Hydroxamate-Based Selective Macrophage Elastase (MMP-12) Inhibitors and Radiotracers for Molecular Imaging

Article abstract of DOI:10.1021/acs.jmedchem.0c01514

Macrophage elastase [matrix metalloproteinase (MMP)-12] is the most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-targeted imaging may predict AAA progression and rupture risk. Here, we report the design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12 selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99mTc-radiotracers, 99mTc-AGA-1 and 99mTc-AGA-2, derived from AGA. 99mTc-AGA-2 displayed faster blood clearance in mice and better radiochemical stability compared to 99mTc-AGA-1. Based on this, 99mTc-AGA-2 was chosen as the lead tracer and tested in murine AAA. 99mTc-AGA-2 uptake detected by autoradiography was significantly higher in AAA compared to normal aortic regions. Specific binding of the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly, this study introduces a novel family of selective MMP-12 inhibitors and tracers, paving the way for further development of these agents as therapeutic and imaging agents.

Full text of DOI:10.1021/acs.jmedchem.0c01514

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Article
Hydroxamate-Based Selective Macrophage Elastase (MMP-12)
Inhibitors and Radiotracers for Molecular Imaging
Kiran Gona, Jakub Toczek, Yunpeng Ye, Nowshin Sanzida, Arvene Golbazi, Parnaz Boodagh,
Mani Salarian, Jae-Joon Jung, Saranya Rajendran, Gunjan Kukreja, Terence L. Wu, Laurent Devel,
and Mehran M. Sadeghi*
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ABSTRACT: Macrophage elastase [matrix metalloproteinase (MMP)-12] is the
most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-
targeted imaging may predict AAA progression and rupture risk. Here, we report the
design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-
12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12
selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99mTc-
radiotracers, 99mTc-AGA-1 and 99mTc-AGA-2, derived from AGA. 99mTc-AGA-2
displayed faster blood clearance in mice and better radiochemical stability compared
to 99mTc-AGA-1. Based on this, 99mTc-AGA-2 was chosen as the lead tracer and
tested in murine AAA. 99mTc-AGA-2 uptake detected by autoradiography was
significantly higher in AAA compared to normal aortic regions. Specific binding of
the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly,
this study introduces a novel family of selective MMP-12 inhibitors and tracers,
paving the way for further development of these agents as therapeutic and imaging
agents.
effects, possibly related to their lack of selectivity and the
complexity of their actions.19 These drawbacks can be
potentially overcome by developing more selective MMP
inhibitors.
MMP-12 or macrophage elastase, a 55 kDa protein best
known for its elastase activity, is produced by macrophages and
a number of other cell types.23 MMP-12 is implicated in the
pathogenesis of abdominal aortic aneurysm (AAA). While
normal arteries express low levels of MMP-12, it is highly
upregulated in aneurysmal arteries in humans and animal
models of AAA.24−26 Accordingly, selective MMP-12 targeting
may be a potential therapeutic, as well as molecular imaging
target in this setting.23,25,27,28
Several hydroxamate-based succinic acid derivatives with α,
P1′, P2′, and P3′ substituents have been developed as
panMMP inhibitors, where the hydroxamate group acts as a
zinc binding group (ZBG).22,29−33 The structure−activity
relationship experiments performed on these series but also
those on other series of MMPIs have elucidated the structural
INTRODUCTION
■
Matrix metalloproteinases (MMPs) are calcium-dependent
zinc endopeptidases that belong to the family of metzincins.
These enzymes are capable of degrading different components
of the extracellular matrix (ECM),1 the regulated breakdown of
which is essential for many biological processes, including
tissue remodeling and resorption.2−4 Dysregulated ECM
degradation contributes to a wide range of diseases such as
cancer, arthritis, nephritis, heart failure, atherosclerosis, and
aneurysm.5−12 MMPs also participate in pathophysiological
processes by regulating the release or activation of chemokines,
cytokines, growth factors, and other bioactive molecules.13−15
Typically, MMPs are secreted as inactive zymogens and require
proteolytic activation by tissue or plasma proteinases to
become active. MMP activity is also regulated by endogenous
regulators called tissue inhibitors of metalloproteinases. Owing
to their important role, MMPs are considered as promising
targets for the development of new therapeutic and diagnostic
tools. Based on this, several broad-spectrum MMP inhibitors
(MMPIs) have been developed as therapeutic agents.6,16−19 In
addition, single-photon emission computed tomography
(SPECT) and positron emission tomography (PET) tracers
are under development and evaluation in preclinical animal
models for molecular imaging of cardiovascular and other
diseases.20−22 Several MMPIs have failed as therapeutic agents
in late-phase clinical trials, mainly due to musculoskeletal side
Received: September 26, 2020
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Figure 1. Chemical structures of the panMMP inhibitor RYM (A) and novel cyclic MMP-12 inhibitors CGA-1 (B) and CGA (C), as well as the
novel acyclic MMP-12 inhibitor AGA (D), radiolabeling precursors AGA-1 (E) and AGA-2 (F), and their corresponding 99mTc-labeled SPECT
imaging agents 99mTc-AGA-1 (G) and 99mTc-AGA-2 (H). The key structural components of AGA and the structure−activity relationship used to
develop novel selective MMP-12 inhibitors and SPECT imaging agents are shown in (I).
requirements to achieve potency and selectivity. In this respect,
the hydroxamate function as a strong ZBG mainly drives
MMPI potency, while the side chain at the P1′ segment, which
interacts within the hydrophobic S1′ pocket, allows the
modulation of their selectivity, with significant differences
when the length and flexibility of this side chain are modified.
The impact of P1′ on MMPI selectivity is even more
pronounced when weaker ZGBs than the hydroxamate moiety
are used.34 Similarly, substituents at α, P2′, and P3′ positions
have often minimal effects in hydroxamate series but have
more impact when other ZBGs are employed. Accordingly,
two glutamate residues in P2′ and P3′ positions within a
pseudo-peptide scaffold containing a phosphinic function as a
weak ZBG and clearly drive the inhibitor selectivity for MMP-
12.35,36 However, our recent studies on the complex role of the
ZBG36 clearly showed that subtle modulations between the
latter and the different substituents along the pseudo-peptide
backbone have to be carefully considered to achieve selectivity,
not excluding the possibility to identify selective binders in the
hydroxamate series.
considerable selectivity over other metalloproteinases. Anti-
succinate-based acyclic or cyclic hydroxamate analogues have
been extensively studied in the literature as panMMP
inhibitors and their chemistry is well established. While
keeping the hydrophobic P1′ fragment unchanged on the
anti-succinate hydroxamate core, cyclizing the α and P2′
positions, and modifying the P3′ segment with a free acid side
chain (CGA and CGA-1, Figure 1), we observed selective
binding toward MMP-12 when compared with several other
MMPs. Keeping in mind the challenges involved in preparing
these multistep, multichiral cyclic analogues, a similar method-
ology (keeping a free acid side chain) was used to prepare an
easily accessible succinyl hydroxamate-based acyclic analogue
(AGA, Figure 1), which also displayed selective MMP-12
inhibition. Two precursors for 99mTc conjugation (AGA-1 and
AGA-2, Figure 1) developed based on AGA retained MMP-12
selectivity. The corresponding single-photon computed
tomography (SPECT) tracers (99mTc-AGA-1 and 99mTc-
AGA-2, Figure 1) were synthesized and their radiochemical
stability, ex vivo binding, pharmacokinetics, and biodistribution
were evaluated and compared. Finally, the tracer displaying the
best performance, 99mTc-AGA-2, was tested in a murine model
of AAA in which MMP-12 is upregulated.
Given the role of MMP-12 in AAA and other pathological
processes, we have sought to develop selective MMP-12
inhibitors and their corresponding radiotracers with a
B
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a
Scheme 1. Synthetic Scheme for the Preparation of CGA and CGA-1
a
Reagents and conditions: i. BnBr/DBU/toluene/RT/2 h and 60 °C/1 h/71% yield; ii. (a) 9-BBN/THF; (b) H2O2/H2O/RT/12 h/70% yield; iii.
CBr4/Ph3P/DCM/RT/63% yield; iv. Pd/C (10%)/H2/CH3OH/RT/30 min/85% yield; v. H-Tyr-OBz/EDCI/HOBT/DMF/RT/12 h/74% yield;
vi. Cs2CO3/acetonitrile/RT/6 h/55% yield; vii. TFA/DCM/RT/2 h/100% yield; viii. Bz-ONH2/HOAT/HATU/DIPEA/DMF/RT/18 h/62%
yield; ix. Pd/C (10%)/H2/CH3OH/RT/2 h/58% yield; x. Glu-di-t-butyl ester/EDCI/HOBT/DMF/RT/18 h/25% yield; xi. TFA/DCM/RT/2 h/
8% yield.
hydrolysis of 13 using a 30% hydrogen peroxide solution and
LiOH-H2O in THF/H2O at 0 °C resulted in 14. L-
Phenylalanine-OBz ester was coupled with intermediate 14
using EDCI and HOBT in DMF to obtain 15. The t-butyl
group of intermediate 15 was hydrolyzed using TFA in DCM
to result in 16. The acid obtained was coupled with O-benzyl
hydroxylamine hydrochloride using EDCI and HOBT in DMF
to result in intermediate 17. Both benzyl groups on 17 were
deprotected using 10% Pd/C and hydrogen gas in methanol to
obtain AGA (18).
The Fmoc-L-glutamate benzylester was coupled with N-Boc-
ethylenediamine using EDCI and HOBT in DMF to obtain 19.
Deprotection of the Boc-protecting group using TFA and
DCM resulted in 20, which was coupled with 6-Boc-
hydrazinonicotinic acid (Boc-HYNIC acid) to obtain 21.
The Fmoc group was deprotected using piperidine in DCM to
obtain intermediate 22, which was coupled with intermediate
18 using EDCI and HOBT in DMF to obtain 23. The benzyl
ester was hydrolyzed using 10% Pd/C and hydrogen gas in
methanol and deprotection of N-Boc using TFA in DCM
resulted in AGA-1 (24).
The synthetic strategy used for the preparation of AGA-2 is
shown in Scheme 3. D-Tyrosine alanine O-benzyl ester was
coupled with the intermediate 14 using EDCI and HOBT in
DMF to obtain 25. The t-butyl group of 25 was hydrolyzed
using TFA in DCM and led to 26. The resulting acid was
coupled with O-benzyl hydroxylamine hydrochloride using
EDCI and HOBT in DMF to access 27. N-Boc-protected
ethanolamine was coupled with 27 using Cs2CO3 in DMF to
obtain 28. The N-Boc group was deprotected using TFA in
DCM to obtain 29, which was coupled with N-Boc-protected
HYNIC acid using HATU and HOAT in DMF to obtain 30.
The N-Boc group was deprotected using TFA in DCM to
obtain 31, which following deprotection of di-benzyl groups
using 10% Pd/C under hydrogen gas, resulted in AGA-2 (32).
MMP Affinity Determination. The inhibition constants
(Ki) of CGA, CGA-1, and AGA, as well as the radiolabeling
precursors AGA-1 and AGA-2 were evaluated in comparison
RESULTS
■
Synthesis of Selective MMP-12 Inhibitors. The
synthetic strategy used for the preparation of CGA and
CGA-1 is shown in Scheme 1. The anti-succinic acid derivative
(2R,3S)-3-(tert-butoxycarbonyl)-2-isobutylhex-5-enoic acid
was used as the starting material and converted into its benzyl
ester (1) using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and
benzyl bromide in toluene. The intermediate 2 was prepared
by hydroboration of 1 using 9-borabicyclo[3.3.1]nonane (9-
BBN) and H2O2 in tetrahydrofuran (THF). The hydroxyl
intermediate (2) was converted into its bromide derivative
using CBr4 and triphenylphosphine (Ph3P) in dichloro-
methane (DCM) to obtain 3. The benzyl ester group on 3
was deprotected using 10% palladium on carbon (Pd/C) and
hydrogen gas in methanol to obtain 4. L-Tyrosine benzyl ester
was coupled to the acid 4 using 1-ethyl-3-(3-dimethylamino-
propyl) carbodiimide hydrochloride (EDCI) and 1-hydrox-
ybenzotriazole hydrate (HOBT) in N,N-dimethyl formamide
(DMF) to obtain 5. The bromo group from the α-position and
the hydroxyl group from P2′ were cyclized using Cs2CO3 in
anhydrous acetonitrile to give the cyclized intermediate 6. The
t-butyl group on 6 was deprotected using trifluoroacetic acid
(TFA) in DCM to obtain 7, which was coupled with O-benzyl
hydroxylamine hydrochloride using HBTU in DMF to obtain
8. Both benzyl groups on 8 were deprotected using 10% Pd/C
and hydrogen gas in methanol to obtain the acid CGA (9).
Coupling of L-glutamic acid di-tert-butyl ester with 9 using
EDCI and HOBT in DMF followed by hydrolysis of acid esters
resulted in CGA-1 (11).
The synthetic strategy used for the preparation of AGA and
AGA-1 is shown in Scheme 2. The synthesis started from 4-
methylpentanoic acid, which was converted to 4-methylpenta-
noyl chloride using oxalyl chloride in THF at 0 °C. The acid
chloride obtained was then converted to intermediate 12 by
reacting with (S)-(−)-4-benzyl-2-oxazolidinone in THF at −78
°C. Reacting intermediate 12 with 2.0 M sodium bis-
(trimethylsilyl)amide solution (NaHMDS) at −78 °C followed
by tert-butyl bromoacetate in THF resulted in 13. The
C
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a
Scheme 2. Synthetic Scheme for the Preparation of AGA and AGA-1
a
Reagents and conditions: i. pivCl/LiCl/0 °C/2 h/S-benzyl oxazolidinone/THF/−78 °C to RT/12 h/55% yield; ii. NaHMDS (1.9 M)/Br-
CH2CO2-t-Bu/THF/−78 °C to RT/12 h/62% yield; iii. LiOH/H2O2/THF/0 °C to RT/2 h/75% yield; iv. L-phenylalanine benzyl ester/EDCI/
HOBT/DMF/RT/18 h/72% yield; v. TFA/DCM/RT/2 h/100% yield; vi. Bz-ONH2/BOP/DIEA/DMF/RT/18 h/80% yield; vii. Pd/C (10%)/
H2/CH3OH/RT/2 h/62% yield; viii. Boc-NH-CH2CH2-NH2/Fmoc-Glu(O-Bz)-OH/EDCI/HOBT/DMF/0 °C to RT/2.5 h/72% yield; ix. TFA/
DCM/RT/30 min/72% yield; x. N-Boc-HYNIC acid/EDCI/HOBT/DIEA/DMF/RT/18 h/55% yield; xi. piperidine/DCM/RT/30 min/65%
yield; xii. 2.7/2.11/EDCI/HOBT/DIPEA/DMF/RT/18 h/45% yield; xiii. (a) TFA/DCM/RT/2 h; (b) Pd/C (10%)/H2/CH3OH/RT/2 h/14%
yield.
a
Scheme 3. Synthetic Scheme for the Preparation of AGA-2
a
Reagents and conditions: i. H-Tyr-OBz/EDCI/HOBT/DMF/RT/18 h/65% yield; ii. TFA/DCM/RT/2 h/100% yield; iii. Bz-ONH2/HBTU/
DIEA/DMF/RT/18 h/66% yield; iv. 2-(BOC-amino)ethyl bromide/Cs2CO3/acetonitrile/60 °C/4 h/58% yield; v. TFA/DCM/RT/2 h/100%
yield; vi. N-Boc-HYNIC acid/HOAT/HATU/DIEA/DMF/RT/18 h/53% yield; vii. TFA/DCM/RT/100% yield; viii. Pd/C (10%)/H2/CH3OH/
RT/2 h/9% yield.
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a
Table 1. Inhibition Constants (Ki) of AGA, CGA, CGA-1, and RYM
Ki (nM)
rhMMP-2
rhMMP-7
rhMMP-9
rhMMP-12
rhMMP-13
CGA
CGA-1
RYM
AGA
AGA-1
AGA-2
300 81
396 28
5.8 0.4
164 43
222 17
>5 μM
39 2.0
14.6 0.1
17.2 0.1
323 129
615 53
>5 μM
1101 289
2107 454
20.4 2.0
125 14
1305 204
155 14
1.1 0.6
1.2 0.1
1.0 0.5
31 12
89 18
15.3 5.4
81 33
11.8
1
7.5 1.4
8.9 0.2
78
5
>5 μM
a
Ki values represent the mean SD of three experiments.
with the panMMP inhibitor RYM for a set of recombinant
human MMPs (rhMMPs) using an assay involving the
cleavage of a fluorogenic substrate, Mca-Lys-Pro-Leu-Gly-
Leu-Dpa-Ala-Arg-NH2. All newly developed inhibitors showed
great affinity and selectivity toward rhMMP-12 when
compared to a select group of rhMMPs. Accordingly, the
cyclic inhibitors CGA and CGA-1 displayed Ki values of <2
nM, and acyclic inhibitors AGA, AGA-1, and AGA-2 displayed
Ki values ranging from 7.5 to 12 toward rhMMP-12 (Table 1).
The selectivity of cyclic inhibitors for rhMMP-12 relative to
other MMPs tested ranged from at least 12-fold (for CGA-1
against rhMMP-7) to 28-fold (for CGA against rhMMP-13).
Among acyclic inhibitors, AGA-2 displayed the highest
selectivity (17-fold against rhMMP-9). Finally, like their
binding to rhMMP-12, all inhibitors showed high affinity for
murine MMP-12 (Table S1).
Figure 2. Ex vivo binding of 99mTc-AGA-1 (A) and 99mTc-AGA-2 (B)
to human normal aorta and AAA tissue. n = 3 in each group; **P <
0.01 and ***P < 0.001. AU, arbitrary units.
Radiolabeling. 99mTc-labeling of precursors AGA-1 and
AGA-2 was performed according to an established method-
ology.37,38 Both 99mTc-AGA-1 and 99mTc-AGA-2 showed more
showed faster blood clearance with a residual blood level of 1.2
0.2% injected dose (ID)/mL at 2 h post injection (p.i.),
while the residual blood activity for 99mTc-AGA-1 was 3.0
0.2% ID/mL at 2 h p.i. (Figure 3). The high activity in bile and
kidney indicated mixed hepatobiliary and renal clearance for
both tracers. Importantly, both tracers displayed low uptake
(2.0 0.4 and 2.0 0.9% ID/g, respectively) in the normal
aorta, supporting the possibility of using these tracers for
vascular imaging applications (Figure 3).
than 95% conversion of 99mTcO4 to its radiolabeling product
−
with retention times of 18.0 and 22.6 min, respectively. Both
99mTc-AGA-1 and 99mTc-AGA-2 were consistently obtained
with >95% radiochemical yield and radiochemical purity
without a need for any purification. To further confirm the
radiolabeling efficiency, when AGA-1 or AGA-2 (HYNIC
precursors) was not added to the labeling solution, only one
major peak of 99mTc-colloid or 99mTc-coligand product at 3.3
min was observed, and there was no radioactive peak
corresponding to radiolabeled products at 18.0 or 22.6 min
(Figure S1). The specific activity values of the radiotracers
were in the range of 540−810 MBq/nmol.
Binding to Human AAA Tissue. MMP-12 is highly
upregulated in human AAA and several preclinical studies have
indicated its important role in the development and potentially
rupture of AAA.22,24−26,39 Therefore, as a prelude to
performing in vivo imaging studies, we evaluated the binding
of 99mTc-AGA-1 and 99mTc-AGA-2 to human AAA and normal
aorta. Both 99mTc-AGA-1 and 99mTc-AGA-2 displayed
significantly higher binding to human AAA as compared to
normal aortic tissue (P < 0.001 for 99mTc-AGA-1 and P <
0.001 for 99mTc-AGA-2, n = 3 per group, Figure 2).
Stability Studies. 99mTc-AGA-1 and 99mTc-AGA-2 were
incubated in murine blood at 37 °C and their stability was
monitored by radio-HPLC analysis at 1 and 2 h post
incubation. 99mTc-AGA-1 exhibited poor stability, with the
radio-HPLC peak corresponding to the intact tracer decreasing
to ∼70% of total radioactivity at 1 h and ∼50% of that activity
at 2 h. Conversely, 99mTc-AGA-2 showed excellent radio-
chemical stability during the 2 h blood incubation period
(Figure 4).
Evaluation in Murine Model of AAA. Based on its
favorable selectivity, pharmacokinetics, and stability in blood,
99mTc-AGA-2 was selected for further evaluation in a murine
model of AAA that is associated with significant MMP-12
upregulation. Apolipoprotein E-deficient (Apoe−/−) mice (n =
11) were infused with angiotensin (Ang) II (1000 ng/kg/min;
Calbiochem) through a subcutaneous minipump for 4 weeks.
As expected, this led to suprarenal AAA development and AAA
rupture in a subset of animals.40 The surviving animals after 4
weeks of Ang II infusion (n = 8) were injected with 99mTc-
Radiotracer Biodistribution. The biodistribution and
blood clearance of 99mTc-AGA-1 and 99mTc-AGA-2 were
investigated in C57BL/6J mice following intravenous admin-
istration of each radiotracer (18.5
3.7 MBq). These and
AGA-2 (37
6 MBq) retro-orbitally under anesthesia,
other animal studies were performed under protocols approved
by Yale University and Veteran Affairs Connecticut institu-
tional animal care and use committees. Serial blood samples
were collected over a 2 h period, following which the animals
were euthanized, different tissue samples and body fluids were
collected and weighed, and their radioactivity was measured by
gamma-well counting. Relative to 99mTc-AGA-1, 99mTc-AGA-2
maintained for 1 h post injection. At 2 h post injection, the
aorta and carotid arteries were dissected from the surrounding
tissues and placed on a phosphor screen (MultiSensitive
Phosphor Screen, PerkinElmer) along with standard references
of known activity for quantitative autoradiography. The
phosphor screen was scanned with a phosphorimager
(Typhoon Trio, GE Healthcare Life Sciences) to obtain
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Figure 3. MMP-12 tracer biodistribution and clearance. Blood kinetics (A) and biodistribution (B) at 2 h after 99mTc-AGA-1 and 99mTc-AGA-2
administration in C57BL/6J mice. ID, injected dose. n = 3 in each group; *P < 0.05, **P < 0.01, and ***P < 0.001 (t-test).
software, NIH). This showed significantly higher 99mTc-AGA-2
uptake in the remodeled suprarenal abdominal aorta (srAA),
where AAA is typically located, compared to the adjacent,
normal-sized infrarenal abdominal aorta (irAA) (n = 8, P <
0.01, paired t-test, Figure 5). 99mTc-AGA-2 in suprarenal aorta
was also significantly higher in Ang II-treated animals with
AAA than those animals with low remodeling (LR) that had
not developed AAA (n = 4 in each group, P < 0.05, Mann−
Whitney U test, Figure 5).
99mTc-AGA-2 Binding Specificity. To investigate 99mTc-
Figure 4. MMP-12 tracer stability in mouse blood. Representative
radiochromatograms of Na99mTcO4 (green lines) and 99mTc-AGA-1
AGA-2 binding specificity, we evaluated the effect of MMP
(A) and 99mTc-AGA-2 (B) immediately after radiolabeling (blue
inhibitors on tracer binding to AAA and normal-sized irAA
lines) after 1 h (red lines) and after 2 h (gray lines).
tissues from Ang II-infused animals, as described above. Co-
incubation with 500-fold molar excess of either the panMMP
inhibitor GM6001 or the MMP-12-specific inhibitor AGA
significantly reduced 99mTc-AGA-2 binding to AAA tissue (P <
0.001 for both inhibitors), indicating the MMP-12-specificity
of 99mTc-AGA-2 binding in AAA (Figure 6). Interestingly, co-
digitalized images of radiotracer uptake, where focal tracer
uptake was readily detectable in animals with AAA (Figure 5).
Regions of interest (ROIs) were drawn over different segments
of the aorta to quantify the 99mTc-AGA-2 signal (Fiji/ImageJ
Figure 6. 99mTc-AGA-2 binding specificity. Ex vivo binding of 99mTc-
AGA-2 to mouse infrarenal aorta (irAA) and AAA tissue without
(gray columns) and with co-incubation with 500-fold molar excess
MMP-12-specific inhibitor (AGA, yellow columns) or panMMP
inhibitor (GM6001, green columns). n = 3 for each group. ns, not
significant; ***P < 0.001, one-way ANOVA.
incubation with MMP inhibitors had no effect on 99mTc-AGA-
2 binding to irAA tissue, suggesting that the low level of tracer
binding to the normal aorta is nonspecific (Figure 6).
DISCUSSION AND CONCLUSIONS
Figure 5. MMP-12 imaging in AAA. Examples of ex vivo photographs
(A, B) and corresponding autoradiographs after 2 h post injection of
99mTc-AGA-2 (C, D) of aortae from Ang II-induced Apoe−/− mice
with abdominal aortic aneurysm (A, C) and low remodeled aorta (B,
D). (E) Quantification of the 99mTc-AGA-2 signal in suprarenal
abdominal aorta (srAA) and infrarenal abdominal aorta (irAA) of Ang
II-infused mice. n = 8; **P < 0.01, paired t-test. (F) Quantification of
the 99mTc-AGA-2 signal in animals with AAA and low remodeled
(LR) aorta. n = 4 in each group; *P < 0.05, Mann−Whitney U test.
ID, injected dose.
■
Here, we report the design, synthesis, and preclinical
evaluation of first generation of succinate-based hydroxamate
analogues as novel selective MMP-12 inhibitors and their
99mTc-labeled HYNIC-conjugated analogues as novel MMP-
12-selective SPECT radiotracers. Our data show that
modifying the P3′ segment of hydroxamate MMP inhibitors
with a free acid side chain leads to selective MMP-12 binding.
Of the two acyclic MMP-12 tracers developed based on this
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design, 99mTc-AGA-2 displayed better stability and pharmaco-
kinetics, as well as binding to human and murine AAA tissue.
Autoradiography studies confirmed the higher in vivo uptake
of the tracer in AAA relative to normal size aorta, setting the
stage for future SPECT studies.
according to the literature and coupling of o-benzyl
hydroxamate to the free acid followed by deprotection of
both benzyl groups resulted in CGA. To synthesize CGA-1, a
di-protected glutamate was added to CGA, followed by
deprotection of both ester groups. The strategy used for the
synthesis of the acyclic AGA followed a previously reported
synthetic methodology with modifications.36 The intermediate
15 was synthesized starting from 4-methylvaloric acid and S-4-
benzyl-2-oxazolidinone. The hydrolysis of ester and coupling
of o-benzyl hydroxamate to the free acid followed by
deprotection of both benzyl groups resulted in AGA. AGA-1
was synthesized using a multiprotected glutamate with a
selective deprotection strategy and addition of HYNIC at the
end, as shown above. AGA-2 was synthesized by replacing the
phenylalanine ester from the AGA synthetic scheme with L-
tyrosine benzyl ester. The HYNIC group was added by
functionalizing the hydroxyl group on the phenyl to generate a
free amine, which in turn was coupled to HYNIC acid to
obtain AGA-2 through peptide coupling. The synthetic
strategy introduced here is novel for this class of anti-succinate
hydroxamate derivatives, where the P2′ position was used for
adding an HYNIC group (a chelator for 99mTc-radiolabeling).
This synthetic strategy reduced the number of synthetic steps
(8 vs 13 for AGA-1 and AGA-2, respectively), increasing the
yields and feasibility of synthesis. Also, this synthetic strategy
allowed us to keep the free acid on the P3′ position, which
resulted in better MMP-12 binding selectivity than AGA-1.
All these molecules with an acid side chain in their C-
terminal end displayed selective nanomolar inhibition toward
rhMMP-12. When both acid groups on the side chain of CGA-
1 were replaced by amide groups, the resulting molecule
(RYM) lost its selectivity toward MMP-12 and displayed
nanomolar inhibition toward all the rhMMPs tested. Hence, it
is understood that the presence of an acid side chain around
the P3′ segment of succinyl hydroxamate analogues is one of
the key reasons for achieving MMP-12 selectivity. Such an
impact was rather unexpected in hydroxamate series but was,
however, consistent with previous observations made on
selective MMP-12 inhibitors for which two glutamate residues
in P3′ and P2′ positions induce a favorable MMP-12 selectivity
profile.35,52 Since the analysis of the crystal structure of those
inhibitors in the interaction with MMP-12 did not reveal any
specific interaction of glutamate residues within the catalytic
cleft, it was postulated that the MMP-12 selectivity of those
inhibitors was more likely related to the presence of two
negative charges, better tolerated in the case of MMP-12 than
in that of other tested MMPs. In any event, the presence of a
carboxylic function in the C-terminal end of inhibitors
described in the present study has significant effects on their
selectivity profile, but several other structural differences must
be considered. Accordingly, the ring opening from cyclic CGA
to acyclic AGA inhibitor has almost no impact on binding to
MMP-2 and MMP-13, while a drop in potency (10-fold) is
observed for MMP-7 and MMP-12. Interestingly, further
structural modifications on the AGA scaffold (AGA-1 and
AGA-2) have no effect on binding to MMP-12 but impact to
variable extents the inhibitors’ potency toward other MMPs
tested. Particularly, the position of the bulky HYNIC moiety
relative to the pseudo-peptide backbone seemed to have major
effects on inhibitor binding, with a conjugation of this group
onto the P2′ side chain that was particularly well tolerated by
MMP-12 while rejected by MMP-2/7 and MMP-13. Overall,
AGA-2 that combined a carboxylic function in its C-terminal
MMP-12 plays a key role in several inflammatory diseases
such as chronic obstructive pulmonary disease (COPD),
atherosclerosis, and AAA.41 Considering the importance of
MMP-12 in several pathophysiological processes, this study
aimed at developing and evaluating novel selective MMP-12
inhibitors and their corresponding SPECT radiotracers. To
date, several panMMP inhibitors and a few selective MMP
inhibitors have been reported.22,25,28,37,42−45 Among these,
several are acyclic and macrocyclic hydroxamate analogues that
contain a ZBG and a lipophilic side chain at the P1′ segment
for interacting with the hydrophobic S1′ pocket of MMPs.
Large differences in MMP bindings are observed by changing
the side chain at the P1′ segment and, hence, the S1′ pocket is
considered as the “selectivity pocket”.46 A few hydroxamate-
based MMP inhibitors, e.g., ilomastat (GM-6001), RO-32-
3555, CGS-27023A, marimastat, BB-1101, prinomastat, RO-
32-3555, CGS-27023A, and batimastat (BB-94), have been
evaluated as therapeutic agents. However, the clinical use of
many of these inhibitors is hampered by musculoskeletal
toxicity and other side effects, attributed in part to the lack of
targeting specificity or selectivity, or the high concentrations of
inhibitors required for the therapeutic effects.19,47 As an
alternative, more selective MMP inhibitors are under develop-
ment. To access such selective inhibitors, as the structural
differences between different MMP families mainly occur at
the S1′ subsite, the investigators have utilized the P1′ group to
introduce the inhibitor specificity.48,49 Accordingly, a selective
MMP-13 inhibitor has been developed, which is characterized
by the absence of a catalytic zinc binding site and binding into
the S1′ pocket while extending into an additional S1′ side
pocket (S1′*).50 Other investigators have developed thiirane-
containing selective MMP-2 and MMP-9 inhibitors, which first
coordinate with the active site zinc ion and exploit the deep
S1′ pocket of MMP-2 and MMP-9. Considerable MMP-2
selectivity over MMP-9 was also achieved by exploiting the
more constricted pocket of MMP-9 than MMP-2 and by
introducing a bigger group with unfavorable steric inter-
actions.51 Phosphinic peptides are known to behave as potent
inhibitors of zinc metalloproteases. By exploiting the
phosphinic peptide chemistry, a highly selective MMP-12
inhibitor, RXP-470, has been developed.52
Succinyl hydroxamate-based small molecules that use the
hydroxamate group as their ZBG may be considered as the
most successful panMMP inhibitors. However, except for
hydroxamate-based selective MMP-2 inhibitors,53 not much
work has been reported on developing single MMP-selective
succinyl hydroxamate inhibitors, and specifically, there is no
report of selective hydroxamate MMP-12 inhibitors to date.
We sought to address this gap by modifying the P3′ segment to
achieve selectivity toward a single MMP while maintaining
high binding affinities. Accordingly, several succinyl hydrox-
amate analogues, i.e., CGA, CGA-1, AGA, AGA-1, and AGA-2,
were designed and successfully synthesized by incorporating an
acid side chain within the P3′ segment. The macrocyclic
hydroxamate MMP-12 inhibitors, CGA and CGA-1, were
synthesized starting from anti-succinate derivatives, following a
synthetic strategy reported previously with some modifica-
tions.33 The synthesis of the intermediate 7 was achieved
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purity of the final compounds and key intermediates were assessed by
analytical HPLC (see the Supporting Information for representative
chromatograms) and HRMS and 1H NMR (see the Supporting
Information for the representative HRMS and NMR spectra). Each
final compound displayed a purity of >95%.
Synthetic Chemistry. Intermediate 6. One gram (1.65 mmol) of
5 was dissolved in 20 mL of acetonitrile, 1.9 g (5.96 mmol) of
Cs2CO3 was added, and the mixture was stirred at room temperature
for 6 h. The solution was concentrated, 50 mL of water was added,
and the solution was extracted with ethyl acetate (50 mL × 3). The
combined organic layers were dried over MgSO4, filtered,
concentrated, and purified by flash column chromatography to give
460 mg (55%) of the title compound. ES−MS: observed [MH]+,
524.2.
end and an HYNIC moiety in its P2′ position appeared to be
the most selective agent among the set of inhibitors designed
in this study.
HYNIC, along with two coligands such as TPPTS and
tricine, is a widely used chelator for 99mTc-labeling.
Importantly, AGA and its HYNIC-conjugated radiolabeling
precursors AGA-1 and AGA-2 displayed a selective inhibition
profile toward MMP-12. This demonstrated that adding an
HYNIC moiety does not have a significant effect on MMP-12
binding and inhibition. This confirmed previous results with a
C-terminal extension on the pseudo-peptide scaffold that did
not significantly impact the MMP-12 selectivity profile of
optical probes or radiotracers compared to their parent
molecule.25,28,54 Both AGA-1 and AGA-2 were conveniently
radiolabeled with 99mTc using TPPTS and tricine formulation
in high radiochemical purity and yield to access 99mTc-AGA-1
and 99mTc-AGA-2.
1H NMR (400 MHz, chloroform-d) δ 7.99 (dt, 1H), 7.62−7.47
(m, 2H), 7.43−7.28 (m, 6H), 7.02−6.91 (m, 2H), 6.80−6.71 (m,
2H), 6.05 (d, 1H), 5.24−4.93 (m, 3H), 4.47−4.26 (m, 2H), 3.18 (dd,
1H), 3.04−2.78 (m, 3H), 2.35 (dtd, 2H), 1.77−1.61 (m, 3H), 1.42 (s,
10H), 1.15 (dtd, 1H), 1.00 (ddd, 1H), 0.81 (dd, 6H) (Figure S2).
Intermediate 7. Two hundred fifty milligrams of 6 was dissolved in
4 mL of TFA and 1 mL of DCM. After stirring at room temperature
for 2 h, the solution was concentrated and dried under vacuum to get
the title compound in a quantitative yield. ES−MS: observed [MH]+,
468.2.
Intermediate 8. A mixture of 7 (220 mg, 0.47 mmol), (2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) (214 mg, 0.56 mmol), and DIEA (0.4 mL, 2.35 mmol)
was dissolved in 2.5 mL of anhydrous DMF. The mixture was stirred
at room temperature for 20 min, followed by addition of Bn-ONH2
(247.0 mg, 1.55 mmol). The mixture was further stirred at room
temperature for 18 h. Twenty-five milliliters of cold H2O was added,
the mixture was stirred, and the solid formed was filtered. The solid
was vacuum-dried and used for the next step. Yield, 165 mg (62%);
ES−MS: observed [MH]+, 573.2. 1H NMR (400 MHz, chloroform-d)
δ 7.60−7.28 (m, 10H), 6.95 (dt, 2H), 6.81−6.67 (m, 2H), 5.32−4.94
(m, 4H), 4.51−4.32 (m, 2H), 3.53−3.32 (m, 2H), 2.43−2.24 (m,
2H), 1.87−1.47 (m, 6H), 1.47−1.30 (m, 2H), 1.22−0.98 (m, 1H),
0.86 (td, 6H) (Figure S3).
Intermediate 9 (CGA). A mixture of 8 (100 mg, 0.17 mmol) and
Pd/C (0.02 g, 20%, wet) in 10 mL of methanol was stirred under
hydrogen gas at room temperature for 2 h. The mixture was filtered,
followed by washing of Pd/C with methanol (5 mL × 4). The
combined methanol filtrate was concentrated, and the product was
further purified by flash column chromatography to give 40 mg (58%)
of the title compound. ES−MS: observed [M − H]−, 391.2. 1H NMR
(400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.67 (s, 1H), 7.73 (d, 1H),
7.21−6.96 (m, 3H), 6.79 (dd, 1H), 4.70 (s, 1H), 4.09 (s, 3H), 2.07−
1.91 (m, 1H), 1.69−1.55 (m, 1H), 1.39−1.10 (m, 4H), 0.89−0.66
(m, 6H) (Figures S4 and S5).
Intermediate 10. To a stirred solution of 9 (30 mg, 0.07 mmol) in
1 mL of anhydrous DMF were added EDCI (30 mg, 0.157 mmol),
HOBT (22 mg, 0.157 mmol), and L-glutamic acid di-tert-butyl ester
(40 mg, 0.157 mmol), and the mixture was stirred at room
temperature overnight. To the resulting mixture, ice-cold water
(100 mL) was added and the mixture was stirred for 30 min. The
resulting solid was filtered, dried, and purified by chromatography
(silica gel, 5% methanol/DCM) to obtain 12 mg (25%) of the title
compound. ES−MS: observed [MH]+, 634.2.
MMP-12 gene expression is significantly upregulated in
human AAA and both 99mTc-AGA-1 and 99mTc-AGA-2
displayed higher binding to human AAA than normal aortic
tissue. The stability studies performed in murine blood
revealed that 99mTc-AGA-1 is not stable and disintegrates
over time, whereas 99mTc-AGA-2 was found to be stable at
least for 2 h. From the biodistribution studies, we found that
tracer 99mTc-AGA-2 clears faster from the blood when
compared to 99mTc-AGA-1 (1.2
0.2 and 3.0
0.2 %ID/
mL, respectively). Based on favorable blood kinetics and
stability in murine blood, 99mTc-AGA-2 was chosen for further
evaluation. For preclinical evaluation of our new tracers, we
used an established animal model of AAA with significant
upregulation of MMP-12 in aneurysm, i.e., Apoe−/− mice
infused with Ang II.40 In vivo evaluation of 99mTc-AGA-2 in
Ang II-infused mice revealed significantly higher tracer uptake
in AAA, where MMP-12 expression is upregulated. Blocking
experiments with panMMP and MMP-12 inhibitors performed
with mouse tissues established the MMP-12 specific binding of
99mTc-AGA-2 in AAA by reducing tracer binding in AAA to the
nonspecific low levels observed in normal aortae. Combined,
these data support the potential of 99mTc-AGA-2 for molecular
imaging of MMP-12 activation, not only in AAA, but also other
diseases that are associated with MMP-12 upregulation.
In conclusion, this study reports the development and
evaluation of novel succinyl hydroxamate-based selective
MMP-12 inhibitors and their corresponding 99mTc-labeled
radiotracers. MMP-12 selectivity was achieved by introducing
an acid around the P3′ segment of succinyl hydroxamate
analogues. The lead tracer 99mTc-AGA-2 has favorable
pharmacokinetics for vascular imaging and binds selectively
to murine AAA in vivo. This sets the stage for future in vivo
SPECT imaging studies in AAA and other inflammatory
diseases where MMP-12 plays a key role.
EXPERIMENTAL SECTION
■
Intermediate 11 (CGA-1). A mixture of 10 (12 mg, 0.018 mmol) in
100 mL of DCM was cooled down to 0 °C and 20 mL of TFA was
added dropwise. The mixture was stirred for 2 h at room temperature
and concentrated under vacuum to remove the solvent and excess
TFA. The reaction mixture was further dried by codistilling with
toluene twice (10 mL × 2) and purified by preparative HPLC. ES−
Commercially available reagents and solvents were purchased from
Sigma-Aldrich (USA) and used as received without further
purification. The following instruments were used for quality control:
(1) HPLC: Waters HPLC system 2489, UV−Vis detector, 600
controller, and Empower Pro software; (2) LC−MS: Agilent LC−MS
6120B Quadrupole, 6550A iFunnel Q-TOF; (3) HRMS: Agilent
QToF 6546 instrument (over 30,000 resolution at ∼200 MW) was
used with a gradual increase in buffer composition of 0−80% MeOH
in water in 10 min; (4) NMR: Agilent NMR DD2 400 MHz with
OneProbe (Agilent Technologies, Santa Clara, CA, USA); (5)
fluorescent plate reader (BioTek/Synergy HT). The identity and
1
MS: observed [M − H]−, 520.2. H NMR (400 MHz, DMSO-d6) δ
10.34 (s, 1H), 8.68 (s, 1H), 7.92 (d, 1H), 7.51 (d, 1H), 7.29−6.97
(m, 3H), 6.82 (dd, 1H), 6.56 (s, 1H), 4.74 (ddd, 1H), 4.28−3.92 (m,
4H), 2.27 (q, 2H), 2.00 (td, 1H), 1.92−1.75 (m, 2H), 1.60 (td, 1H),
1.44−1.04 (m, 4H), 0.89−0.53 (m, 6H) (Figures S6 and S7).
H
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1
Intermediate 16. Five hundred milligrams of 15 was dissolved in 4
mL of TFA and 5 mL of DCM. After stirring at room temperature for
2 h, the solution was concentrated and dried under vacuum to get the
title compound in a quantitative yield. ES−MS: observed [MH]+,
412.2.
Intermediate 17. A mixture of 16 (180 mg, 0.42 mmol),
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP) (0.22 g, 0.506 mmol), and DIEA (290 mg, 1.68
mmol) was dissolved in 4 mL of anhydrous DMF. The mixture was
stirred at room temperature for 20 min, followed by addition of Bn-
ONH2 (62 mg, 0.506 mmol). The mixture was further stirred at room
temperature overnight and concentrated. The residue was triturated
with 1 N HCl, and the solid obtained was filtered and washed with 1
N Na2CO3, water, and brine. The product was further purified by
flash column chromatography to give 180 g (80%) of the title
compound. ES−MS: observed [MH]+, 517.2.
Intermediate 18 (AGA). A mixture of 17 (100 mg, 0.193 mmol)
and Pd/C (0.01 g, 10%, wet) in 10 mL of methanol was stirred under
hydrogen gas at room temperature for 2 h. The mixture was filtered,
followed by washing of Pd/C with methanol (5 mL × 4). The
combined methanol filtrate was concentrated, and the product was
further purified by flash column chromatography to give 40 mg (62%)
of the title compound. ES−MS: observed [M − H]−, 335.1. The
representative NMR and HRMS spectra are shown in the Supporting
Information. 1H NMR (400 MHz, methanol-d4) δ 7.35−7.16 (m,
5H), 4.75−4.62 (m, 1H), 3.21 (dd, 1H), 3.10−2.94 (m, 1H), 2.94−
2.74 (m, 1H), 2.05 (qd, 2H), 1.53 (ddt, 2H), 1.11 (ddt, 1H), 0.88
(dd, 6H) (Figures S8 and S9).
Intermediate 19. To a stirred solution of Fmoc-Glu(O-Bn)-OH
(2.0 g, 4.35 mmol), HOBT (0.7 g, 5.22 mmol), and N-Boc-
ethylenediamine (0.77 g, 4.79 mmol) in 20 mL of anhydrous DMF
cooled in an ice bath was added EDCI (1 g, 5.224 mmol), and the
mixture was stirred at room temperature for 2.5 h. Ice-cold water was
added to the resulting mixture, the mixture was stirred, and the solid
obtained was filtered. The resulting residue was purified by
chromatography (silica gel, 60% ethyl acetate/hexane) to obtain 1.8
g (72%) of the title compound. ES−MS: observed [MH]+, 602.2. 1H
NMR (400 MHz, chloroform-d) δ 7.81−7.60 (m, 2H), 7.51 (d, 2H),
7.38−7.18 (m, 9H), 5.11−4.97 (m, 2H), 4.33 (q, 2H), 4.13 (t, 2H),
3.43−3.04 (m, 4H), 2.57−2.26 (m, 2H), 2.20−1.99 (m, 1H), 2.00−
1.82 (m, 1H), 1.34 (s, 9H) (Figure S10).
MS: observed [MH]+, 515.2. H NMR (400 MHz, methanol-d4) δ
8.55 (dd, 2H), 8.27−8.12 (m, 1H), 8.02 (dd, 1H), 7.44−7.27 (m,
2H), 6.71 (dd, 2H), 4.17 (dd, 2H), 3.72−3.37 (m, 9H), 2.59−2.20
(m, 7H), 2.18−2.00 (m, 3H), 1.50 (s, 9H) (Figure S13).
Intermediate 23. A mixture of 18 (20 mg, 0.06 mmol), 22 (37 mg,
0.07 mmol), and HOBT (10 mg, 0.07 mmol) was dissolved in 2 mL
of DMF and cooled down to 0−5 °C in an ice bath, followed by
addition of EDCI (14 mg, 0.07 mmol). The mixture was stirred at
room temperature overnight and concentrated. The residue was
dissolved in 10 mL of CH2Cl2 and washed with 1 N HCl, H2O, and
brine. The DCM solution was dried over MgSO4, filtered, and
concentrated. The product was purified by flash column chromatog-
raphy (2% methanol/DCM) to give the title compound (21 mg,
45%). ES−MS: observed [MH]+, 799.9.
Intermediate 24 (AGA-1). Twenty milligrams of 23 was dissolved
in 2 mL of TFA and 1 mL of DCM. After stirring at room
temperature for 2 h, the solution was concentrated and dried under
vacuum to get crude t-butyl-deprotected acid product. To the acid,
Pd/C (4 mg, 20%, wet) in 1 mL of methanol was stirred under
hydrogen gas at room temperature for 2 h. The mixture was filtered,
followed by washing of Pd/C with methanol (1 mL × 4). The
combined methanol filtrate was concentrated, and the product was
further purified by flash column chromatography followed by
preparative HPLC purification to give 2 mg (14%) of the title
compound. ES−MS: observed [MH]+, 643.3. H NMR (400 MHz,
1
chloroform-d) δ 7.38−7.19 (m, 6H), 6.80 (d, 2H), 6.69−6.47 (m,
2H), 5.20−4.88 (m, 2H), 4.74 (d, 3H), 2.99 (dd, 2H), 2.84 (dd, 2H),
2.65 (tt, 2H), 2.11 (ddd, 2H), 1.41 (q, 2H), 1.30−1.12 (m, 2H), 1.02
(ddd, 1H), 0.71 (m, 6H) (Figures S14 and S15).
Intermediate 27. A mixture of 26 (2 g, 4.7 mmol), (2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) (2.14 g, 5.6 mmol), and DIEA (1.6 mL, 9.4 mmol) was
dissolved in 20 mL of anhydrous DMF. The mixture was stirred at
room temperature for 20 min, followed by addition of Bn-ONH2 (988
mg, 6.2 mmol). The mixture was further stirred at room temperature
for 18 h. Two hundred fifty milliliters of cold H2O was added, the
mixture was stirred, and the solid formed was filtered. The solid was
vacuum-dried and used for the next step. Yield, 1.65 g (66%); ES−
1
MS: observed [MH]+, 533.6. H NMR (400 MHz, methanol-d4) δ
7.47−7.25 (m, 9H), 7.08−6.94 (m, 2H), 6.77−6.59 (m, 2H), 5.10 (d,
2H), 4.90−4.74 (m, 7H), 4.65 (dd, 1H), 3.12−2.75 (m, 3H), 2.23−
1.90 (m, 2H), 1.56−1.36 (m, 2H), 1.09−1.00 (m, 1H), 0.83 (dd, 6H)
(Figure S16).
Intermediate 20. One gram of 19 obtained above was dissolved in
10 mL of DCM and 3 mL of piperidine. The mixture was stirred at
room temperature for 30 min and concentrated under vacuum. The
product was purified by flash column chromatography (2% methanol/
DCM) to give the title compound (0.6 g, 72%). ES−MS: observed
Intermediate 28. To a stirred solution of 2-(Boc-amino)ethyl
bromide (0.31 g, 1.4 mmol) and Cs2CO3 (0.92 g, 2.82 mmol) in 10
mL of anhydrous acetonitrile at 60 °C was added dropwise a solution
of 27 (0.5 g, 0.94 mmol) in 5 mL of acetonitrile over a period of 1 h.
The resulting mixture was stirred at 60 °C for another 3 h and
concentrated under vacuum. The product was redissolved with ethyl
acetate and filtered, followed by washing of the solid with ethyl
acetate for five times (10 mL × 5). The combined ethyl acetate filtrate
was washed with 1 N HCl solution, water, and brine. The ethyl
acetate solution was dried over anhydrous MgSO4, filtered, and
concentrated. The resulting residue was purified by silica gel
chromatography using (silica gel, 40% ethyl acetate/hexane) to give
the product (0.4 g, 58%). ES−MS: observed [MH]+, 676.3. 1H NMR
(400 MHz, DMSO-d6) δ 7.47−7.18 (m, 10H), 7.15−7.08 (m, 1H),
7.03−6.93 (m, 1H), 6.87−6.71 (m, 1H), 6.67−6.62 (m, 1H), 5.18−
4.96 (m, 2H), 4.90−4.67 (m, 2H), 4.51−4.37 (m, 1H), 3.87 (td, 1H),
3.11 (d, 1H), 3.01−2.82 (m, 2H), 2.78 (d, 1H), 1.38 (s, 9H), 1.14−
1.05 (m, 1H), 0.86−0.68 (m, 6H) (Figure S17).
Intermediate 29. Four hundred milligrams of 28 was dissolved in 4
mL of TFA and 6 mL of DCM. After stirring at room temperature for
2 h, the solution was concentrated and dried under vacuum to get
crude Boc-deprotected free amine product in quantitative amounts,
which was used directly for the next step. ES−MS: observed [MH]+,
576.3.
Intermediate 30. To a stirred solution of 29 (100 mg, 0.174
mmol) in 5 mL of anhydrous DMF were added 1-hydroxy-7-
azabenzotriazole (HOAt) (47 mg, 0.35 mmol), hexafluorophosphate
1
[MH]+, 502.2. H NMR (400 MHz, methanol-d4) δ 7.91−7.53 (m,
5H), 7.49−7.20 (m, 8H), 5.14 (s, 2H), 4.53−4.40 (m, 1H), 4.37 (dd,
1H), 4.24 (dt, 1H), 4.09 (dd, 1H), 3.59−3.37 (m, 3H), 3.17−2.94
(m, 2H), 2.44 (dt, 2H), 2.22−2.04 (m, 1H), 2.05−1.86 (m, 1H)
(Figure S11).
Intermediate 21. To a stirred solution of 20 (100 mg, 0.2 mmol)
in 1 mL of anhydrous DMF were added EDCI (40 mg, 0.24 mmol),
HOBT (32 mg, 0.24 mmol), and 6-Boc-hydrazinonicotinic acid (50
mg, 0.2 mmol), and the mixture was stirred at room temperature
overnight. To the resulting mixture, ice-cold water (100 mL) was
added and the mixture was stirred for 30 min. The resulting solid was
filtered, dried, and purified by chromatography (silica gel, 1%
methanol/DCM) to obtain 80 mg (55%) of the title compound.
ES−MS: observed [MH]+, 737.2. 1H NMR (400 MHz, DMSO-d6) δ
8.99−8.80 (m, 1H), 8.52−8.35 (m, 2H), 8.24 (s, 1H), 8.00−7.84 (m,
2H), 7.79 (d, 2H), 7.63 (t, 2H), 7.49−7.38 (m, 1H), 7.37−7.15 (m,
9H), 6.52 (dd, 1H), 4.99 (s, 2H), 4.31−4.06 (m, 3H), 3.90 (td, 1H),
2.47 (d, 7H), 2.29 (t, 2H), 1.87 (ddd, 1H), 1.79−1.64 (m, 1H), 1.33
(s, 9H) (Figure S12).
Intermediate 22. Eighty milligrams of 21 obtained above was
dissolved in 10 mL of DCM and 3 mL of piperidine. The mixture was
stirred at room temperature for 30 min and concentrated under
vacuum. The product was purified by flash column chromatography
(2% methanol/DCM) to give the title compound (35 mg, 65%). ES−
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azabenzotriazole tetramethyl uronium (HATU) (132 mg, 0.38
mmol), 6-Boc-hydrazinonicotinic acid (88 mg, 0.35 mmol), and the
mixture was stirred at room temperature overnight. To the resulting
mixture, ice-cold water (50 mL) was added and the mixture was
stirred for 30 min. The resulting solid was filtered, dried, and purified
by chromatography (silica gel, 1% methanol/DCM) to obtain 74 mg
Tracer Binding to Human Tissues. Human tissues were
collected from anonymous donors under a protocol approved by
the Yale University institutional review board (#12481). Frozen tissue
sections of human AAA and normal aorta were incubated in triplicates
with ∼1.8 MBq of 99mTc-AGA-1 or 99mTc-AGA-2 at 37 °C for 2 h.
Following washing of the unbound radiotracer, the bound activity in
each sample was measured using a gamma counter (WIZARD2,
PerkinElmer). Next, 200 μL of protein lysis buffer [0.3 M NaCl, 50
mM Tris, 1% Triton X-100, and cOmplete Protease Inhibitor Cocktail
(Sigma-Aldrich)] was added to each sample, aortic tissue was lysed,
and protein concentration was measured using a colorimetric assay
[Protein Assay Dye Reagent Concentrate (Bio-Rad) and BioMate 3
(Thermo Scientific)]. The bound activity in each sample was
normalized to protein concentration.
1
(53%) of the title compound. ES−MS: observed [MH]+, 737.2. H
NMR (400 MHz, DMSO-d6) δ 8.96−8.28 (m, 8H), 8.01−7.73 (m,
3H), 7.48−7.03 (m, 12H), 6.83 (d, J = 8.6 Hz, 2H), 6.51 (t, J = 9.5
Hz, 2H), 5.13−4.93 (m, 3H), 4.70 (d, J = 2.9 Hz, 2H), 4.45 (q, J =
7.5, 7.0 Hz, 2H), 4.04 (t, J = 5.9 Hz, 5H), 3.80 (d, J = 7.8 Hz, 6H),
3.09 (d, J = 7.4 Hz, 2H), 2.92 (dd, J = 11.9, 7.3 Hz, 3H), 2.72 (s, 2H),
2.06 (tt, J = 14.5, 5.8 Hz, 3H), 1.45−1.35 (m, 11H), 0.85−0.60 (m,
6H) (Figure S18).
Intermediate 31. Fifty milligrams of 30 was dissolved in 1 mL of
TFA and 2 mL of DCM. After stirring at room temperature for 2 h,
the solution was concentrated and dried under vacuum to get crude
Boc-deprotected free amine product in quantitative amounts, which
was used directly for the next step. ES−MS: observed [MH]+, 637.2.
Intermediate 32 (AGA-2). A mixture of 31 (40 mg, 0.193 mmol)
and Pd/C (0.01 g, 25%, wet) in 4 mL of methanol was stirred under
hydrogen gas at room temperature for 2 h. The mixture was filtered,
followed by washing of Pd/C with methanol (5 mL × 4). The
combined methanol filtrate was concentrated, and the product was
further purified by flash column chromatography and preparative
HPLC to give 2.9 mg (9%, after two steps) of the title compound.
ES−MS: observed [MH]+, 530.7. 1H NMR (400 MHz, methanol-d4)
δ 8.53−8.35 (m, 2H), 7.99−7.79 (m, 2H), 7.04 (dq, J = 7.0, 3.3 Hz,
2H), 6.82−6.67 (m, 2H), 6.58 (dd, J = 8.8, 6.3 Hz, 2H), 3.99 (p, J =
5.5 Hz, 2H), 3.78−3.54 (m, 3H), 3.39 (q, J = 7.0 Hz, 2H), 3.02 (d, J
= 4.8 Hz, 1H), 2.87−2.75 (m, 1H), 2.65 (s, 1H), 2.23−1.93 (m, 2H),
0.86−0.56 (m, 6H) (Figures S19 and S20).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01514.
Inhibition constants (Ki) of CGA, CGA-1, RYM, AGA,
AGA-1, and AGA-2 for recombinant murine (rm)
MMP-9 and MMP-12 (Table S1); H NMR assign-
ments of compound 6 (Table S2); H NMR assign-
1
1
1
ments of compound 8 (Table S3); H NMR assign-
ments of compound CGA (Table S4); 1H NMR
assignments of compound CGA-1 (Table S5); 1H
1
NMR assignments of compound AGA (Table S6); H
NMR assignments of compound intermediate 19 (Table
1
S7); H NMR assignments of compound intermediate
MMP Affinity Determination. The MMP inhibition constants
for newly synthesized selective MMP-12 inhibitors were measured
based on the kinetic effects of an inhibitor on MMP-mediated
catalytic cleavage of a fluorogenic substrate, namely, Mca-Lys-Pro-
Leu-Gly-Leu-Dpa-Ala-Arg-NH2, in an assay buffer of 50 mM Tris/
HCl buffer (pH 6.8) and 10 mM CaCl2 at 25 °C. The inhibition
constants were measured toward MMP-2, MMP-7, MMP-9, MMP-
12, and MMP-13 using 0.2−0.5 nM MMP at four different
concentrations of the fluorogenic substrate (8, 16, 32, and 64 μM)
and at five different concentrations of the inhibitors (31.25, 125, 0.5,
2.5, and 5 μM). The assays were performed in black flat-bottom 96-
well plates (Microfluor 1 Black, Thermo Fisher Scientific). The
fluorescence signals were monitored using a BioTek Synergy HT
spectrophotometer equipped with a plate shaker and temperature
device controller. From the fluorescence measured, the Ki values were
determined using the method previously proposed by Horovitz and
Leviski.55
1
20 (Table S8); H NMR assignments of compound
1
intermediate 21 (Table S9); H NMR assignments of
compound intermediate 22 (Table S10); 1H NMR
assignments of compound AGA-1 (Table S11); 1H
NMR assignments of compound intermediate 27 (Table
1
S12); H NMR assignments of compound intermediate
1
28 (Table S13); H NMR assignments of compound
1
intermediate 30 (Table S14); H NMR assignments of
compound AGA-2 (Table S15); 99mTc-AGA-1 and
99mTc-AGA-2 stability in blood (Figure S1); H NMR
1
spectrum of compound 6 (Figure S2); 1H NMR
spectrum of compound 8 (Figure S3); 1H NMR
spectrum of compound CGA (Figure S4); HPLC and
HRMS spectra of CGA (Figure S5); 1H NMR spectrum
of compound CGA-1 (Figure S6); HPLC and HRMS
Radiolabeling. 99mTc-labeling of precursors AGA-1 and AGA-2
was performed according to an established methodology.37,38 Briefly,
the coligand kits for radiolabeling of HYNIC precursors with
1
spectra of CGA-1 (Figure S7); H NMR spectrum of
compound AGA (Figure S8); HPLC and HRMS spectra
1
of AGA (Figure S9); H NMR spectrum of compound
−
99mTcO4 were prepared by lyophilizing the solution containing
1
intermediate 19 (Figure S10); H NMR spectrum of
tricine (6.5 mg/mL), 3,3′,3″-phosphanetriyltris(benzenesulfonic acid)
trisodium salt (TPPTS) (5.0 mg/mL), pluronic F-127 (0.1 mg/mL),
succinic acid (12.7 mg/mL), sodium succinate (38.5 mg/mL), and
mannitol (40 mg/mL).33 Each kit was mixed with 2 μg (in 2 μL of
DMSO) of HYNIC precursor (AGA-1 or AGA-2) and 200−400 μL
compound intermediate 20 (Figure S11); 1H NMR
spectrum of compound intermediate 21 (Figure S12);
1H NMR spectrum of compound intermediate 22
1
(Figure S13); H NMR spectrum of compound AGA-
−
(740−1110 MBq) of 99mTcO4 solution and heated at 95 °C for 20
1 (Figure S14); HPLC and HRMS spectra of AGA-1
(Figure S15); 1H NMR spectrum of compound
min. The radiolabeled solution was cooled down to room temperature
before subjecting it to quality control by instant thin-layer
chromatography (ITLC) and radio high-performance liquid chroma-
tography (HPLC) analyses. ITLC analysis was performed using either
a silica gel plate or TLC paper as the stationary phase and acetonitrile
as the mobile phase. HPLC analysis was performed using a C-18
reverse-phase analytical column as the stationary phase and aqueous
acetonitrile (containing 0.16% ammonium formate) and water
(containing 0.16% ammonium formate) as the mobile phase for
gradient elution.
1
intermediate 27 (Figure S16); H NMR spectrum of
compound intermediate 28 (Figure S17); 1H NMR
spectrum of compound intermediate 30 (Figure S18);
1H NMR spectrum of compound AGA-2 (Figure S19);
HPLC and HRMS spectra of AGA-2 (Figure S20)
(PDF)
SMILES and Ki values of compounds (CSV)
J
https://dx.doi.org/10.1021/acs.jmedchem.0c01514
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Medicine, New Haven, Connecticut 06511, United States;
Veterans Affairs Connecticut Healthcare System, West Haven,
Connecticut 06516, United States
Gunjan Kukreja − Cardiovascular Molecular Imaging
Laboratory, Section of Cardiovascular Medicine and Yale
Cardiovascular Research Center, Yale University School of
Medicine, New Haven, Connecticut 06511, United States;
Veterans Affairs Connecticut Healthcare System, West Haven,
Connecticut 06516, United States
Terence L. Wu − Yale West Campus Analytical Core, Yale
University, West Haven, Connecticut 06516, United States
Laurent Devel − CEA, INRAE, Medicaments et Technologies
pour la Sante (MTS), SIMoS, Université Paris-Saclay, 91191
Gif-sur-Yvette, France; orcid.org/0000-0002-4464-5826
AUTHOR INFORMATION
■
Corresponding Author
Mehran M. Sadeghi − Cardiovascular Molecular Imaging
Laboratory, Section of Cardiovascular Medicine and Yale
Cardiovascular Research Center, Yale University School of
Medicine, New Haven, Connecticut 06511, United States;
Veterans Affairs Connecticut Healthcare System, West Haven,
Connecticut 06516, United States; orcid.org/0000-0002-
5074-3510; Phone: 203 737 6954;
Email: mehran.sadeghi@yale.edu; Fax: 203 937 3884
Authors
Kiran Gona − Cardiovascular Molecular Imaging Laboratory,
Section of Cardiovascular Medicine and Yale Cardiovascular
Research Center, Yale University School of Medicine, New
Haven, Connecticut 06511, United States; Veterans Affairs
Connecticut Healthcare System, West Haven, Connecticut
06516, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01514
Notes
Jakub Toczek − Cardiovascular Molecular Imaging
Laboratory, Section of Cardiovascular Medicine and Yale
Cardiovascular Research Center, Yale University School of
Medicine, New Haven, Connecticut 06511, United States;
Veterans Affairs Connecticut Healthcare System, West Haven,
Connecticut 06516, United States
Yunpeng Ye − Cardiovascular Molecular Imaging Laboratory,
Section of Cardiovascular Medicine and Yale Cardiovascular
Research Center, Yale University School of Medicine, New
Haven, Connecticut 06511, United States; Veterans Affairs
Connecticut Healthcare System, West Haven, Connecticut
06516, United States
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH grant R01HL138567 and VA
Merit grant I01BX004038. P.B. and M.S. were supported by
NIH training grants (5T32HL98069 and 5T32HL007950).
We thank Dr. Mousumi Ghosh for the assistance in chemical
analyses. The resources of the Yale Translational Research
Imaging Center, funded in part by NIH grant 1S10RR018039,
and West Campus Analytical Core at Yale University were
used for these studies.
Nowshin Sanzida − Cardiovascular Molecular Imaging
Laboratory, Section of Cardiovascular Medicine and Yale
Cardiovascular Research Center, Yale University School of
Medicine, New Haven, Connecticut 06511, United States;
Veterans Affairs Connecticut Healthcare System, West Haven,
Connecticut 06516, United States
Arvene Golbazi − Cardiovascular Molecular Imaging
Laboratory, Section of Cardiovascular Medicine and Yale
Cardiovascular Research Center, Yale University School of
Medicine, New Haven, Connecticut 06511, United States;
Veterans Affairs Connecticut Healthcare System, West Haven,
Connecticut 06516, United States
Parnaz Boodagh − Cardiovascular Molecular Imaging
Laboratory, Section of Cardiovascular Medicine and Yale
Cardiovascular Research Center, Yale University School of
Medicine, New Haven, Connecticut 06511, United States;
Veterans Affairs Connecticut Healthcare System, West Haven,
Connecticut 06516, United States
Mani Salarian − Cardiovascular Molecular Imaging
Laboratory, Section of Cardiovascular Medicine and Yale
Cardiovascular Research Center, Yale University School of
Medicine, New Haven, Connecticut 06511, United States;
Veterans Affairs Connecticut Healthcare System, West Haven,
Connecticut 06516, United States
ABBREVIATIONS
■
AAA, abdominal aortic aneurysm; ECM, extracellular matrix;
TIMP, tissue inhibitors of metalloproteinases; srAA, suprarenal
abdominal aorta; irAA, infrarenal abdominal aorta; ZBG, zinc
binding group; EDC-I, 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide; HOBT, 1-hydroxybenzotriazole hydrate; RT,
room temperature; pivCl, pivaloyl chloride; BOP, benzotriazol-
1-yloxytris(dimethylamino)phosphonium hexafluorophos-
phate; DIEA, N,N-diisopropylethylamine; HATU, hexafluor-
ophosphate azabenzotriazole tetramethyl uranium; HOAT, 1-
hydroxy-7-azabenzotriazole; rh, recombinant human; Bq,
becquerel; ID, injected dose; Ang II, angiotensin II; ROI,
region of interest; TPPTS, 3,3′,3″-phosphanetriyltris-
(benzenesulfonic acid) trisodium salt; HYNIC, 6-hydrazinoni-
cotinyl
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