and a hydrophobic
interaction with the deep P1’ pocket. The more potent of the two
triazole regioisomers exhibits and IC50 of 3.7 nM versus MMP-2 and
IC50 of 46 nM versus MMP-9.
tumor-specific nor do they accumulate in tumor cells. Carboranes
including borocaptate are icosahedral boron cage molecules that
have found use in the treatment of diseases including various
cancers and rheumatoid arthritis most notably through BCNT, and
also are distinguished with
a rich and diverse synthetic
chemistry.[5] Yet even these agents have demonstrated
therapeutic efficacy in patients with high grade gliomas, recurrent
tumors of the head and neck region, and a much smaller number
with cutaneous and extra-cutaneous melanomas.[6] There is a
critical need for boron-containing therapeutics that both contain a
higher density of boron atoms with a high neutron-capture cross
section, and which are selectively taken up in tumor cells. Agents
similarly are needed for boron neutron capture synovectomy for
the treatment of severe cases of rheumatoid arthritis, which works
through the same principle and mechanism as BNCT.[5] Boron-
containing agents designed for neutron capture therapy have
recently been reviewed.[1] Boron agents have played an
increasingly important role in medical applications,[7] and recent
literature reports of BNCT agents include a membrane-permeable
boron cluster that was developed using the cell-penetrating
lipopeptide pepducin as the vehicle for intracellular delivery of
boron clusters.[8] Cellular uptake has been evaluated for
pentagamaboronon-0 (PGB-0) to treat breast cancer with
BNCT.[9] A receptor-mediated uptake of boron-rich Neuropeptide
Y analogues has recently been described for BNCT.[10] Relying on
the accumulation of folate in fast-growing cancer cells, a folate
receptor-targeted novel boron compound for BNCT has been
investigated in F98 glioma-bearing rats.[11] Very recent reports
include selectively targeting tumor cells via transportation by the
membrane transport protein LAT1[12] and the synthesis and
evaluation of tris-carborane ɑ-D-mannopyranoside derivatives
prepared using click chemistry.[13]
Introduction
Boron neutron capture therapy (BNCT) is
a binary
radiotherapeutic cancer treatment that has shown great promise
in clinical trials against glioblastoma, glioblastoma multiforme,
melanoma, malignant gliomas, and glioblastomas as reviewed
recently.[1] In BNCT, a drug containing 10B atoms is transported
into tumor cells and then irradiated with thermal neutrons.[2]
Thereupon, a 10B nucleus absorbs a neutron to form an excited
11B nucleus that undergoes decay via fission, emitting an α-
particle (4He2+) as well as a 7Li3+ ion, both with high kinetic energy.
The highly charged particles have a range of only about one cell
diameter (5-9 μm) which limits the radiation damage to the cancer
cell in which they arise, thus avoiding damage to the surrounding
healthy tissue.[3] The ultimate success of BNCT depends upon the
availability of neutron sources, and the advent of accelerator
based neutron sources (ABNS) is encouraging, as well as the
need for new and more effective boron delivery agents.[4]
Currently, the clinically investigated[1] BNCT drugs include
boronophenylalanine and sodium borocaptate that are neither
Matrix metalloproteinases (MMPs) are a family of zinc-
dependent endopeptidases that are involved in the remodeling
1
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Table 1. Previously reported potent and selective α-sulfone hydroxamate MMP inhibitors
MMP
Enzyme Inhibitory Potency, IC50 (nM)
Inhibitor
SC-76276
SC-78080
SC-77964
MMP-1
9,000
>10K
MMP-2
0.2
<0.1
<0.1
MMP-3
13.0
28.7
MMP-7
>10K
7,000
7,000
MMP-8
1.8
1.7
MMP-9
1.5
0.2
MMP-13
0.4
<0.1
0.1
4,000
22.0
1.2
0.1
and degradation of all components of the extracellular matrix
(ECM).[14] MMP enzymes play a key role in normal development,
morphogenesis, bone remodeling, wound healing, and
angiogenesis, yet inappropriately high MMP activity has been
implicated in a number of disease states including tumor growth
and metastasis and in the degradation of articular cartilage in
arthritis.[15] MMPs in particular gelatinases MMP-2 and MMP-9 are
known to be overexpressed in tumors as well as in articular
cartilage in patients suffering from rheumatoid and
osteoarthritis.[16] In order to halt disease progression resulting
from exaggerated matrix remodeling mediated by MMPs, MMP
inhibitors (MMPi's) have been extensively explored.[17] Elegant
EGFR inhibitor[18] and kinase inhibitor[19,20] target-directed BNCT
agents have recently been reported by Viñas and colleagues.
Importantly, MMP inhibitors bearing fluorescent dyes have also
been used in imaging of cancer cells by binding tightly to MMP
enzymes,[21] and a hydrogel-based delivery system to treat
glioblastoma multiforme using upregulated MMP-2 and MMP-9 to
deliver chemotherapeutics has been described.[22]
We have previously described[23,24] α-sulfone hydroxamate
MMP inhibitors that are highly potent for MMPs that are over-
expressed in tumors and are critically involved in tumor growth
and accompanying angiogenesis, and in metastasis. Clinical
candidates arising from these efforts are illustrated in Table 1
including SC-276 (SC-76276),[23] SC-78080/SD-2590, and SC-
77964,[24] shown with their respective potencies at various MMP
enzymes. These α-sulfone MMP inhibitors exhibited high levels of
selectivity versus MMP-1, which has been implicated in the
musculoskeletal syndrome (MSS) side effect in patients treated
clinically with broad-spectrum MMP inhibitors, and also exhibited
oral bioavailability and efficacy in several in vivo models of cancer
and congestive heart failure. Because the piperidine-N-
substituent is directed into solvent and is outside of the enzyme
active site, very large groups may be placed in this position
without a significant loss of potency[21] and it is this position where
we intended to install a carborane moiety. Utilizing this approach,
we now report MMP inhibitors employing the pharmacophore of
these clinical candidates that bind with high potency to gelatinase
enzymes MMP-2 and MMP-9 that are overexpressed[16] in tumors
and in arthritic tissues that should be capable of delivering a high
density of boron atoms to tumor and/or arthritic tissue, thus
moiety in SC-78080 and SC-77964 contains fluorine (19F) atoms
that will enable the use of magnetic resonance spectroscopy for
the detection and localization of fluorinated drugs in tumors, which
is essential to demonstrating the localization of drug in target
tissues.[25]
For
example,
4-10B-borono-2-18F-fluoro-l-
phenylalanine (18F-FBPA) was developed for monitoring the
pharmacokinetics of 4-10B-boronol-phenylalanine (10B-BPA) used
in BNCT with positron emission tomography (PET).[26] For these
reasons we have selected to pursue analogs that contain the
trifluoromethoxy ether linkage, in addition to the fact that these
analogs were among the most potent and selective among the ɑ-
sulfone hydroxamates that were previously described.[23,24]
Results and Discussion
The synthetic route to these carborane-bearing MMP
inhibitors for BNCT is an extension of our earlier procedures[23,24]
and the route was improved during process development. The
preparation of key intermediate
9 as the THP-protected
hydroxamate is illustrated in Scheme 1. Ethyl N-Boc-piperidine-4-
carboxylate (1) was treated with LDA under cryogenic conditions
to produce the enolate anion that subsequently reacts with
disulfide 2 to produce the sulfide adduct 3. The preparation of
disulfide 2 was accomplished by the reaction of 4-fluorothiophenol
in DMSO in markedly improved yields by isolating the disulfide at
0-5°C providing crystalline material as a low melting solid. The
sulfide adduct 3 was reported to have been oxidized to sulfone 4
using MCPBA which required chromatographic purification.
Utilization of the use of the recently-described safe, efficient, and
scalable conditions using urea hydrogen peroxide (UHP)/phthalic
anhydride system[27] in ethyl acetate enabled this oxidation to be
carried out on a 180 g scale with facile isolation of highly pure
sulfone 4 via an extractive workup without chromatography. The
N-Boc sulfone 4 was deprotected with hydrogen chloride gas in
isopropyl acetate to produce the piperidine hydrochloride salt 5,
which was then alkylated with propargyl bromide and potassium
carbonate in DMF to cleanly produce the propargylamine adduct
6 in high yield. SNAr reaction of propargylamine 6 with 4-
(trifluoromethoxy)phenol and potassium carbonate in DMF
produced the diphenyl ether adduct 7 in high yield. Alkaline
hydrolysis of ethyl ester 7 in ethanol with an excess of NaOH and
overnight heating to achieve 98% yield of ester to carboxylic acid
8. Lastly, the reaction of carboxylic acid 8 with O-(tetrahydro-2H-
enabling
a high neutron-capture cross section and binary
treatment of tumors utilizing BNCT as well as the treatment of RA
with neutron therapy. In addition, these potent inhibitors exhibit a
much lower potency at MMP-1, which has been implicated in the
muscular skeletal syndrome. The trifluoromethoxyphenyl ether
2
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Scheme 1. Synthesis of THP-protected hydroxamate 9
pyran-2-yl)hydroxylamine using EDC and HOBt with DMAP as
base in DMF afforded pure THP-hydroxamate 9 in 90% yield after
normal phase chromatographic purification.
complex mixtures. We postulate that the hydroxamate moiety was
undergoing chelation with decaborane or the corresponding
decaborane activated complex. Indeed, THP removal by
decaborane has been previously reported.[30]
Scheme 2. Preparation of TBDMS Propyl Azido Carborane 15
We therefore elected to employ [3+2] click chemistry to
attach boron clusters to the alkyne moiety of 9.[31,32] Others have
employed click chemistry to append carboranes toward the
preparation of new BNCT agents.[13,33] The preparation of the
requisite carboranyl propyl azide is shown below in Scheme 2.
Formation of 1-azido-iodopropane was accomplished based on
literature precedent[34] which entailed SN2 reaction of sodium
azide and 1-bromo-3-chloropropane in DMF providing a 95%
mass balance of 1-azido-3-chloropropane as the major product
along with 1H NMR showing a 85/15 mixture of chloro/bromo
which was taken forward without further purification. The crude 1-
azido-3-halopropane was subjected to Finkelstein conditions
using sodium iodide in acetone that generated pure 1-azido-3-
iodopropane in 81% isolated yield after filtration through a plug of
silica gel. The next reaction involved preparation of the tert-
butyldimethylsilyl (TBDMS)-carborane derivative 14 which
entailed deprotonation of commercially-available o-carborane
with n-butyllithium generating the carborane anion that
subsequently attacks TBDMSCl affording highly pure TBDMS
carborane 14 in 94% isolated yield. The last step was
accomplished using lithium hexamethyldisilazide (LiHMDS) to
generate the carboranyl anion that sequentially attacks 1-azido-
3-iodo-propane to furnish pure TBDMS propyl azido carborane 15
in 98% isolated yield.
Reactions involving alkynes and decaborane have been a
useful method of covalently attaching boron clusters to carrier
molecules[28] for a wide range of chemical including electronics,
nanomaterials, and medicine. It was envisioned that direct
reaction of the alkyne moiety of N-propargyl piperidine 9 with B-
10H12(MeCN)2[28] would furnish the corresponding closo-carborane
for BNCT after deprotection of the hydroxamate. Many sets of
conditions were evaluated including variations in temperature,
solvent, in situ generation of the B10H12(MeCN)2 complex,
stoichiometry of B10H12(MeCN)2, and catalytic addition of silver
nitrate,[29] but reactions with either THP-protected derivative 9 or
free hydroxamate 10, formed[24] by deprotection of 9 yielded
There are a number of methods of employing click
chemistry[13,32,33] coupling azides to alkynes either thermally or via
copper or ruthenium catalysis (CuAAC or RuAAC, respectively).
We initially performed a thermal click reaction to obtain both
triazole regioisomers, thus the thermal click reaction without metal
catalysis was used to prepare both the 1,4-disubstituted and 1,5-
disubstitued triazole click products for evaluation. Scheme 3
3
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Scheme 3. Thermal click reaction of propargyl piperidine 9 with carboranyl propylazide 15
outlines the preparation of the 1,4-disubstituted and 1,5-
disubstitued triazole click products was accomplished by
preparation of protected carboranyl propyl azide and subsequent
click reaction with 9 in toluene at elevated temperatures (120-
140°C). The thermal heating of an azide and an alkyne without
copper and ruthenium agents generates a mixture of 1,4-
disubstituted and 1,5-disubstituted triazole products. Executing
the click chemistry on TBDMS propyl azido carborane 15 and
propargyl piperidine 9 confirmed formation of 1,4-disubstituted
triazole and 1,5-disubstituted triazole products. Two separate
reactions were carried out using copper to facilitate the selective
formation of the 1,4-disubstiuted triazole click product and
ruthenium catalysis to promote selective formation of the 1,5-
disubstituted click product.
Carrying out the CuAAC reaction using stoichiometric
copper sulfate and excess sodium ascorbate in aqueous THF at
room temperature produced solely the 1,4-disubstituted triazole.
Performing the RuAAC reaction generated the 1,5-disubstitued
triazole with only trace amounts (<2% AUC) of the 1,4-triazole
when performing the reaction at ambient temperature.
140 °C for 19 hours performed slightly better with a total
conversion of 69% (sum of both triazole regioisomers) and
contained less starting material alkyne (22%). The formation of
presumed nido complex, based on high polarity since the nido
complex would be negatively charged, was lower at 120°C even
after 42 hours and slightly elevated at 140°C after 19 hours,
however heating the mixture for longer periods of time at 140°C
does consume more starting material alkyne, but at the expense
of forming significantly higher amount of nido-like components.
Batch preparation of the click regioisomers was executed on
a gram scale using batch-type pressure vessels at 120°C for 58
hours. HPLC analysis of the reaction after 58 hours showed ~65%
conversion (sum of click regioisomers) with ~20% unreacted
alkyne. HPLC analysis confirmed that the thermal Huisgen 1,3-
dipolar cycloaddition reaction gives 1,4-triazole and 1,5-triazole
products with a ratio of 1.6 to 1, respectively, which agrees with
Sharpless' study[35] of the component ratio of the triazole
regioisomers. Cleavage of the TBDMS group was accomplished
using 1M tetrabutylammonium fluoride (TBAF) in THF for the 1,4-
triazole regioisomer and for the 1,5-triazole regioisomer. Lastly,
deprotection of the THP protecting group was executed using 4N
HCl in 1,4-dioxane/methanol at room temperature. Once the
reaction was complete, the reaction mixture was concentrated,
redissolved in dichloromethane, and subsequently precipitated
using diethyl ether to furnish both carborane containing
regioisomers as hydrochloride salts.
Given the work to identify which component was either 1,4-
triazole or 1,5-triazole, the next phase was to optimize the thermal
click reaction in toluene as the solvent medium. The temperatures
screened for the thermal click reaction were 100°C, 120°C, and
140°C. The click reaction at 100°C is significantly slower
compared to the reactions at 120°C and 140°C. The reaction at
120°C was capable of progressing to 62% conversion (sum of
both triazole regioisomers) after 42 hours whereas the reaction at
4
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The MMP inhibitory potencies of click isomers 18 and 19 were
evaluated with IC50 data tabulated in Table 2. Gelatinases MMP-
2 and MMP-9 were selected as target enzymes because these
enzymes are upregulated in a number of tumor types. MMP-1 was
selected because its inhibition in broad-spectrum MMP inhibitors
has been suggested to be involved in causing the musculoskeletal
syndrome (MSS).[24] These data give an indication of the potency
of binding of these BNCT agents to MMP enzymes that are
upregulated in tumor cells. Deprotected alkyne 10 was assayed
as a direct comparator lacking the carborane cluster, and NNGH
(BML-205)[36] was employed as a standard. Both 1,4-triazole 18
and 1,5-triazole 19 exhibited low nM potency at MMP-2 (37 nM
and 9.8 nM) as well as at MMP-9 (46 uM and 13 nM, respectively),
while neither compound showed any significant inhibition of MMP-
1, as expected. 1,5-Triazole 19 was ~3X more potent than triazole
18. Triazole 19 was 49X less potent than propargyl piperidine 10
at MMP-2, and 43X less potent than propargyl piperidine 10 at
MMP-9. The loss in potency of the carboranes relative to the
simple propargyl piperidine 10 may be due to a physicochemical
effect such as hydrophobic collapse, nevertheless both triazoles
exhibit very good, low nM potency.
inbuilt parameters or empirical potential energy functions for
various types of boron-atoms, yet alone for structure/ligand-based
drug design.[37,38] While there is an urgent need to develop
accurate force field parameters for the entire carborane cluster, in
order for the molecular modeling/docking to work successfully,
several strategies have been implemented including substitution
of boron atoms within the carborane, with hexa-coordinated
carbon atoms defined with special force field parameters using
PyMOL, AutoDock, Glide, FlexX and Surfflex software
programs.[38-44] This strategy leads to forced simplifications that
result in approximate calculations, which have limitations in the
design of new molecules due to lack of accuracy. Nevertheless,
computational methods are often useful for predicting or
explaining experimentally observed results.[37] Carboranes can
act as a bioisosteric replacement for phenyl rings, as can
adamantane, due to their similarity in size and lipophilicity, which
has been extremely useful in medicinal chemistry.[45,46] There are
many examples of closo-carboranes having been used as
bioisosteric replacements for heteroaromatic or heteroaliphatic
rings.[47-56] Due to the complexity of substituting each individual
boron atom within the carborane cluster with carbon atoms, we
considered an alternative approach, where the entire carborane
cluster is replaced with a simple phenyl ring for molecular docking
experiments, given the similar 3-dimensional sweep volume and
high lipophilicity of both moieties.
The reported solution structure of the known hydroxamate
MMP inhibitor 20 (SC-74020, Figure 1) bound to MMP-2 (PDB
accession code: 1HOV)[57] was used as a model for the docking
experiments. Molecular models of compounds 21 and 22 were
developed using the Molecular Operating Environment (MOE)
computational suite’s Builder utility as the ionized species 21 and
22 as specified in Figure 1.
The docking results of isomers 21 and 22 suggest that both
compounds form a similar intricate web of contacts to the catalytic
zinc ion and surrounding amino acids (Figure 2A and 2B). In 1,4-
triazole 21, important coordination between oxygen atom of the
hydroxamate and the metal is observed, and supplementary
interactions are formed with side chains of His 120 and His 130,
respectively. Two hydrogen bonds are made between sulfone
moiety and Leu 83 on the protein backbone (Figure 2A). An
additional close interaction (2.4 Å) is observed between another
hydrogen atom within piperidine substituent and Ala 83 residue.
The diphenyl ether tail protrudes deeply into the S1’ subsite. The
piperidine N-substituent extends into the solvent and typically has
little or no impact on potency at MMP-2. The capacity of the P1’
pocket to accommodate such large moieties as the
Table 2. MMP-2 and MMP-9 Inhibitory Potency and
Selectivity vs. MMP-1
IC50 (nM) or % inhibition
Compounds
MMP-1
MMP-2
MMP-9
N-propargyl
piperidine
10
860 ± 48
0.24 ± 0.025
0.30 ± 0.06
1,4-triazole
18
1,5-triazole
19
>10,000
*NI at 10,000 nM
>10,000
37 ± 1.3
46 ± 4.6
9.8 ± 0.96
2.7 ± 0.51
13 ± 1.2
20% at 10,000 nM
120 ± 4.0
5.1 ± 0.65
NNGH
*NI denotes no significant Inhibition. IC50 values were determined in
duplicate. Reported[24] IC50 values for N-propargyl piperidine 10, MMP-
1 IC50 = 2,600 nM, MMP-2 IC50 = <0.1 nM, MMP-9 IC50 = 0.1 nM. NNGH
(BML-205)[36]
is
N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl
hydroxamic acid used as a standard MMP inhibitor in the assay with
reported IC50 values, MMP-1 = 220 nM, MMP-2 = 6.9 nM and MMP-9 =
2.9 nM.
Carboranes, which are icosahedral boron-rich clusters, are
exceptionally difficult to model computationally in the same way
as organic moieties due to their unique electronic properties,
different hydrogen bonding patterns, hexacoordinate carbon and
boron atoms, and different properties for ionic forms.[37] As a
consequence of its non-classical bonding interactions, molecular
modeling of carboranes is extremely difficult. Many currently
available software packages for molecular modeling, do not have
phenyloxyphenylsulfonate
is
rather
remarkable,
and
demonstrates that the pocket is both flexible and open at the
bottom. The trifluoromethoxy substituent at the terminus of the
biphenyl ring in the P1’ pocket is surrounded by the hydroxyl of
Thr 143, hydrophobic chain of Leu 137, and guanidine group of
Arg 149. Docking of hydroxamate 22 shows all of the same
interactions with MMP-2, displayed by 21, with the exception of
Figure 1. MMP inhibitor 20 (SC-74020) constructed for use as a model for docking; ionized form of model 1,4-triazole isomer 21
used in docking experiments, ionized forms of model 1,5-triazole isomer 22 used for docking experiments
5
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Figure 2. Hydrogen bonds and metal (pink sphere) coordination is depicted as dotted lines. (A) Modeled complex of 1,4-triazole
regioisomer 21 bound at the MMP-2 active site (PDB accession code: 1HOV). (B) Modeled complex of 1,5-triazole regioisomer 22
bound at the MMP-2 active site (PDB accession code: 1HOV).
generating two hydrogen bonds between the sulfone moiety and
Leu 83, as well as with Leu 82, residues (Figure 2).
0.05% formic acid in water and mobile phase B was 0.05% formic acid in
acetonitrile, and a gradient of 5 to 90% B in Mobile phase A over 5 min
was applied.
Bis-(4-Florophenyl) disulfide (2). 4-Fluorothiophenol (215.2 g, 1.679
mole) was heated in DMSO (1.4 liters) at 65°C for 8 hours. The mixture
was cooled to 15°C and this mixture was added via vacuum transfer using
a PTFE transfer line to a separate reactor equipped with an overhead
mechanical stirrer and containing 5 kg mixture of ice/USP purified water to
afford a white slurry. The slurry was allowed to stir overnight at 0-5°C. The
following day, the slurry was filtered and the wetcake solids were washed
with cold USP water (1.5 liters), and pulled dry overnight under a stream
of nitrogen to provide disulfide 2 as a white crystalline solid (207.4 g,
97.1%). mp = 50-52°C. HPLC purity: 100% AUC. The product was stored
in the refrigerator under dry nitrogen. 1H NMR (CDCl3, 400 MHz): δ 7.44
(m, 4H), 7.00 (m, 4H). 13C NMR (CDCl3, 100 MHz): δ 163.96, 161.49,
132.32, 132.29, 131.43, 131.34, 116.51, 116.29, 77.47, 77.16, 76.84.
Spectral data were consistent with reported values.[23]
Conclusion
In summary we report the synthesis of potent gelatinase (MMP-2
and MMP-9) ligands bearing isosahedral carborane clusters to
enable the delivery a high cross-section of boron atoms to tumor
cells through click attachment of the carborane moiety to the
previously-described MMP inhibitors SC-78080 and SC-77964.
The presence of fluorine (19F) atoms will enable the use of
magnetic resonance spectroscopy for the detection and
localization of these fluorinated drugs in tumors.[25] Evaluation of
in vivo efficacy of these derivatives is in progress and will be
reported in due course.
1-tert-Butyl
4-ethyl
4-(4-fluorophenylthio)piperidine-1,4-dicar-
boxylate (3). A solution of N-Boc-ethylisonipecotate (1) (150.0 g, 0.583
moles) in anhydrous THF (1.8 L) was cooled to -78°C. To this mixture was
slowly added a solution of LDA (2.0 M in THF/ethylbenzene, 350 mL, 0.700
mole) over 22 minutes such that temperature did not exceed -70°C. The
resulting mixture was allowed to warm to 0°C, then cooled to -40°C, and
subsequently treated with a solution of disulfide 2 (148.75 g, 0.583 mole)
in anhydrous THF (600 mL) dropwise over 30 minutes. The mixture was
allowed to warm to ambient temperature overnight. The following day, the
reaction was deemed complete by HPLC analysis. The reaction mixture
was quenched with a solution of acetic acid (7.7 g) in USP water (200 mL),
and concentrated under reduced pressure at 35-40°C to yield an aqueous
residue that was extracted with ethyl acetate (3 x 450 mL). The combined
organic layers were successively washed with USP water (300 mL), brine
(500 mL), dried with sodium sulfate, and concentrated under reduced
pressure to provide the crude sulfide adduct (232.4 g). Filtration through a
silica plug (1.2 kg, 60-200 micron, 5:1 ratio) eluting with ethyl
acetate/heptane provided pure sulfide 3 as a light yellow oil (192.26 g,
86.0%). HPLC purity: 99% AUC. 1H NMR (CDCl3, 400 MHz): δ 7.41 (m,
2H), 7.02 (m, 2H), 4.13 (q, 2H, J = 14.4 Hz), 3.80 (bd, 2H), 3.09 (m, 2H),
2.09 (m, 2H), 1.76 (m, 2H), 2.12-2.06 (m, 2H), 1.75 (m, 2H), 1.45 (s, 9H),
1.22 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3, 100 MHz): δ 171.72, 165.16,
162.67, 154.71, 139.17, 139.08, 125.16, 125.13, 116.12, 115.90, 79.81,
77.48, 77.16, 76.84, 61.33, 53.79, 33.17, 28.48, 14.16. Spectral data were
consistent with reported values.[23]
Experimental Section
All solvents were distilled prior to use and all reagents were used without
further purification unless otherwise noted. All synthetic reactions were
conducted under an atmosphere of nitrogen. Silica gel 60A, 40−75 μm
(200 × 400 mesh), was used for column chromatography. Aluminum-
backed silica gel 200 μm plates were used for TLC. For carborane
chemistry, TLC analysis was accomplished using glass-backed TLC plates
and visualizing with UV and 0.4% PdCl2 in 3M HCl. 1H NMR spectra were
obtained using either a 400 MHz or a 500 MHz spectrometer with
trimethylsilane (TMS) as the internal standard. 13C NMR spectra were
obtained using a 75 or 125 MHz spectrometer. 11B NMR spectra were
obtained using a 100 MHz spectrometer. NMR spectra were processed
using the Mnova NMR software program produced by Mestrelab Research.
The purity of all compounds was determined to be ≥95% unless otherwise
noted by high performance liquid chromatography (HPLC) employing a
mobile phase A = 5% acetonitrile B in water and a mobile phase B = 0.1%
TFA in acetonitrile with a gradient of 60% B increasing to 95% over 10 min,
holding at 95% B for 5 min, then returning to 60% B and holding for 5 min.
HRMS spectra were measured on a TOF instrument by electrospray
ionization (ESI). HRMS spectra were collected using a Waters Acquity I
class UPLC and Xevo G2-XS QTof mass spectrometer with Waters
Acquity BEH C18 column (1.7 µm, 2.1x50 mm). Mobile phase A was
6
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10.1002/cmdc.202000470
ChemMedChem
FULL PAPER
1-tert-Butyl 4-ethyl 4-(4-fluorophenylsulfonyl)piperidine-1,4-dicar-
boxylate (4). To a 3-necked 5 liter glass reactor equipped with an
overhead mechanical stirrer, J-Kem thermocouple, cooling bath at 15-
20°C , nitrogen inlet, and an additional funnel, was charged with ethyl
acetate (1.5 liters), urea hydrogen peroxide (UHP, 132.61 g, 1.41 moles),
and sulfide 3 (180.00 g, 0.4694 moles). Solid phthalic anhydride (209.16
g, 1.41 moles) was added over 5 minutes and ethyl acetate (700 mL) was
used as a rinse. The resulting mixture was allowed to stir overnight while
the cooling bath remained in place. After 18 h, HPLC analysis showed
complete conversion to sulfone. The reaction slurry was filtered to remove
urea and phthalic acid and the filtrate was washed with a solution of
aqueous 10% sodium sulfite (1.27 L). The organic layer was digested with
aqueous 10% sodium carbonate (800 mL), washed with brine, dried with
sodium sulfate, concentrated and finally chased with heptane to provide
the sulfone 4 as an off-white solid (193.61 g, 99.3%), HPLC purity: 100%
AUC, mp 91-95°C. 1H NMR (CDCl3, 400 MHz): δ 7.81 (m, 2H), 7.25 (m,
2H), 4.20 (m, 4H), 2.63 (vbs, 2H), 2.30 (bs, 2H), 2.04 (td, 2H, J = 12.0, 4.0
Hz), 1.45 (s, 9H), 1.25 (t, 3H, J = 8.0 Hz); 13C NMR (CDCl3, 100 MHz): δ
167.66, 166.7, 165.10, 154.40, 133.26, 133.16, 131.11, 131.08, 116.41,
116.18, 80.26, 77.48, 77.16, 76.84, 72.58, 62.77, 28.41, 27.80, 13.99, 0.05.
HRMS (ESI-ToF): m/z calcd for C19H26FNNaO6S+ [M+Na]+: 438.1363,
found 438.1361. Spectral data were consistent with reported values.[23]
sulfone 6 (24.86 g, 70.34 mmol), anhydrous DMF (125 mL), milled
potassium
carbonate
(19.54
g,
141.4
mmol),
and
4-
(trifluoromethoxy)phenol (25.10 g, 140.7 mmol). The reaction was heated
at 90°C for 19 h after which time the reaction was deemed complete by
HPLC. The reaction mixture was cooled to rt and DMF was removed on a
rotary evaporator under reduced pressure at 40-60°C. The residue was
diluted with MTBE/toluene (1:1, 300 mL) and washed with 1M NaOH (2 x
250 mL), USP purified water (250 mL), brine (250 mL), dried with sodium
sulfate, filtered, and concentrated to furnish diphenyl ether 7 (36.06 g,
100% yield, yellow oil). HPLC analysis: 98.03% AUC. 1H NMR (CDCl3, 400
MHz): δ 7.76 (m, 2H), 7.27 (m, 2H), 7.10 (m, 2H), 7.06 (m, 2H), 4.23 (m,
2H), 3.25 (s, 2H), 2.90 (bs, 2H), 2.40 (bd, 2H), 2.19 (m, 5H), 1.27 (m, 3H).
13C NMR (CDCl3, 100 MHz): δ 167.0, 162.5, 153.3, 146.1, 132.8, 129.6,
123.0, 121.6, 117.3, 78.3, 77.4, 77.1, 76.7, 73.4, 72.0, 62.4, 49.3, 46.7,
28.3, 14.0. HRMS (ESI-ToF): m/z calcd for C24H25F3NO6S+ [M+H]+:
512.1355, found 512.1353.
Ethyl 1-(prop-2-ynyl)-4-(4-(4-(trifluoromethoxy)phenoxy) phenyl-sul-
fonyl)piperidine-4-carboxylate (8). To a 2-L reactor equipped with an
overhead mechanical stirrer, J-Kem thermocouple, heating mantle, reflux
condenser with nitrogen inlet, and an additional funnel, was charged with
a solution of ethyl ester 7 (28.50 g, 55.72 mmol) in EtOH/THF (1:1, 850
mL) and a solution of NaOH (22.30 g, 557.17 mmol) in USP water (425
mL). The reaction mixture was heated to 65°C and allowed to stir for 18
hours which HPLC analysis showed complete consumption of ester 7. The
mixture was allowed to cool to ambient temperature, diluted with USP
water (300 mL), and concentrated at 45-50°C on a rotary evaporator to
remove EtOH and THF. The aqueous residue was diluted with USP water
(600 mL) and the pH was adjusted to pH 2.25 using 2M HCl (282 mL)
providing a thick white slurry. The slurry was filtered, washed with USP
water (750 mL), pulled dry, and dried further in a vacuum oven at 40°C to
provide carboxylic acid 8 (26.30 g, 97.6% yield, white solid). HPLC
analysis: 94.4% AUC, mp 188-190°C (decomp.). 1H NMR (CDCl3, 400
MHz): δ 7.78 (m, 2H), 7.49 (d, 2H, J = 8.0 Hz), 7.31 (m, 2H), 7.19 (m, 2H),
3.31 (s, 2H), 3.17 (s, 1H), 2.85 (d, 2H, J = 12.0 Hz), 2.19 (d, 2H, J = 12.0
Hz), 2.11 (t, 2H, J = 12.0 Hz), 1.92 (td, 2H, J = 12.0, 4.0 Hz). 13C NMR
(CDCl3, 100 MHz): δ 167.5, 161.7, 153.3, 145.03, 145.01, 132.8, 129.2,
123.32, 122.0, 117.4, 78.5, 76.3, 71.0, 48.4, 45.7, 27.5. HRMS (ESI-ToF):
m/z calcd for C22H21F3NO6S+ [M+H]+: 484.1042, found 484.1042.
Ethyl
4-(4-fluorophenylsulfonyl)piperidine-4-carboxylate
hydro-
chloride (5). Into a solution of sulfone 4 (10.25 g, 24.7 mmol) in
isopropylacetate (iPAc, 120 mL) was added HCl gas (10.00 g) via sparge
and the reaction was allowed to stir overnight at room temperature. HPLC
analysis confirmed that the absence of starting material. The mixture was
concentrated under reduced pressure to afford piperidinium hydrochloride
salt 5 (8.72 g, 100% yield). HPLC purity: 100% AUC. mp 181-184°C. 1H
NMR (DMSO-d6, 400 MHz): δ 9.65 (bs, 1H), 9.42 (bs, 1H), 7.89 (m, 2H),
7.58 (t, 2H, J = 8.8 Hz), 4.13 (q, 2H, J = 14.4 Hz), 3.41 (d, 2H J = 13.2 Hz),
2.76 (bs, 2H), 2.32 (m, 4H), 1.10 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3, 100
MHz): δ 170.74, 167.94, 165.97, 165.36, 133.46, 133.36, 130.33, 130.30,
116.79, 116.56, 77.47, 77.15, 76.84, 70.05, 67.69, 63.58, 41.32, 25.12,
21.88, 21.50, 13.92, 0.05. HRMS (ESI-ToF): m/z calcd for C14H19FNO4S+
[M+H]+: 316.1019, found 316.1018. Spectral data were consistent with
reported values.[23]
Ethyl 4-(4-fluorophenylsulfonyl)-1-(prop-2-ynyl)piperidine-4-carbox-
ylate (6). A 2-L reactor was charged with hydrochloride salt 5 (30.00 g,
85.27 mmol), anhydrous DMF (600 mL), and potassium carbonate (23.8 g,
172.2 mmol) and the resulting mixture was stirred under mechanical
stirring for 10 minutes. Propargylamine (97% purity, 10.35 g, 87.00 mmol)
was then added and the reaction was allowed to stir for 23 h at room
temperature after which time HPLC analysis revealed 11.75% AUC of
unreacted piperidine 5 and 87.3% AUC of propargyl amine 6. The reaction
was charged with an additional portion of propargylamine (1.58 g, 1.00 mL,
8.41 mmol) and the reaction was allowed to stir for an additional 1 h at
room temperature after which time the reaction was deemed complete by
HPLC (2.3% AUC of unreacted piperidine 5 was noted along with 96.3%
AUC of propargyl amine 6. The reaction mixture was added dropwise to a
5-L reactor containing ice-cold USP purified water (2.5 L) to initiate
crystallization. The resulting slurry was stirred overnight at 0-5°C via
mechanical stirring, filtered, washed with USP water (150 mL), and pulled
dry under a stream of nitrogen to provide N-propargyl piperidine 6 (28.44
g, 94.4%) as a light beige solid, HPLC analysis: 99.81% AUC, mp 100-
102oC. 1H NMR (CDCl3, 400 MHz): δ 7.82 (m, 2H), 7.23 (m, 2H), 4.21 (q,
2H, J = 12.0 Hz), 3.26 (d, 2H, J = 4.0 Hz), 2.90 (m, 2H), 2.39 (m, 2H), 2.20
(m, 5H), 1.25 (t, 3H, J = 12.0 Hz). 13C NMR (CDCl3, 100 MHz): δ 167.5,
166.8, 165.0, 133.2, 133.1, 131.4, 116.3, 116.1, 78.2, 77.4, 77.1, 76.8,
73.5, 71.9, 62.5, 49.1, 46.6, 49.1, 46.6, 28.1, 14.0. HRMS (ESI-ToF): m/z
calcd for C17H21FNO4S+ [M+H]+: 354.1175, found 354.1176. Spectral data
were consistent with reported values.[23]
1-Prop-2-ynyl)-N-(tetrahydro-2H-pyran-2-yloxy)-4-(4-(4-(trifluoro-
methoxy)phenoxy)phenylsulfonyl)piperidine-4-carboxamide (9). To a
500-mL reactor, equipped with a large magnetic stirring bar, J-Kem
thermocouple, cooling Dewer, and nitrogen inlet, was charged with
carboxylic acid 8 (20.00 g, 41.37 mmol) and anhydrous DMF (180 mL).
The mixture was allowed to stir for 10 minutes at ambient temperature then
cooled the mixture to < 5°C. To the cooled mixture was sequentially added
O-(Tetrahydro-2H-pyran-2-yl)hydroxylamine (7.50 g, 64.02 mmol), EDCI-
HCl (10.00 g, 81.85 mmol), and DMAP (8.00 g, 65.36 mmol). The mixture
was allowed to stir overnight and gradually warmed to room temperature
while the reactor remained in the cooling bath. After 17 hours, HPLC
analysis revealed 16.1% of unreacted carboxylic acid 8. The cooling bath
was removed and the reaction mixture was allowed to stir an additional 33
hours at room temperature whereby HPLC analysis confirmed that the
reaction was deemed complete. The reaction was quenched with USP
water (5 mL) and concentrated under high vacuum at 30°C on a rotovap
evaporator to remove DMF. The residue was diluted with ethyl acetate
(350 mL) and subsequently washed with saturated aqueous sodium
bicarbonate solution (200 mL), brine (200 mL), dried with magnesium
sulfate, filtered, and concentrated under reduced pressure to provide
crude THP-hydroxamate 9 (34.31 g), which was purified by silica gel
chromatography using a glass gravity column (24 in x 3 in) and silica gel
(680 g, 60-200 micron, Silicycle) and eluted with an EtOAc/heptane
gradient (30% to 70%) to provide Compound 9 (21.78 g, 90.4%) as white
solids. HPLC analysis: 97% AUC, mp 89-91oC. MS [M+H]+ obs 583.46 m/z.
1H NMR (CDCl3, 400 MHz): δ 9.40 (s, 1H), 7.80 (dd, 2H, J = 6.9, 2.0 Hz),
7.27 (m, 2H), 7.12 (dd, 2H, J = 6.8, 2.3 Hz), 7.05 (dd, 2H, J = 6.9, 2.0 Hz),
5.00 (t, 1H, J = 2.8 Hz), 4.00 (td, 1H, J = 11.2, 2.4 Hz), 3.69 (m, 1H), 3.23
(d, 2H, J = 2.4 Hz), 2.92 (m, 2H), 2.35-2.30 (m, 3H), 2.25-2.20 (m, 4H),
Ethyl 1-(prop-2-ynyl)-4-(4-(4-(trifluoromethoxy)phenoxy)phenyl-sul-
fonyl)piperidine-4-carboxylate (7). A 500-mL reactor equipped with an
overhead mechanical stirrer, J-Kem thermocouple, heating mantle,
nitrogen inlet, and an additional funnel was charged with fluorophenyl
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000470
ChemMedChem
FULL PAPER
1.88-1.76 (m, 3H), 1.68-1.58 (m, 3H). 13C NMR (CDCl3, 100 MHz): δ 164.0,
162.8, 153.0, 146.1, 146.0, 132.6, 128.0, 123.0, 121.8, 121.7, 119.2, 117.4,
102.0, 78.6, 73.4, 70.3, 62.3, 49.0, 48.9, 46.6, 28.6, 28.4, 27.8, 25.0, 18.3.
HRMS (ESI-ToF): m/z calcd for C27H30F3N2O7S+ [M+H]+: 583.1726, found
583.1723.
trace amount of starting material and the reaction was deemed complete.
The reaction mixture was quenched with 30 mL of USP purified H2O, then
extracted with Et2O (3x30 mL). The combined organic layers were dried
with MgSO4, filtered and concentrated to give crude product 7.07 g as a
pale yellow oil. The crude oil was purified over silica gel (140 g, 60-200
micron) eluting with n-hexane and 10% Et2O/hexane to give the silyl-
protected carborane 14 (5.05 g, 93.9%) as a white crystalline solid, mp 64-
66°C. 1H NMR (CDCl3, 400 MHz): δ 3.44 (bs,1H), 2.87-1.54 (m, 10H), 1.02
(s, 9H), 0.23 (s, 6H). 11B NMR (Decoupled, 100 MHz): δ = 0.34, -1.76, -
7.02, -10.73, -12.31, -13.26. 11B NMR (Coupled, 100 MHz): δ = 1.01, -0.94,
-2.57, -6.29, -7.87, -9.99, -11.62, -12.41, -13.2, -14.26.
N-hydroxy-1-(prop-2-yn-1-yl)-4-((4-(4-(trifluoromethoxy)phenoxy)-
phenyl)sulfonyl)piperidine-4-carboxamide (10). Hydroxamate 10 was
prepared according to the method reported.[24]
1-azido-3-chloropropane (12). Safety Note on Handling Azido
Compounds. Sodium azide is extremely toxic (LC50 Inhalation = 37 mg/m3
for rats, LD50 Dermal = 20mg/kg for rabbits) and very soluble in water (>30
g/100 mL at 0°C). Sodium azide can be easily absorbed dermally and
consequently must be handled with appropriate personal protection
equipment (PPE). Sodium azide decomposes above 275°C, generating
highly reactive sodium metal. Sodium azide is not compatible with any acid
as it spontaneously forms highly explosive hydrazoic acid on contact, even
in dilute solution. Low molecular weight organic azides are potentially
explosive substances that can decompose with a slight input of external
energy (heat, friction, pressure, etc.). Although there has not been any
documented explosions on this end of work, any organic azides where the
weight attributed to the azido group exceeds 25% of the molecular weight
should be handled with significant caution. It is recommended that a blast
shield be used during synthesis and avoid of very large-scale reactions
TBDMS Propyl Azido Carborane 15. According to
a general
precedent,[34] to a dry 100 mL round bottomed flask under a nitrogen
atmosphere was added anhydrous THF (18 mL) and 1M LiHMDS (9.7 mL).
The mixture was cooled to -78°C. A solution of TBDMS carborane (2.00 g)
in anhydrous THF (10 mL) was added to the cryogenic mixture via syringe
over 5 min such that the temperature was maintained < -65°C. The
reaction mixture was allowed to stir an additional 5 minutes at -78°C then
allowed to warm to 0°C, stirred an additional 1.25 h at 0°C, and cooled to
-78°C. A solution of 1-azido-3-iodopropane (2.15 g) in anhydrous THF (12
mL) was added over 3 minutes at -78°C. The reaction was allowed reaction
to stir at -78°C for 10 minutes then allowed to warm to room temperature
and stirred an additional 1.25 hours at ambient temperature. The reaction
was cooled to 0°C, quenched with USP purified water (5 mL), concentrated
under reduced pressure, extracted with diethyl ether (2x 20mL). The
combined organic layers were dried with sodium sulfate, filtered and
concentrated under reduce pressure to give a crude yellow oil (3.13 g).
The crude oil (3.13 g) was dissolved in DCM/n-hexane (3.5 mL, 25/75, v/v)
and passed through a large silica plug (40 g) packed in DCM/n-hexane
(25/75, v/v). The silica plug was flushed with DCM/n-hexane (200 mL,
25/75, v/v) to collect eight fractions (each about 10-15 mL). Fractions 2-6
were combined and concentrated to give carborane azide 15 (2.58 g,
97.7%) as a white solid, mp 41-43°C. 1H NMR (CDCl3, 400 MHz): δ 3.32
(t, 2H), 3.15-1.5 (m, 14H), 1.07 (s, 9H), 0.34 (s, 6H). 13C NMR (CDCl3, 20
MHz): δ 80.6, 76.5, 50.9, 35.3, 29.7, 27.7, 20.5, -2.3. 11B NMR (Decoupled,
100 MHz): δ = 0.29, -3.76, -7.29, -10.18. 11B NMR (Coupled, 100 MHz): δ
= 0.99, -0.56, -3.13, -4.62, -6.57, -8.16, -9.48, -11.24.
when dealing with these substances.[58,59] According to
a general
precedent,[60] to a 250 mL round bottomed flask was added 100 mL of
anhydrous DMF and 10.02 g of 1-bromo-3-chloropropane and then 4.2 g
of sodium azide. The reaction was placed in an ambient temperature water
bath and stirred overnight (16 h) at room temperature. The reaction mixture
was diluted with 50 mL of Et2O and 50 mL USP purified water, stirred 2-3
minutes then separated the organic layer (top). Extracted the bottom
aqueous layer with Et2O (2x 60 mL). The combined organic layers were
washed with USP purified water (3x 50 mL), dried with sodium sulfate,
filtered and concentrated at 25-30°C under reduced pressure to give a
colorless oil (7.21g, 95% mass balance) which was taken forward without
any further purification to give a mixture of 1-azido-3-chloropropane as the
major product (81%) that contained ~15% of 1-azido-3-bromopropane,
and which was carried on as such into the next step. 1H NMR (CDCl3, 400
MHz): δ 3.64 (t, 2H, J = 8.0 Hz), 3.51 (t, 3H, J = 8.0 Hz), 2.02 (q, 2H, J =
12.0, 4.0 Hz)
1,4-Triazole 16 and 1,5-Triazole 17 via Thermal Click Reaction. To a
100 mL round bottomed flask was added carboranyl azide 15 (1.00 g, 3.30
mmol), alkyne 9 (1.54 g, 2.95 mmol) and anhydrous toluene (50 mL). The
mixture was stirred for 10 minutes at ambient temperature and the mixture
was equally portioned into six different pressure vials (ChemGlass, 40 mL)
where each vial contained 8.3-8.5 mL of mixture. Each mixture was purged
with argon, placed on a heating block at 120°C, and allowed to stir at
120°C for 58 hours. HPLC analysis of each reaction vial indicated that
there was ~20% unreacted alkyne and the reaction was taken forward at
this point to avoid degradation of product and introduction of impurities that
would be difficult to remove downstream. All six reactions after cooling to
room temperature were combined and concentrated under reduced
pressure to provide a crude oil (2.76 g). The crude oil was dissolved in
DCM (30 mL), treated with silica gel (6.9 g, 60-200 micron, Silicycle), and
concentrated to dryness to give dry-loaded material on silica which was
placed and packed in a 65 g solid loading cartridge. An 80 g RediSep Rf
Gold silica gel column cartridge was equilibrated with ethyl acetate/n-
hexane (40/60, 2CV), 100% ethyl acetate (1CV), and ethyl acetate/n-
hexane (40/60, 2CV). The purification was accomplished using an ethyl
acetate/n-hexane step-gradient from ethyl acetate/n-hexane (40/60) to
100% ethyl acetate. The elution of components were in the following order:
recovered unreacted starting material 9, 1,5-triazole 17, and lastly 1,4-
triazole 16. Appropriate fractions were combined and concentrated to
isolate recovered starting material (compound 9, 0.36 g, colorless oil, TLC
Rf = 0.51 using 100% EtOAc), 1,5-click product (colorless glass solid, 0.60
g, 32.1% yield, corrected yield for recovered SM, TLC Rf = 0.35 using
100% EtOAc), and 1,4-click product (white solid, 0.90 g, 48.1% yield,
corrected yield for recovered SM, TLC Rf = 0.15 using 100% EtOAc).
1-Azido-3-iodopropane (13). According to the general precedent,[34] to
a 1000 mL 3-necked round bottomed flask was added 19.35 g of sodium
iodide, 7.5 g of crude 1-azido-3-chloropropane and 190 mL of acetone.
The vessel was purged with dry nitrogen, covered with aluminum foil, and
heated to 52°C. After 40 h, the reaction was allowed reaction to cool to
room temperature. The reaction mixture as a yellow slurry was filtered over
a pad of Celite, and the funnel and flask were with acetone (~100 mL),
then concentrated the yellow filtrate on a rotovap at 25-30°C to remove
acetone. After concentration, an orange-yellow residue was obtained (oily
solids, 26.2 g). Hexane (50 mL) was added to the oil/solid residue
(yellowish-orange) which changed the color to a greenish solid. The slurry
was stirred overnight at room temperature then passed over a short silica
plug (65 g) packed in n-hexane, then the plug was eluted with n-hexane to
collect fractions (each 50-70 mL). Appropriate fractions were combined
and concentrated at 25°C to give iodo azide 13 (10.77 g, 81.3%) as a
colorless oil. 1H NMR (CDCl3, 400 MHz): δ 3.44 (t, 2H), 3.25 (t, 2H), 2.04
(quintet, 2H).
TBDMS Carborane 14. According to a general procedure[61] was added
3.00 g of o-carborane, 12 mL anhydrous toluene, and 6 mL anhydrous
Et2O. Stirred at room temperature until completely dissolved then cooled
to < 5°C. Added n-BuLi solution (1.66 M, 13.2 mL) over about 5 minutes
to give a murky turbid white mixture. Removed cooling bath after 5 minutes
and allowed to store at room temperature. After 2.5 hours, solid TBDMSCl
(3.47 g) was added at room temperature as one portion which addition was
endothermic. Reaction is a murky white solution. After 22.5 h, the reaction
slurry was analyzed by TLC (80% hexane and 20% Et2O) and showed a
Characterization data for 1,4-triazole click product 16
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000470
ChemMedChem
FULL PAPER
HPLC purity: 96% AUC (retention time: 26.5 min). 1H NMR (CDCl3, 400
MHz): δ 9.38 (s, 1H), 7.80 (d ,2H, J = 8.0 Hz), 7.41 (s, 1H), 7.28 (d, 2H, J
= 8.0 Hz), 7.11 (d, 2H, J = 8.0 Hz), 7.05 (d, 2H, J = 8.0 Hz), 4.99 (s, 1H),
4.33 (t, 2H, J = 6.4 Hz), 3.99 (t, 1H, J = 10.3 Hz), 3.68 (bd, 1H), 3.59 (s,
2H), 2.93 (bs, 2H), 2.40-1.20 (m, 26H), 0.98 (s, 9H), 0.23 (s, 6H). 13C NMR
(CDCl3, 100 MHz): δ 164.1, 162.9, 152.9, 146.1, 145.3, 132.5, 128.5,
123.0, 122.3, 121.8, 117.4, 102.1, 79.8, 77.4, 77.2, 77.0, 76.7, 76.2, 70.5,
62.4, 52.9, 49.8, 49.8, 49.2, 34.9, 30.7, 28.5, 28.3, 27.8, 27.4, 24.9, 20.3,
18.24, -2.60. 11B NMR (Decoupled, 100 MHz): δ = 0.27, -3.86, -7.47, -
10.51.11B NMR (Coupled, 100 MHz): δ = 1.09, -0.59, -3.17, -4.34, -6.64, -
8.41, -9.41, -10.81. HRMS (ESI-ToF): m/z calcd for C38H61B10F3N5O7SSi+
[M+H]+: 926.4944, found 926.5007. FTIR: 2941.1, 2863.3, 2575.8, 1686.5,
1587, 1501.4, 1488.6, 1316.7, 1244.2, 1220.2, 1185.8, 1149.4, 1133.2,
1086.5, 1038.0, 944.4, 896.8, 858.2, 838.3, 752.8, 731.2, 677.0 cm-1.
temperature, TLC analysis (100% EtOAc) showed complete consumption
of starting material. The reaction mixture was concentrated to a crude oil
residue which was dissolved in ethyl acetate (2 mL) and washed with water
(1 mL, pH 7-7.5). The aqueous phase was extracted ethyl acetate (1 mL).
The combined organic layers were washed with water (pH 7-7.5, 1 mL),
dried with sodium sulfate, filtered, and concentrated under reduced
pressure to give crude product (108 mg). The crude oil (108 mg) was
dissolved in ethyl acetate (0.5 mL) and passed through a silica plug (0.27
g) in ethyl acetate. The plug was flushed with ethyl acetate. Appropriate
fractions were combined and concentrated to afford desired THP-
protected 1,4-triazole (88 mg, 78.6% yield, colorless oil) which was taken
forward without any further purification. HPLC purity: 97.4% AUC
(retention time: 24.1 min). HRMS (ESI-ToF): m/z calcd for
C32H47B10F3N5O7S+ [M+H]+: 812.4079, found 812.4131. FTIR: 2946.6,
2850.4, 2586.7, 1682.0, 1587.0, 1501.5, 1488.4, 1461.3, 1293.7, 1219.9,
1244.5, 1185.9, 1132.6, 1149.0, 1086.1, 1037.5, 1021.5, 956.2, 944.2,
896.0, 873.1, 835.1, 816.0, 754.5, 722.7, 678.9, 645.0 cm-1. To a 10 mL
vial containing THP-protected 1,4-triazole (88 mg, 0.109 mmol)) under
nitrogen atmosphere was added anhydrous 1,4-dioxane (0.9
mL)/methanol (0.1 mL) and the mixture was allowed to stir until complete
dissolution was achieved. To the solution was added 4N HCl in 1,4-
dioxane (0.14 mL) and the reaction was allowed to stir for 2 h at room
temperature where HPLC analysis revealed that the reaction was deemed
complete. The reaction mixture was concentrated under reduced pressure
at 30°C to give a crude oil. The crude oil was dissolved in dichloromethane
(1 mL) and diethyl ether (3 mL) was added to generate a white slurry. The
slurry was allowed to stir at ambient temperature for 1.5 hours, filtered, and
the filter cake was washed with diethyl ether (2 mL) and n-heptane (5 mL),
pulled dry under nitrogen, and further dried in vacuo at room temperature
to provide the title 1,4-triazole carborane hydrochloride salt 18 (60 mg,
72.2%) as a white solid. HPLC purity: 97.4% AUC (retention time: 10.4
min), mp 206oC (decomp.). 1H NMR (DMSO-d6, 500 MHz): δ 11.18 (bs,
0.6H), 10.90 (bs, 0.4H), 9.33 (bs, 0.5H), 8.27 (bs, 0.7H), 7.75 (d, 2H, J =
10.0 Hz), 7.48 (d, 2H, J = 10.0 Hz), 7.32 (d, 2H, J = 10.0 Hz), 7.19 (d, 2H,
J = 10.0 Hz), 5.25 (bs, 1H), 4.39 (t, 4H), 3.47 (vbs, 2H), 3.16 (m, 2H), 3.01-
2.52 (complex, 3H), 2.30-1.40 (complex, 17H). 13C NMR (DMSO-d6, 125
MHz): δ 163.0, 161.9, 153.4, 145.0, 132.9, 123.3, 121.9, 121.1, 119.0,
117.8, 75.5, 63.2, 57.5, 57.5, 57.5, 51.6, 48.4, 33.4, 29.5, 24.9, 23.1, 19.4,
19.2, 13.5. HRMS (ESI-ToF): m/z calcd for C27H39B10F3N5O6S+ [M+H]+:
728.3504, found 728.3540. 11B NMR (Decoupled, 100 MHz): δ = 18.56, -
2.94, -6.09, -9.67, -12.20, -13.17. 11B NMR (Coupled, 100 MHz): δ = 18.56,
-2.15, -3.64, -5.21, -6.32, -8.91, -11.08, -12.95, -13.86.
Characterization data for 1,5-triazole click product 17
HPLC purity: 95% AUC (retention time: 27.4 min). 1H NMR (CDCl3, 400
MHz): δ 9.43 (s, 1H), 7.80 (d, 2H, J = 12.0 Hz), 7.49 (s, 1H), 7.28 (d, 3H,
J = 8.0 Hz), 7.11 (d, 2H, J = 8.0Hz), 7.05 (d, 2H, J = 8.0 Hz), 5.01 (bs, 1H),
4.39 (t, 2H, J = 8.0 Hz), 3.99 (t, 1H, J = 8.0 Hz), 3.70 (d, 1H, J = 8.0 Hz),
3.49 (s, 2H), 2.80 (t, 2H, J = 8.0 Hz), 1.58-2.30 (c, 24H), 1.25 (s, 1H), 1.00
(s, 9H). 13C NMR (CDCl3, 100 MHz): δ 164.16, 162.91, 152.91, 1461,
134.74, 132.63, 132.42, 127.82, 123.03, 121.80, 121.71, 117.50, 101.87,
80.13, 77.23, 76.35, 70.15, 62.30, 50.20, 49.93, 49.74, 47.42, 35.04, 30.11,
28.30, 28.10, 27.77, 27.43, 24.92, 20.32, 18.12, -2.59. 11B NMR
(Decoupled, 100 MHz): δ = 0.37, -3.85, -7.43, -10.47. 11B NMR (Coupled,
100 MHz): δ = 0.94, -0.47, -4.55, -6.70, -8.43, -10.83. HRMS (ESI-ToF):
m/z calcd for C38H61B10F3N5O7SSi+ [M+H]+: 926.4944, found 926.4999.
FTIR: 2939.8, 2869.6, 2579.3, 1685.9, 1586.8, 15014, 1488.7, 1319.0,
1295.5, 1220.0, 1243.6, 1185.7, 1149.9, 1134.3, 1086.8, 1037.2, 957.7,
944.6, 896.9, 873.3, 858.7, 838.3, 818.4, 732.1, 677.3 cm-1
Preparation of 1,4-triazole 16 via CuAAC-Catalyzed Click Reaction. To
a 100 mL round bottomed flask was added alkyne (690 mg, 1.18 mmole),
azide (400 mg, 1.18 mmol), magnetic stir bar, and THF (30 mL). A freshly
prepared solution of copper sulfate pentahydrate (303 mg, 1.21 mmol) and
sodium ascorbate (234 mg, 1.18 mmol) in deionized water (30 mL) was
added to the organic mixture over 4 minutes, and during the addition of
aqueous solution, the mixture was sparged with nitrogen. The reaction was
stirred for 12 hours at room temperature which TLC analysis (eluting with
100% EtOAc on silica gel) indicated full consumption of alkyne starting
material. The reaction mixture was concentrated under reduced pressure
and the residue was dissolved into ethyl acetate (40 mL). The organic layer
was washed with 10% ammonium chloride (40 mL), brine (40 mL), dried
with sodium sulfate, filtered, and concentrated to provide crude 1,4-triazole
click product. The crude product was dissolved in 5 mL of ethyl acetate
and loaded onto two separate preparative thin layer chromatography
plates (20 x 20 cm) and eluting with 100% ethyl acetate to give purified
1,4-triazole click product 16 (650 mg, 59.6% yield) as a pale green solid.
1,5-Triazole Hydroxamate HCl Salt 19. To TBDMS-protected 1,5-triazole
17 (71 mg, 0.077 mmol) was added anhydrous THF (0.9 mL) and the
resulting mixture was cooled solution to –78⁰C. To the cryogenic mixture
was added a solution of 1M TBAF in THF (90 µL). After 5 minutes, the
cooling bath was removed and then the reaction was allowed to warm to
room temperature. After 75 minutes at room temperature, TLC analysis
(100% EtOAc) showed complete consumption of starting material. The
reaction mixture was concentrated to a crude oil residue which was
dissolved in ethyl acetate (2 mL) and washed with water (1 mL, pH 7-7.5).
The aqueous phase was extracted ethyl acetate (2 mL). The combined
organic layers were washed with water (pH 7-7.5, 1 mL), dried with sodium
sulfate, filtered, and concentrated under reduced pressure to give crude
desired product (82 mg) which was taken forward without any further
purification. HPLC purity: 95.2% AUC (retention time: 26.4 min). HRMS
(ESI-ToF): m/z calcd for C32H47B10F3N5O7S+ [M+H]+: 812.4079, found
812.4139. To a 10 mL vial containing crude THP-protected 1,5-triazole
from the desilylation step (82 mg) under a nitrogen atmosphere was added
anhydrous 1,4-dioxane (0.9 mL)/methanol (0.1 mL) and allowed mixture to
stir until complete dissolution was achieved. To the solution was added 4N
HCl in 1,4-dioxane (0.14 mL) and the reaction was allowed to stir for 2.5
hours at room temperature where HPLC analysis revealed that the
reaction was deemed complete. The reaction mixture was concentrated
under reduced pressure at 30 + 5°C to give a crude oil. The crude oil was
dissolved in dichloromethane (0.5 mL) and diethyl ether (3 mL) was slowly
added to generate a white slurry. The slurry was allowed to stir at ambient
temperature for 20 min, filtered, and the filter cake was washed with diethyl
Preparation of 1,5-triazole 17 via RuAAC-Catalyzed Click Reaction.
To a 20 mL round bottomed flask was added alkyne (750 mg, 1.29 mmol),
carboranyl azide 15 (460 mg, 1.35 mmol), Cp*RuCl(cod) (65 mg, 0.17
mmol), magnetic stir bar, and THF (13 mL). The mixture was sparged with
nitrogen for 5 minutes, and then allowed to stir at room temperature for 24
hours after which TLC and HPLC analyses showed complete consumption
of alkyne starting material. The reaction mixture was concentrated under
reduced pressure to give crude product. The crude product was dissolved
in ethyl acetate (6 mL) and loaded onto three separate preparative thin
layer chromatography plates (20 x 20 cm) and eluting with 100% ethyl
acetate to give purified 1,5-triazole 17 (665 mg, 55.9% yield) as a light
beige solid.
1,4-Triazole Hydroxamate 18. To TBDMS-protected 1,4-triazole 16 (128
mg, 0.139 mmol) was added anhydrous THF (1.25 mL) and the resulting
mixture was cooled solution to –78°C. To the cryogenic mixture was added
a solution of 1M TBAF in THF (0.17 mL) over approximately 30 seconds.
After 5 minutes, the cooling bath was removed and then the reaction was
permitted to warm to room temperature. After 30 minutes at room
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000470
ChemMedChem
FULL PAPER
ether (2.5 mL) and n-hexane (5 mL), concentrated to dryness under
nitrogen, and further dried in vacuo at room temperature to provide the title
1,5-triazole carboranyl hydrochloride salt 19 (58 mg, 99%) as a white solid.
HPLC purity: 99.1% AUC (retention time: 10.9 min), mp 203°C (decomp.).
1H NMR (DMSO-d6, 500 MHz): δ 11.28-11.20 (complex, 1.8H), 9.38 (bs,
0.6H), 7.98 (bs, 0.8H), 7.75 (d, 2H, J = 10.0 Hz), 7.48 (d, 2H, J = 10.0 Hz),
7.33 (d, 2H, J = 10.0 Hz), 7.19 (d, 2H, J = 10.0 Hz), 5.25 (s, 1H), 4.70-4.40
(complex, 4H), 4.20-3.20 (complex, 6H), 3.16 (m, 0.6H), 3.05-2.70
response curve generated with the equation Y=B+(T-B)/1+10((LogEC50-X)×Hill
Slope), where Y=percent activity, B=minimum percent activity, T=maximum
percent activity, X= logarithm of compound and Hill Slope=slope factor or
Hill coefficient. The IC50 value was determined by the concentration
causing a half-maximal percent activity.
Acknowledgements
(complex, 2H), 2.45-2.18 (complex, 7H), 2.10-1.80 (6H), 1.57 (m, 2H). 13
C
NMR (DMSO-d6, 125 MHz): δ 163.0, 161.8, 153.4, 145.0, 132.9, 123.3,
121.8, 121.1, 119.1, 117.8, 75.6, 62.9, 57.5, 57.5, 57.5, 48.4, 46.7, 46.6,
46.6, 46.6, 46.6, 33.5, 29.4, 23.1, 19.2, 13.5. HRMS (ESI-ToF): m/z calcd
for C27H39B10F3N5O6S+ [M+H]+: 728.3504, found 728.3538. 11B NMR
We thank Loyola University Chicago and Northern Illinois
University for support of this work.
(Decoupled, 100 MHz): δ = 18.55, -2.90, -6.26, -9.74, -12.24, -13.14. 11
B
Keywords: matrix metalloproteinase, MMP, boron neutron
NMR (Coupled, 100 MHz): δ = 18.58, -2.13, -3.71, -8.99, -11.11, -12.88.
capture therapy, BNCT, carborane
Molecular Docking Protocol. Molecular models of 21 and 22 were
developed using the Molecular Operating Environment (MOE)
computational suite’s Builder utility followed by minimization in the gas
phase using the MMFF94X force field. The solution structure of compound
20 bound to MMP-2 was then uploaded into MOE and prepared for docking
using MOE’s Structure Preparation utility. The hydrogen-bonding network
of the docking model was further optimized at pH of 7.4 by automatically
sampling different tautomer/protomer states using Protonate3D, which
calculates optimal protonation states, including titration, rotamer, and “flips”
using a large-scale combinatorial search. The binding pocket of MMP-2
was surveyed using MOE’s Site Finder utility, which employs an alpha
shape construction algorithm to identify regions of tight atomic packing in
which are filled inactivated dummy atoms that ligand docking can be
directed to. Following the preparation of the MMP-2 docking model and
inactivation of other atoms, molecular docking using the previously
generated ligand conformation database was carried out at the docking
site specified by the dummy atoms populating the binding pocket of MMP-
2 of the docking active site. Ligand placement employed the Alpha Triangle
method with Affinity dG scoring to generate 100 data points per unique
ligand that were further refined using the Induced Fit method with
GBVI/WSA dG scoring to obtain the top 50 docking poses per ligand. The
Amber12:EHT force field was used to perform these calculations.
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Enzyme Assays. Enzyme assays were performed under contract by BPS
Bioscience of San Diego, CA. For MMP-1, MMP-2, and MMP-9, the
substrate employed was 390 MMP FRET and was purchased from
Anaspec. NNGH was purchased from Enzo Life science. Enzymes
activator APMA (p-aminophenylmercuric acetate) was purchased from
Millipore. MMP-1, MMP-2, and MMP-9 enzymes were purchased from
Sigma. For MMP-1, 6 ng of enzyme was employed with 5 uM 390 MMP
Substrate. For MMP-2, 0.7 ng of enzyme was employed with 2 uM 390
MMP Substrate. For MMP-9, 0.5 ng of enzyme was employed with 1 uM
390 MMP Substrate. A series of dilutions of each compound was prepared
with 10% DMSO and 5 µl of the dilution was added to a 50 µl reaction so
that the final concentration of DMSO was 1% in all of reactions.
The enzymes were diluted in 50 mM HEPES buffer pH 7.4, 10 mM CaCl2,
0.05% Brij-35, and 1 mM APMA for activation at 37°C for 2 hours. The
enzymatic reactions were conducted in duplicate at room temperature for
30 minutes in a 50 µl mixture containing 50 mM HEPES buffer, pH7.4,
10mM CaCl2, 0.05% Brij-35, the MMP substrate, MMP enzyme, and a test
compound. Fluorescence intensity was measured at an excitation of 328
nm and an emission of 393 nm using a Tecan Infinite M1000 microplate
reader. Phosphatase activity assays were performed in duplicate at each
concentration. The fluorescent intensity data were analyzed using the
computer software, Graphpad Prism. In the absence of the compound, the
fluorescent intensity (Ft) in each data set was defined as 100% activity. In
the absence of enzyme, the fluorescent intensity (Fb) in each data set was
defined as 0% activity. The percent activity in the presence of each
compound was calculated according to the following equation: %activity =
(F-Fb)/(Ft-Fb), where F= the fluorescent intensity in the presence of the
compound.
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ChemMedChem
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10.1002/cmdc.202000470
ChemMedChem
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Potent gelatinase (MMP-2 and MMP-9) ligands that spare MMP-1 have been prepared for use in BNCT based on previously reported
MMP inhibitors SC-78080/SD-2590 and SC-77964. Click chemistry was employed to install the carborane moiety yielding two triazole
regioisomers. Molecular docking versus MMP-2 using MOE was performed with phenyl as a bioisosteres for the carborane moiety
suggesting active site binding similar to the SC-model compounds.
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