Synthesis and in Vivo Biological Evaluation of 68Ga-Labeled Carbonic Anhydrase IX Targeting Small Molecules for Positron Emission Tomography

Article abstract of DOI:10.1021/acs.jmedchem.6b00623

Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [⁶⁸Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing ⁶⁸Ga for preclinical CA IX imaging are scarce, and this is one of the first effective ⁶⁸Ga compounds reported for PET imaging of CA IX.

Full text of DOI:10.1021/acs.jmedchem.6b00623

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Article
Synthesis and in vivo Biological Evaluation of 68Ga Labelled Carbonic
Anhydrase IX Targeting Small Molecules for Positron Emission Tomography
Deborah Sneddon, Raymon Niemans, Matthias Bauwens, Ala Yaromina, Simon J.A. van Kuijk, Natasja
Gabi Lieuwes, Rianne Biemans, Ivo Pooters, Paul A. Pellegrini, Nigel A Lengkeek, Ivan Greguric,
Kathryn F. Tonissen, Claudiu T Supuran, Philippe Lambin, Ludwig Dubois, and Sally-Ann Poulsen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00623 • Publication Date (Web): 20 Jun 2016
Downloaded from http://pubs.acs.org on June 21, 2016
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Abstract
Tumor hypoxia contributes resistance to chemoꢀ and radiotherapy, while oxygenated tumors
are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by
targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with
sulfonamideꢀbased imaging agents. In this study, we present the design and synthesis of novel
gallium radiolabelled small molecule sulfonamides targeting CA IX. The compounds display
favorable in vivo pharmacokinetics (PK) and stability. We demonstrate that our lead
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compound, [ Ga]ꢀ2, discriminates CA IX expressing tumors in vivo in a mouse xenograft
model using positron emission tomography (PET). This compound shows specific tumor
accumulation, low uptake in blood and clears intact to the urine. These findings were
reproduced in a second study using PET/CT (computed tomography). Small molecules so far
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investigated utilizing Ga for preclinical CA IX imaging are scarce and this is one of the first
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effective Ga compounds reported for PET imaging of CA IX.
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Introduction
Molecular imaging with positron emission tomography (PET) has had a profound impact on
primary diagnosis, management, therapy monitoring and prognosis in cancer; it is
noninvasive and provides personalized care to patients by informing treatment decisions and
evaluating treatment response. Hypoxia (low oxygen concentration) is a characteristic feature
of solid tumors. Hypoxic cells coꢀopt adaptive mechanisms to switch to a glycolytic
metabolism, promote cell proliferation, evade immune attack, induce angiogenesis, invade
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and metastasize. Tumor hypoxia is a negative prognostic factor associated with a more
aggressive phenotype, specifically with resistance to chemoꢀ and radiotherapy. For example,
up to a 3ꢀfold higher radiation dose is needed to achieve the same level of tumor cell death in
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hypoxic tumors compared to oxygenated tumors. The implementation of a hypoxiaꢀguided
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clinical management strategy, such as hypoxia radiation sensitizers (e.g. nimorazole ) or
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hypoxia specific cytotoxic therapy e.g. THꢀ302 (Figure 1A) to those patients most likely to
benefit is currently not possible as there is no established method in routine clinical practice
that is: (i) non invasive, (ii) routine to prepare, and (iii) indicative of the hypoxic cell
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population Most current methods to detect hypoxia are invasive (e.g. require surgery) and
are subject to technical issues causing sampling errors.
Small molecule molecular probes for imaging of hypoxia with PET may be split into two
broad categories, ‘direct’ or ‘indirect’ imaging probes. Nitroimidazoles are direct imaging
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probes for the detection of hypoxia with PET, with one compound, Fꢀfluoromisonidazole
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5
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(
FꢀFMISO) in limited clinical use. Second and third generation nitroimidazoles, FꢀFAZA
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and FꢀHX4, respectively, have been shown by us to address the PK problems of FꢀFMISO
(slow tumorꢀspecific accumulation and nonspecific washout), however, better probes for
5
ꢀ8
hypoxia are still required.
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A critical cellular response to hypoxia is the stabilisation and activation of the transcription
factor hypoxia inducible factorꢀ1α (HIF1α). HIF1α regulates the expression of genes required
for survival under hypoxia. In principle, the gene products may be used as targets for imaging
of tumor hypoxia with indirect probes and bypass the drawbacks associated with
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nitroimidazole probes. Carbonic anhydrase IX (CA IX) is one of the most highly induced
HIF1α responsive genes and is proposed as the “gold standard” endogenous marker of cellular
10ꢀ13
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hypoxia.
CA IX expression is a negative prognostic factor in several types of cancer.
Additionally CA IX (over)expression is thought to predict the therapeutic effect of CA IXꢀ
targeting anticancer therapies. CA IX is overexpressed and sustained in many solid tumors
including breast, brain (glioblastoma), clear cell renal, colorectal, head and neck, bladder and
nonꢀsmall cell lung carcinomas but expression in normal tissues is restricted to the stomach
10, 15
and GI tract.
CA IX is a transmembrane zinc metalloenzyme that catalyzes the reversible hydration of CO2
ꢀ
+
12, 13, 16ꢀ20
to HCO and H , enabling the tumor to regulate pH allowing its spread and survival.
3
Expression of CA IX is commonly used as a histologic marker of tissue hypoxia, with
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detection using M75 or G250 , two different monoclonal antibodies specific for CA IX.
There are several antibody and antibody fragment based imaging agents with in vivo data that
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indirectly target hypoxic tumors by binding to CA IX.
Our groups have shown that small molecule sulfonamides are able to discriminate oxygen
levels in tissues and bind preferentially to CA IX only in hypoxic cells, while CA IX targeting
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antibodies also bind upon reoxygenation. Therefore our attention has turned to small
molecules to develop radiopharmaceuticals to detect CA IX positive tumors with PET. Very
few indirect small molecules that that incorporate a primary sulfonamide functional group,
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required for tight binding to the active site zinc CA IX (see examples in Figure 1) have so far
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5ꢀ29
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been developed and tested for CA IX imaging with PET in vivo.
Compound [ F]Uꢀ104
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proved ineffective due to poor PK. [ F]VM4ꢀ037 was found to be safe for use in healthy
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volunteers , but no CA IX dependent uptake was found in vivo.
In a recent phase II pilot
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study of two patients with clear cell renal cell carcinoma (RCC) primary tumors with this
agent uptake was observed in both healthy and cancerous kidney as well as metastases, and
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CA IX selectivity was not confirmed , limiting the use of this imaging agent in RCC. Only
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the trivalent sulfonamide compound, [ F]AmBF
3
ꢀ(ABS)
, enabled tumor visualisation with a tumor to blood
ratio (TBR) of 2.88 ± 1.81 in CA IXꢀexpressing HTꢀ29 tumors 1 h after injection,
3
, to date demonstrates imaging
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efficacy in vivo.26 [ F]AmBF
ꢀ(ABS)
3 3
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interestingly its monovalent variant, AmBF ꢀABS, was ineffective. No discrimination was
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shown between CA IXꢀexpressing or nonꢀexpressing tumors, however, preꢀinjection of
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3 3
acetazolamide effectively blocked uptake of [ F]AmBF ꢀ(ABS) in the tumor. Recently
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another series of monoꢀ, diꢀ and trivalent sulfonamides based on GaꢀDOTA as the PET
reporter group were tested in a CA IXꢀexpressing HTꢀ29 tumor xenograft, again only the
trivalent sulfonamide had a TBR that significantly differed to that of controls (where test
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animals are first treated with acetazolamide as a CA IX blocking sulfonamide).
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Ga (t1/2 = 68 minutes) has been used to label small molecules, biological macromolecules as
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well as nanoꢀ and microꢀparticles. It is a favourable positron emitter because its γ emission
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is negligible and it can be produced in a Ge/ Ga generator, an onꢀsite cyclotron is not
34
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required. As the parent radionuclide Ge has a long half life (t1/2 = 270.8 days) it can be
34
stored for relatively long periods. To make PET imaging with sulfonamides suitable for
eventual use in cancer patients, the purpose of the present work is to design and synthesize
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novel small molecule Ga labelled imaging agents that can selectively target CA IX positive
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tumor cells in vivo. Specifically, agents with improved PK properties, CA IX targeting, TBR
and image contrast than those previously described are sought.
A
O
P
H
O
18F
N
OH
18F
O
N
N
N
18F
N
N
Br
OH
O
O
N
O
N
N
O
NH
N
N
O
N
N+
OH N
N+
O
O-
N
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Br
O
O-
[18F]-MISO
[18F]-FAZA
[18F]-HX4
TH-302
H
N
B
O
N
H2N
N
O
S
18F
O
N
N
O
O
S
NH2
O
HO
O
O
S
O
N
H
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18F
H
[18
F]VM4-037
[18
F]-U-104
O
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n
H
O
N
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S
NH2
N
O
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NH2
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O
O
NH2
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H
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NH
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N
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N
N
N
n
n
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S
N
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N
H2N
O
n= 0 or 2
O
O
N
B
F
F
NH
F
n
[18
F]AmBF3-ABS
[18
F]AmBF3(ABS)3
H2NO2S
O
HN
O
HN
O
N
O
HN
O
SO2NH2
H
O
SO2NH2
O
N
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N
N
N
68
N
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68Ga
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N
O
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Ga
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N
O
O
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O
O
NH
O
HO
H2NO2S
O
NH
[68
Ga]-DOTA-AEBSA
[68Ga]-DOTA-(AEBSA)2
[68Ga]-NOTGA-(AEBSA)3
H2NO2S
Figure 1: A) Nitroimidazole hypoxia targeted cytotoxic drug THꢀ302 and small molecule
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nitroimidazole PET imaging agents for hypoxia: FꢀMISO, FꢀFAZA and FꢀHX4. B)
Small molecule primary sulfonamides investigated in animal models for CA IX imaging with
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PET.
Results and discussion
Compound design and synthesis. Most small molecule CA inhibitors incorporate a primary
sulfonamide functional group, which imparts molecular recognition specificity for the zinc in
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the active site of CAs, but not the metal of other metalloenzymes. The active site of CA IX
is, however, structurally similar to CA I and CA II, the major CA isozymes within red blood
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cells, (CA I: 1.6 ± 2.3 mg/g hemoglobin (Hb); CA II: 1.8 ± 0.3 mg/g Hb). As a consequence
of the binding of sulfonamide based imaging agents to CA I and II in red blood cells,
increased background signal and reduced image contrast have hampered the efforts of others
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in this field.
Our group has contributed substantially to the development of CA
inhibitors with enhanced selectivity for CA IX over CA I and CA II in vitro, and via
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extrapolation, in vivo. We have shown that the different CA active sites have variable
tolerance to the nature of moieties appended to the aromatic sulfonamide CA targeting
3
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group.
This attribute allows fineꢀtuning of the bioactivity, physicochemical and
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toxicological properties of the compound to better target a particular CA isozyme. The CA
nat
67
nat
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IX targeting agents of this study, compounds 1 ( Ga and Ga) and 2 ( Ga, Ga and Ga)
extend on our established design principles. These compounds are primary sulfonamides
tethered to a metal chelator, DOTA (1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7,10ꢀtetraacetic acid)
via either an intervening aliphatic triazole linker (1) or hydrophilic triazole PEG linker (2),
Figure 2. The DOTA macrocycle is the workhorse metal ion chelator for molecular imaging
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44 68
agents, forming stable complexes with the PET imaging isotope Galliumꢀ68, Ga.
Ga is
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becoming a relevant isotope for routine clinical examinations with Ga PET imaging agents
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such as GaꢀDOTATATE and GaHBEDꢀPSMA in clinical use.
Ga is a common
radionuclide for use with Single Photon Emission Computed Tomography (SPECT). The
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most widely used application is of Gaꢀcitrate for inflammation and infection imaging. The
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relatively long halfꢀlife (t1/2 = 3.26 days) makes Ga a useful tool for the assessment of key
parameters of galliumꢀbased radiopharmaceuticals; including radiochemical stability,
metabolic stability and plasma protein binding, this in turn informs subsequent decisions on in
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vivo protocols. The preparation of Ga complexes also permits the optimization of
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radiolabelling conditions, purification methodology and reformulation procedures prior to
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using the shorter halfꢀlife PET radionuclide Ga. We first synthesised the ‘cold’ compounds,
nat
nat
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Gaꢀ1 and Gaꢀ2, followed by the corresponding radiolabelled compounds Gaꢀ1 and Gaꢀ
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2
to establish optimized radiolabelling conditions. Gaꢀ2 was selected as the target compound
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for in vivo PET imaging studies.
O
O
O
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Ga
O
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O
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O
Ga
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O
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N
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O
N
N
SO2NH2
SO2NH2
H
H
N
N
O
O
natGa-1
[67
Ga]-1
natGa-2
67Ga]-2
[
[68
Ga]-2
Figure 2: Target [sulfonamide]ꢀ[triazole linker]ꢀ[DOTA] compounds for use as CA IX
imaging agents.
The compound design as [sulfonamide]ꢀ[variable linker]ꢀ[DOTA] is deliberately modular.
This enables a straightforward synthesis using copper catalyzed azideꢀalkyne cycloaddition
(CuAAC), or ‘click chemistry’, to combine the components. CuAAC is one of the most
accomplished reactions for combining groups to pool their individual properties into a single
47
molecule. The biopharmaceutical stability of the resulting triazole is favourable, it is
resistant to acidic, basic, reductive, oxidative conditions in addition to enzymatic
48
degradation. Scaffold 2 employs a tetraethylene glycol linker, which is a shortened
polyethylene glycol (PEG) chain with good biopharmaceutical properties, polarity and water
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solubility, aiding the eventual formulation. The PEGꢀbased linker was additionally selected
to enhance the likelihood that the agents would have improved specificity for CA IX. The
increased polarity reduces plasma protein binding and membrane permeability and thus may
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lessen the offꢀtarget binding to CA I and II in red blood cells. A previous generation of
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DOTA based scaffolds were designed by Rami et al but to the best of our knowledge, these
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were not radiolabelled or evaluated as CA IX imaging agents in vivo.
Target compounds 1 and 2 are synthesized from three modular components, [sulfonamide],
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[linker] and [DOTA], with incorporation of the gallium cation as the final step (Scheme 1 and
2). The synthesis of the [sulfonamide] component, 4ꢀethynyl benzene sulfonamide 5, has
53
been described previously. The [linker] components, 4 and 11, were designed with
orthogonal end groups. An azide facilitates reaction with 5 via CuAAC, while the bromide
provides an orthogonal leaving group facilitating the S 2 substitution reaction with the
N
[DOTA] component 7. Linkers 4 and 11 were prepared by reaction of bromoacetylbromide
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(
3.3 equiv) with the amino azides 3 and 10, respectively. The [sulfonamide] 5 and [linker]
components 4 and 11 were subjected to CuSO (0.01 equiv), sodium ascorbate (0.1 equiv) and
4
TBTA (0.01 equiv) to generate 6 and 12, respectively. The removal of excess copper ions
from 6 and 12 was achieved by a solid EDTA chase or by washing the organic phases with
EDTA (1.0 M) in ammonium hydroxide (28.0ꢀ30.0%, NH basis) solution. The [DOTA]
3
component 7 was prepared from commercially available cyclen as described by Prashun et
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N
al. S 2 substitution of 6 and 12 with 7 using anhydrous conditions gave the tBuꢀprotected
compounds 8 and 13 in reasonable yields. Treatment of compounds 8 and 13 with either neat
formic acid or TFA/DCM (1:1) removed the tBu protecting groups to provide the nonꢀ
nat
metallated precursor compounds 9 and 14 in high yield. Next, the target Ga complexes,
nat
nat
Gaꢀ1 and Gaꢀ2, were prepared in quantitative yield from 9 and 14 using Ga(NO
in H O with pH adjusted to pH 4.5 with 1.0 M HCl or 1.0 M KOH. Compounds 9 and 14
were purified by reverseꢀphase HPLC prior to biological evaluation and radiolabelling with
3
)
3
2
•xH O
2
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Ga and/or Ga.
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nat
a
Scheme 1. Synthesis of [ Ga]-1.
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a
Reagents and conditions: (i) Bromoacetylbromide (3.3 equiv), 1.0 M aqueous NaOH (3
4
equiv), DCM, rt, 18 h ; (ii) Compound 5 (1.0 equiv), CuSO (0.05 equiv), sodium ascorbate
(0.1 equiv), TBTA (0.05 equiv), 2:1 DMSO:H O, 45 °C, 3 h (iii) Compound 7 (1.2 equiv),
2
K
2
CO
3
(1.2 equiv), anhyd MeCN, rt, 18 h; (iv) 1:1 TFA:DCM, rt, 18 h (v) Ga(NO
3
)
3
2
•xH O
(1.1 equiv), H
2
O, 80 °C, 2ꢀ4 h.
nat
a
Scheme 2. Synthesis of [ Ga]ꢀ2.
O
O
(
i)
(ii)
O
Br
O
Br
O
H2N
N3
N
N3
3
3
N
N
N
SO2NH2
O
H
H
3
SO2NH2
N
10
Br
11
1
2
Br
O
tBu
N
O
5
O
N
OtBu
(iii)
N
HN
OtBu
O
O
7
O
O
HO
O
tBu
N
O
O
O
N
N
N
O
N
N
N
N
(v)
OH
(iv)
O
Ga
O
O
O
O
O
N
O
N
O
tBu
N
O
N
O
N
N
N
N
N
N
H
3
SO2NH2
N
H
N
N
H
3
SO2NH2
OH
N
3
SO2NH2
N
O
tBu
N
O
O
natGa-2
14
13
a
Reagents and conditions: (i) Bromoacetylbromide (3.3 equiv), NaOH (1.0 M, 2 equiv),
DCM, rt, 18 h ; (ii) Compound 5 (1.0 equiv), CuSO (0.01 equiv), sodium ascorbate (0.1
4
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
equiv), TBTA (0.01 equiv), 2:1 DMSO:H
2
O, 30 °C, 18 h (iii) Compound 7 (1.2 equiv),
K CO (1.3 equiv), anh MeCN, 60 °C, 3 h; (iv) 1:1 TFA:DCM, rt, 18 h (v) Ga(NO ) •xH O
2
3
3 3
2
2
(1.1 equiv), H O, 80 °C, 2ꢀ4 h.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Carbonic Anhydrase Binding
nat
nat
The CA binding data for Gaꢀ1, Gaꢀ2 and the reference CA inhibitor acetazolamide were
measured for the cancerꢀassociated CA isozymes, CA IX and XII, and off target CA
nat
nat
isozymes, CA I and II. Compounds Gaꢀ1 and Gaꢀ2 have low affinity for CA I, but bind
equally well to CA II, CA IX and CA XII (K range 59.6 – 84.7 nM) (Table 1). Binding to CA
i
II supports the significance and importance of designing probes to have reduced cell
membrane permeability as this limits access of the probes to the intracellular CA II.
nat
Table 1. Inhibition data for human CA isozymes I, II, IX and XII with compounds Gaꢀ1,
nat
Gaꢀ2 and reference compound acetazolamide.
a,b
c
K
i
(nM)
Selectivity
Compd
hCA I
hCA II
hCA IX hCA XII CA I/CA CA II/CA CA XII/
IX
10
IX
CA IX
n/a
d
acetazolamide
250
387
169
12
25
n/a
0.48
0.85
1.24
nat
Gaꢀ1
72.5
78.3
84.7
63.1
59.6
56.8
4.57
2.67
0.70
0.90
nat
Gaꢀ2
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9
a
b
Errors in the range of ± 5% of the reported value, from three determinations. Measured
using a stopped flow assay that monitors the physiological reaction (CA catalysed hydration
57, 58
2
of CO ).
c
d
Selectivity is determined by the ratio of K s for CA isozymes I, II and XII relative to CA IX.
i
10
11
12
13
14
15
16
17
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23
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44
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49
50
51
52
53
54
55
56
57
58
59
60
5
9
Literature AZA values
Radiochemistry
6
7
Compounds 9 and 14 were successfully radiolabelled with Ga under standard conditions,
.1 M sodium acetate, pH 4.5, 10 min, 95 °C. To test the robustness of the radiolabelling
0
method developed, the compound amount was reduced from 10 nmol progressively to 1 nmol
and the radiochemical yield quantified by reverseꢀphase HPLC (Table 2). It was shown that 9
67
→
[ Ga]ꢀ1 was successfully radiolabelled down to 2 nmol of compound (98%) but at 1 nmol,
67
only 61% radiolabelling was achieved. However, 14 → [ Ga]ꢀ2 radiolabelled efficiently
down to 1 nmol compound (>99%). Purification of radiolabelled products was carried out
67
using either RPꢀHPLC or rapid reverse phase Cꢀ18 solid phase extraction (SPE). Both [ Ga]ꢀ
67
1
and [ Ga]ꢀ2 were reformulated into PBS.
67
67
a
Table 2. Radiochemical yield of [ Ga]ꢀ1 and [ Ga]ꢀ2 at varying compound concentrations.
b
Compound amount
Radiochemical yield (%)
67
67
(
nmol)
9 ꢀ[ Ga]ꢀ1
14 ꢀ [ Ga]ꢀ2
c
2
1
5
0
100
100
d
d
98 ± 1.4
98.5 ± 0.7
5
n/a
>99
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9
1
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1
2
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2
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2
2
2
2
3
3
3
3
3
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3
3
3
3
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4
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5
5
5
5
5
5
5
5
5
5
6
2
1
98
61
100
>99
a
Reaction conditions: Compound 9 or 14 (1 mM in water), sodium acetate (0.1 M, to pH 4.5),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
67
b
GaCl
3
(18ꢀ21 MBq in 0.1 M HCl), 95 °C, 10 min. As determined by RPꢀHPLC (conditions
c
available in supporting information). Labelling in HEPES buffer proceeded with >99%
d
radiochemical yield. ± standard deviation based on two radiolabelling experiments.
6
8
68
68
Compound 14 was radiolabelled with Ga (200ꢀ800 MBq), eluted from a Ge/ Ga generator
IDB Holland, The Netherlands) in about 1 mL, in 400 µL in 3.0 M sodium acetate or
ammonium acetate, pH 4.3, 10 min, 99 °C in high radiochemical purity (> 95%,) as
determined by radioꢀRPꢀHPLC (Inertsil ODS C18, 5 µM, 4.6 × 250 mm, 100:0 → 0:100 H
0.1% TFA/MeCN + 0.1% TFA, 1.0 mL/min).
(
2
O
+
Biopharmaceutical properties
6
7
67
67
The stability of Ga radiolabelled compounds [ Ga]ꢀ1 and [ Ga]ꢀ2 was examined in
phosphate buffered saline (PBS), pH 7.4. The compounds were found to be stable, with ≥95%
of the parent compounds remaining after 18 h incubation at 37.5 °C and ≥90% after 96 h
67
67
incubation. The protein binding of [ Ga]ꢀ1 and [ Ga]ꢀ2 to human serum was minimal (< 7%,
67
n = 3) after 48 h. [ Ga]ꢀ2 exhibited favourable radiochemical purity (data not shown), good
stability and good preliminary physicochemical properties, hence additional
nat
biopharmaceutical properties of cold Gaꢀ2 were assessed (Table 3), by extrapolation these
68
properties should reflect those expected for the radiolabelled analogue [ Ga]ꢀ2. The in vitro
nat
metabolic stability of compound Gaꢀ2 in mouse liver microsomes was measured in the
presence and absence of NADPH, the cofactor required for oxidative metabolism by
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9
nat
CYP450s. Compound Gaꢀ2 exhibited minimal microsomal degradation (t1/2 > 247 min) and
nat
it is expected that compound Gaꢀ2 is subject to low hepatic clearance in vivo. The in vitro
nat
intrinsic clearance of Gaꢀ2 was low (< 7 µL/min/mg protein). The in vitro membrane
nat
nat
permeability (Papp) of Gaꢀ2 in the Cacoꢀ2 cell model (pH 7.4) was measured. Gaꢀ2 was
not detected in the Cacoꢀ2 assay acceptor chamber, while good mass balance (92% ± 6%)
confirmed minimal retention of the compound within the cell monolayer and minimal
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ1
nonspecific adsorption. The experimental value measured, Papp <0.7 cm s , indicates that
nat
Gaꢀ2 has very low cell membrane permeability. The stability and extent of plasma protein
nat
nat
binding of Gaꢀ2 in mouse plasma was analyzed. The measured concentration of Gaꢀ2 in
mouse plasma samples (37 °C) quenched at 2 min was unchanged. However, at 10 min, the
nat
concentration of Gaꢀ2 had dropped but then remained steady over the remainder of the 4 h
nat
incubation. Plasma protein binding of Gaꢀ2 was low (39%) following 4 h incubation.
nat
Cytotoxicity and cell viability of Gaꢀ2 were tested via a standard methyl thiazolyl
nat
tetrazolium assay, with no toxicity observed up to 1 mM of Gaꢀ2 in normoxia (data not
nat
shown). Collectively, these additional properties of Gaꢀ2 are indicative of a safe, well
tolerated compound with physicochemical properties suited to preferential targeting of CA IX
68
over intracellular CAs; hence a favorable TBR ratio of the corresponding Ga compound.
nat
Table 3. Biopharmaceutical properties of compound Gaꢀ2
Compd Degradation
In vitro CLint
Microsomeꢀ
Papp
Plasma
protein
a
b
(
µL/min/mg protein)
Predicted E
H
a
ꢀ1 c
t1/2 (min)
(cm.s )
d
binding (4 h)
nat
Gaꢀ2
>247
<7
<0.13
<0.7
39%
a
Values are represented as mean ± SD (n = 3). Metabolic stability parameters for compound
nat
Gaꢀ2 based on NADPHꢀdependent degradation profiles in mouse liver microsomes. In vitro
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9
1
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1
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2
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2
2
2
2
2
2
3
3
3
3
3
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3
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5
5
5
5
5
5
5
6
b
c
intrinsic clearance value (CLint, in vitro). Predicted in vivo hepatic extraction ratio (E
H
).
d
Papp = apparent permeability across Cacoꢀ2 monolayers. Values are the average of duplicate
determinations.
0
1
2
3
4
5
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7
8
9
0
1
2
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8
9
0
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9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Small animal imaging PET and PET/CT studies
68
Our lead compound, [ Ga]ꢀ2, was chosen for follow up in vivo PET studies and has been
injected intravenously in mice bearing HCT116 tumors with high or low CA IX expression to
assess selectivity of uptake using PET. The efficiency of CA IX genetic silencing was
determined by Western blot analysis and immunofluorescence. In agreement with previous
60, 61
studies,
CA IX levels were significantly lower in CA IXꢀknockdown compared to CA IXꢀ
expressing tumors (Figures 3A & 3B). Additionally, as determined by immunofluorescence
staining, membranous CA IX expression coꢀlocalized with the exogenous hypoxia marker
pimonidazole in CA IXꢀexpressing tumors, whereas in CA IXꢀknockdown tumor hypoxia,
very low or no CA IX expression was present (Figure 3C). This confirms efficient CA IX
68
knockdown and thus a CA IXꢀdependent uptake of [ Ga]ꢀ2.
In order to determine the optimal imaging time point, µPET scans were acquired hourly from
68
1
to 4 h post injection (h p.i.) of [ Ga]ꢀ2. Tumor uptake in the CA IXꢀexpressing model was
clearly observed and found to be highest at 1 h p.i. (Figure 4A). This time point was selected
for all subsequent experiments. Additionally, it was found that the agent was rapidly excreted
renally, as observed by the high presence of the agent in the kidneys and bladder. Mass
spectrometry analysis of the urine confirmed the compound was cleared without metabolism.
To assess compound selectivity, uptake was compared between mice with either CA IXꢀ
expressing or CA IXꢀknockdown tumors. Although CA IXꢀknockdown tumors tend to grow
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slower compared to their CA IXꢀexpressing counterparts, tumor volumes at time of scans
were not statistically different (P = 0.422) between the two groups (315 ± 104 and 277 ± 63
3
mm for CA IXꢀexpressing and CA IXꢀknockdown tumors respectively). The TBR was
significantly higher (P < 0.01) in mice bearing CA IXꢀexpressing (3.87 ± 1.34) as compared
to mice bearing CA IXꢀknockdown (1.99 ± 0.99) tumors (Figures 4B & 4C). Uptake of
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56
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58
59
60
68
[
Ga]ꢀ2 was therefore found to be CA IXꢀdependent. However, lack of anatomical
information in the acquired PET images prompted us to verify coꢀlocalisation with CT.
Therefore experiments were repeated to include CT scans, enabling better tumor delineation
in the fused PET/CT images and more clearly confirming the localization of the agent in the
tumor. Again tumor volumes at time of scans were not statistically different (P = 0.071)
3
between the two groups (492 ± 390 and 125 ± 44 mm for CA IXꢀexpressing and CA IXꢀ
knockdown tumors respectively). Similar to the first experiment, TBR was significantly
higher (P < 0.01) in mice bearing CA IXꢀexpressing (2.36 ± 0.424) tumors as compared to
mice bearing CA IXꢀknockdown (1.30 ± 0.350) tumors (Figures 5A & 5B). Autoradiography
analysis of tumor sections supported the µPET results, showing a higher signal intensity
ꢀ7
ꢀ7
relative to injected dose (ID) in CA IXꢀexpressing tumors (9.12×10 ± 7.25×10 ) as
ꢀ7
ꢀ7
compared to CA IXꢀknockdown (3.84×10 ± 1.53×10 ) tumors (Figure 5C). Low uptake of
68
[ Ga]ꢀ2 has been also observed in CA IXꢀknockdown model, which can be explained by
residual CA IX expression in these tumors. Nevertheless, significantly higher uptake of
68
[
Ga]ꢀ2 in tumors with high CA IX expression confirms selectivity of this imaging
compound.
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0
1
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0
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9
0
1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. CA IX expression in mice bearing CA IXꢀexpressing (shNT) or CA IXꢀknockdown
(shCA IX) tumors. A) Western blot showing CA IX protein levels in representative samples.
B) Quantification of CA IX protein levels as determined by Western blot for all shNT and
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9
shCA IX tumors. C) Immunofluorescence staining of CA IX (red) and the hypoxia marker
pimonidazole (green). Rightꢀside images are magnifications of areas within the white
rectangles in the leftꢀside images. “n” indicates necrotic areas.
10
11
12
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14
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
8
Figure 4. [ Ga]ꢀ2 uptake in mice bearing CA IXꢀexpressing (shNT) or CA IXꢀknockdown
(
shCA IX) tumors. A) Representative µPET scans 1ꢀ4 h p.i. in a shNTꢀtumor bearing mouse.
68
B) Representative µPET scans 1 h p.i. C) TBRs of [ Ga]ꢀ2 uptake determined from PET
scans of shNTꢀtumor bearing mice (n = 11) and shCA IXꢀtumor bearing mice (n = 6). **P <
0.01.
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9
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2
2
2
2
2
2
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3
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3
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3
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3
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0
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0
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9
0
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9
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1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
68
Figure 5: [ Ga]ꢀ2 uptake in mice bearing CA IXꢀexpressing (shNT) or CA IXꢀknockdown
68
(
shCA IX) tumors. A) Representative µPET/CT fusion images 1 h p.i. B) TBRs of [ Ga]ꢀ2
uptake determined from PET scans of shNTꢀtumor bearing mice (n = 4) and shCA IXꢀtumor
bearing mice (n = 5). ** P < 0.01. C) Signal intensity relative to injected dose (ID) as
determined by autoradiography analysis on tumor sections from shNTꢀtumor bearing mice (n
=
6) and shCA IXꢀtumor bearing mice (n = 6).
Conclusion
This study provides the first evidence of nonꢀinvasive, specific detection of CA IX in vivo,
68
using a CA IX targeting small molecule PET radiotracer, [ Ga]-2. The synthesis of the
68
68
unlabeled precursor of [ Ga]-2 (compound 14) and the radiochemistry to introduce Ga
was straightforward, proceeded in good yields and was reproducible while the
biopharmaceutical properties were favorable. This study is a promising step towards a new
predictive tool that will enable testing of the potential of CA IX expression as a biomarker for
selection of patients eligible for CA IXꢀtargeting anticancer therapies.
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9
Experimental
Chemistry ꢀ General Methods and Synthesis
All starting materials and reagents were purchased from commercial suppliers. Where
specified, solvents were available commercially dried or dried prior to use. Reactions took
place open to the atmosphere unless otherwise specified. Reaction progress was monitored
by: TLC using silica gelꢀ60 F254 plates with detection by short wave UV fluorescence (λ =
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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43
44
45
46
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48
49
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51
52
53
54
55
56
57
58
59
60
2
54 nm) and staining with ninhydrin (1 g ninhydrin, 200 mL EtOH, 8mL acetic acid), KMnO
KMnO (0.75g), K CO (5 g), NaOH (75 mg), H O (100 mL) or vanillin stain (5 g of vanillin
in a mixture of EtOH:H O:H SO = 87:10.2:2.8 with subsequent heating; by TLC using RPꢀ
8 silica gelꢀ60 F254s plates with detection by short wave UV fluorescence; by high pressure
liquid chromatography (HPLC) on an Agilent 1100 system using a Thermo Betasil C18 (150
4.6 mm, 5 ꢁm) column. A gradient method was used of 95:5 → 5:95 H O (+ 0.1% TFA):
4
(
4
2
3
2
2
2
4
1
×
2
Acetonitrile (+0.1% TFA) over 10 mins, 1 ml/min flow rate. Silica gel flash chromatography
1
13
1
was performed using silica gel 60 Å (230−400 mesh). NMR ( H, C{ H}, 1Hꢀ1H gCOSY,
1
and HSQC) spectra were recorded on either a 400 or 500 MHz spectrometer at 30 °C. H
NMR spectra were referenced to the residual solvent peak (CDCl
3
δ 7.26 ppm, DMSOꢀd
2.50 ppm). C NMR spectra were referenced to the internal solvent (CDCl δ 77.0 ppm,
DMSOꢀ δ 39.5 ppm). Multiplicity is indicated as follows: s (singlet), d (doublet), t (triplet),
q (quaternary), qn (quintuplet), m (multiplet), dd (doublet of doublet), ddd (doublet of
doublet of doublet), b (broad). Coupling constants ( ) are reported in hertz (Hz). Mestrenova
6
δ
1
3
3
d
6
J
6.1 software was used for NMR analysis. Melting points are uncorrected. Mass spectra (low
and high resolution) were recorded using electrospray as the ionization technique in positive
nat
nat
ion and/or negative ion modes as stated. Purity of all final compounds (8, 9, Gaꢀ1 and Gaꢀ
2
) was ≥ 95% as determined by HPLC with UV detection. Compounds 3, 4, 5, 7 and 10 were
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9
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
3, 54, 56,
synthesised as described elsewhere, with characterisation in agreement with literature.
6
2ꢀ65
Numbering of DOTA compounds used for NMR assignments is shown below.
O
O
tBu
τ
O
O
tBu
τ
N
O
O
τ
τ
εCH
2
O
O
N
N
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
γ
γ
η
ε
tBu
N
N
tBu
N
N
O
O
N
α
N
N
α
N
O
N
τ
ß
tBu
δ
N
τ
ß
tBu
δ
N
H
H
ζ
O
O
2 2
SO NH
2 2
SO NH
Synthesis methods
General Procedure 1: Synthesis of brominated [linker] components
The aminoꢀazide precursor (1 equiv) was suspended in a biphasic DCM/NaOH (aq) solution
(2:1, 2ꢀ3 equiv NaOH) and cooled to 0 °C. Bromoacetyl bromide (3 equiv) was added
dropwise to the DCM layer. The solution was then stirred vigorously overnight at rt. The
reaction mix was diluted with DCM and H
2
O and the aqueous fraction extracted with DCM
CO (50 mM, 3 ),
(2
×
50 mL). The organic fractions were combined and washed with Na
2
3
×
dried with MgSO , filtered and concentrated. The crude compound was sufficiently pure and
4
used crude in the next step.
General Procedure 2: Addition of “cold” Ga
The parent compound (9 or 14, 1 equiv) was suspended in H
2
O and Ga(NO
excess) added. The pH of the reaction was adjusted to ~ pH 4.5 using KOH (1.0 M) or HCl
1.0 M). The reaction mixture was heated at 80 °C and the pH monitored and adjusted
3 3 2
) •xH O (aq)
(
(
accordingly to maintain pH 4.5. The reaction mix stabilised after ~ 2 h. Reaction progress was
monitored by LCꢀMS. Products were purified by RPꢀ18 flash column chromatography
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(H
2
O/MeOH 100:0→5:95%) and the solvent was removed in vacuo leaving a hygroscopic
solid.
N
ꢀ(3ꢀazidopropyl)ꢀ2ꢀbromoacetamide (4). The title compound was synthesized from 3ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
azidoꢀ1ꢀpropanamine HCl salt (3) (1.5 g, 11 mmol) and NaOH (2 equiv) using general
1
procedure 1 and isolated as a yellow oil (1.9 g, 78%). Rf 0.13 (90:10 DCM/MeOH). H NMR
(
500 MHz, CDCl
εCH ), 1.82 (quint, 2H,
εCH or γCH ), 38.1 (εCH or γCH ), 29.3 (βCH ), 28.6 (δCH ). LRMS (ESI ): m/z = 221,
3
)
δ
H
6.72 (br s, 1H, NH), 3.88 (s, 2H, βCH
2
), 3.40ꢀ3.36 (m, 4H, γCH
2
,
13
2
J
= 6.60Hz, δCH
2
). C NMR (125 MHz, CDCl ) 165.8 (αC=O), 49.5
3
ꢀ
(
2
2
2
2
2
2
81
79
ꢀ
+
79
9
+
219 [M–H; Br, Br] . HRMS (ESI) Calcd for C H BrN NaO : 242.9852. Found:
5
4
1
62
2
42.9852. H NMR was in agreement with the data reported in the literature.
2ꢀBromoꢀNꢀ(3ꢀ[4ꢀ(4ꢀsulfamoylphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl]propyl)acetamide (6)
CuSO (7 mg, 0.027 mmol) and sodium ascorbate (10.8 mg, 0.055 mmol) were combined in 1
4
mL H O and added to a solution of alkyne (11) (100 mg, 0.55 mmol), azide (5) (145 mg, 0.66
2
mmol) and TBTA (14.5 mg, 0.055 mmol) in DMSO (2 mL) and the mixture was left to stir at
45 °C and monitored by TLC. Once complete (1.5h), the reaction mixture was filtered
through celite® and washed with H O and the filtrate redissolved in DMF. The DMF was
2
concentrated to a minimum amount before EtOAc (30 mL) was added and the organic phase
rapidly washed with EDTA in ammonium hydroxide (1.0 M, 50 mL). The organic fraction
4
was dried (MgSO ), filtered, and the remaining residue purified by flash column
chromatography (90:10 EtOAc/MeOH). The product was isolated as a white powder (0.04 g,
1
18%). Rf 0.36 (80:20 DCM/MeOH), mp 182ꢀ185 °C. H NMR (500 MHz, DMSOꢀd
6
)
δ
H
8.70
), 7.37
= 6.7 Hz, 2H,
(
s, 1H, triazole CH), 8.39 (t, 1H, NH), 8.02 (m, 2H, 2 × ArCH), 7.89 (m, 2H, 2 × ArCH
(
s, 2H, SO NH ), 4.45 (t,
J
= 7.0 Hz, 2H, εCH ), 3.85 (s, 2H, βCH ), 3.15 (q,
J
2
2
2
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
γCH
2
), 2.08 – 2.02 (quint,
J
= 6.9 Hz, 2H, δCH
2
). C NMR (500 MHz, DMSOꢀd
6 C
) δ 166.2
(αC=O), 145.0 (C ), 143.1 (triazole C
q
q
), 133.9 (Cq
), 126.4 (2 × CHAr), 125.3 (2 × CHAr),
122.6 (triazole CH), 47.4 (εCH2), 36.3 (γCH
2
), 29.5 (βCH
2
2 2
and δCH ), 29.4 (βCH and
ꢀ
81
79
ꢀ
δCH ). LRMS (ESI ): m/z = 402, 400 [MꢀH; Br, Br] . HRMS (ESI) Calcd for
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
C H BrN O S : 402.0228. Found: 402.0208.
13
16
5
3
Triꢀtertꢀbutyl 2,2',2''ꢀ(10ꢀ(2ꢀoxoꢀ2ꢀ((3ꢀ(4ꢀ(4ꢀsulfamoylphenyl)ꢀ1
yl)propyl)amino)ethyl)ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriyl)triacetate (8)
Compound 7 (109 mg, 0.21 mmol), 6 (85 mg, 0.21 mmol) and K CO (58 mg, 0.42 mmol)
Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
2
3
were dissolved in anhyd MeCN (5 mL) and the reaction mixture was stirred overnight at room
temperature. The mixture was filtered through celite® and washed with MeOH. The residue
was purified by flash column chromatography (gradient 100% DCM→ 85:15 DCM/MeOH).
Appropriate fractions were collected together and the product was isolated as a hygroscopic
1
solid (59.3 mg, 33%). Rf 0.04 (95:5 DCM:MeOH). H NMR (400 MHz, DMSOꢀd6, 85 °C) δH
8.63 (s, 1H, CH triazole), 8.17 (t,
J
= 5.38 Hz, 1H, NH), 7.98 (m, 2H, 2
× ArCH), 7.91 (m,
2H, 2 ArCH), 7.20 (s, 2H, SO NH ), 4.45 (t,
×
J
= 7.1 Hz, 2H, εCH ), 3.53 (MeOH), 3.19 (m,
2
2
2
2H, γCH
2
), 3.08 (s, 6H, τCH
2
), 2.94 (dt,
J
= 42.0, 5.2 Hz, 2H, CH
2
Aza), 2.61 (br, 7H, CH
Bu), 1.40 (s,
172.5 (C=O), 172.2 (2 × C=O), 171.6
2
Aza), 2.30 (br, 7H, CH Aza), 2.10 (quint,
J
= 6.9 Hz, 2H, δCH ), 1.44 (s, 9H, t
2
2
13
18H, 2 × tBu). C NMR (75.5 MHz, DMSOꢀd
6
)
δ
C
(C=O), 145.0 (C
q
triazole), 143.1 (C
q
Ar), 133.9 (C
q
Ar), 126.4 (2
×
ArCH), 125.3 (2
×
ArCH), 122.5 (CH triazole), 81.1 (Cq tBu), 80.9 (2 × Cq tBu), 69.8(MeOH), 60.18 (τCH
2
),
5
5.7 (τCH ), 55.3 (τCH ), 54ꢀ49 ꢀ CH Aza in baseline, 47.6 (εCH ), 36.0 (γCH ), 29.6
2
2
2
2
2
+
+
(δCH ), 27.9 (βCH ), 27.5 (CH
t
Bu). LRMS (ESI ): m/z = 836 [M+H] . HRMS (ESI) Calcd
2
3
3
+
65 9 9
for C39H N NaO S :858.4518. Found: 858.4513.
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2,2',2''ꢀ(10ꢀ(2ꢀOxoꢀ2ꢀ((3ꢀ(4ꢀ(4ꢀsulfamoylphenyl)ꢀ1
H
ꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl)propyl)amino)ethyl)ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriyl)triacetic acid (9)
Compound 8 (87.7 mg, 0.1 mmol) was dissolved in DCM/TFA (1:1) (8 mL) and stirred at 40
°
C for 3 h. Reaction progress was monitored by LCꢀMS and RPꢀ18 TLC and conversion was
complete. The solvent was removed in vacuo and then coꢀevaporated with water ( 3)
followed by lyophilization. Rf 0.76 (50:50 MeOH/H O RPꢀ18 TLC). Samples for
radiolabelling were purified using HPLC (isocratic 7:93 MeCN:H O + 0.1% Formic acid –
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
×
2
2
Waters Atlantis® T3 C18, 19 × 150 mm, 10 µM at a flowrate of 12 mL/min). Product
1
fractions were collected and the solvent removed in vacuo (8.5mg, 52% HPLC recovery). H
NMR (500 MHz, DMSOꢀd
m, 2H, 2 ArCH), 7.90 (s, 2H, 2
εCH ), 4.1ꢀ3.0 (m, signals masked by broad H
6
)
δ
H
8.69 (s, 1H, CH triazole), 8.57 (t,
J
= 5.2 Hz, 1H, NH), 8.01
= 6.8 Hz, 2H,
= 5.1 Hz, 2H, γCH ), 2.08
(
×
×
ArCH), 7.38 (s, 2H, SO NH ), 4.48 (t,
J
2
2
2
2
O peak), 3.18 (q,
J
2
1
1
(quint, 2H, δCH ). Correlations under the H O peak were observed in the Hꢀ H gCOSY, and
2
2
+
+
+
HSQC spectra. LRMS (ESI ): m/z = 668 [M+H] . HRMS (ESI) Calcd for C H N O S :
2
7
42
9
9
668.2821. Found: 668.2819.
Nꢀ[2ꢀ[2ꢀ[2ꢀ(2ꢀAzidoethoxy)ethoxy]ethoxy]ethyl]ꢀ2ꢀbromoacetamide (11). The title
compound was synthesized from 11ꢀazidoꢀ3,6,9ꢀtrioxaundecanamine (10) (450 mg, 2.08
mmol) using general procedure 1 and isolated as a yellow oil (0.54 g, 76%). Rf 0.35 (95:5
1
DCM/MeOH). H NMR (500 MHz, CDCl
3
)
δ
H
6.92 (s, 1H, NH), 3.87 (s, 2H, βCH
2
), 3.71 –
3.63 (m, 10H, εCH
2
, ζCH
2
), 3.59 (t,
J
= 5.1 Hz, 2H, δCH ), 3.49 (m, 2H, γCH ), 3.39 (t, J =
2
2
1
3
5.0 Hz, 2H, ηCH
2
N
3
). C NMR (125 MHz, CDCl
3
) 165.8 (αC=O), (70.9, 70.8, 70.7, 70.5,
ꢀ
7
3
3
0.2, εCH ζCH ), 69.5 (δCH ), 50.8 (ηCH ), 40.1 (γCH ), 29.2 (βCH ). LRMS (ESI ): m/z
,
2
2
2
2
2
81
79
ꢀ
+
79
+
39, 337 [MꢀH, Br, Br] . HRMS (ESI ) Calcd for C H BrN O : 339.0662. Found:
1
0
19
4
4
1
66
39.0684. H NMR was in agreement with the data reported in the literature.
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2
2
2
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2
ꢀBromoꢀ
Nꢀ[[2ꢀ(2ꢀ[2ꢀ[4ꢀ(4ꢀsulfamoylphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl]ethoxy]ethoxy)
ethoxy]methyl]acetamide (12)
CuSO (2 mg, 0.007 mmol) and sodium ascorbate (14 mg, 0.071 mmol) were combined in 1
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mL H O and added to a solution of azide (11) (264 mg, 0.78 mmol), alkyne (5) (128 mg, 0.71
2
mmol) and TBTA (4 mg, 0.007 mmol) in DMSO (2 mL) and the mixture was left to stir at 45
°
C and monitored by TLC. Once complete, the reaction mixture was diluted with H
EtOAc and the aqueous phase extracted with EtOAc (4 30 mL). The organic fractions were
combined and washed with EDTA (1.0 M) in ammonium hydroxide (28.0ꢀ30.0%, NH3 basis)
solution, dried (Na SO ), filtered and purified by flash column chromatography (95:5
2
O and
×
2
4
EtOAc/MeOH or DCM/MeOH). The product was isolated as a yellow gum (0.148g, 40%). Rf
1
0
.2 (95:5 DCM/MeOH), mp 85ꢀ90 °C. H NMR (500 MHz, DMSOꢀd
6
)
δ
H
8.66 (s, 1H,
ArCH), 7.90 (m, 2H, 2
= 5.2 Hz, 2H, ηCH ), 3.88 (t, = 5.2 Hz, 2H, ζCH2),
= 5.7 Hz, 2H, δCH NH), 3.20 (q,
166.0 (αC=O), 144.9 (C
triazole CH), 8.28 (br t,
J
= 5.90 Hz 1H, NH), 8.02 (m, 2H, 2
×
×
ArCH), 7.36 (s, 2H, SO NH ), 4.60 (t,
J
J
2
2
2
3
.84 (s, 2H, βCH
.67 Hz, 2H, γCH
ArꢀSO
2
), 3.58 – 3.45 (m, 8H, εCH
2
), 3.39 (t,
J
2
J =
1
3
5
2
). C NMR (500 MHz, DMSOꢀ
d
6
)
δ
C
q
Arꢀ
trizole), 143.0 (C
q
2
NH
2
), 133.9 (C
q
triazole), 126.3 (2
×
ArCH), 125.25 (2
×
ArCH),
1
22.84 (CH triazole), 69.7ꢀ69.5 (εCH , 4C), 68.7 (δCH or ζCH ), 68.6 (δCH or ζCH ), 54.8
2
2
2
2
2
+
81
(DCM), 49.7 (ηCH
2
), 48.6 (γCH
2
), 29.4 (βCH
2
). LRMS (ESI ): m/z = 544, 542 [M+Na; Br,
7
9
+
81
79
+
79
+
Br] ; 522, 520 [M+H; Br, Br] . HRMS (ESI) Calcd for C18
H
26 BrN
5
NaO
6
S : 542.0679.
Found: 542.0681.
Triꢀtertꢀbutyl 2,2',2''ꢀ(10ꢀ(2ꢀoxoꢀ14ꢀ(4ꢀ(4ꢀsulfamoylphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)ꢀ6,9,12ꢀ
trioxaꢀ3ꢀazatetradecyl)ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriyl)triacetate (13)
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2 3
Compound 12 (0.196 g, 0.37 mmol), 7 (253 mg, 0.49 mmol) and K CO (68 mg, 0.49 mmol)
were dissolved in anh MeCN (8 mL) and stirred at 60 °C for 3h. The reaction mixture was
filtered through celite® and washed (MeCN), concentrated and the remaining residue purified
by column chromatography (90:10 DCM: MeOH). Fractions were monitored by HPLC and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
positive fractions combined to give the title compound as a hygroscopic offꢀwhite solid
1
(
0.268 g, 76%). Rf 0.28 (90:10 DCM/MeOH). H NMR (500 MHz, DMSOꢀ
d
6
)
δ
H
8.67 (s,
1
H, CH triazole), 8.18 (t,
J
= 5.9 Hz, 1H, NH), 8.02 (m, 2H, 2
×
ArCH), 7.90 (m, 2H, 2
= 5.1 Hz, 2H, ζCH
×
ArCH), 7.37 (s, 2H, SO
2
NH
2
), 4.59 (t, = 5.1 Hz, 2H, ηCH ), 3.88 (t,
J
2
J
2
),
3.50 (m, 8H, εCH ), 3.41 (t,
J = 5.94 Hz, 2H, δCH ), 3.21 (m, 2H, γCH ), 3.1 ꢀ 2.0 (br, m,
2 2
2
1
3
2
4H, Aza CH
MHz, DMSOꢀd
Ar), 134.0 (C
2
, βCH
2
, 3
172.5 (C=O), 172.1 (2 × C=O), 171.6 (C=O), 144.9 (C
Ar), 126.3 (2 ArCH), 125.2 (2 ArCH), 122.8 (CH triazole), 81.1 (Cq
×
τCH
2
), 1.43 (s, 9H,
t
Bu), 1.41 (s, 18H, 2 × tBu). C NMR (125
6
)
δ
C
q
triazole), 143.1
(C
q
q
×
×
t
Bu), 80.9 (2× Cq tBu), 69.6 ꢀ69.5 (εCH , βCH ), 68.8 (ζCH ), 68.5 (δCH ), 59.7 (τCH ), 55.7
2
2
2
2
2
(τCH
2
), 55.3 (τCH
2
), 49.7 (ηCH
2
), 38.5 (γCH
2
), 27.5 (CH
3
tBu), CH
2
Aza peaks masked in
+
+
+
baseline. LRMS (ESI ): m/z = 954 [M+H] . HRMS (ESI) Calcd for C44
H
75
N
9
NaO12S :
976.5148. Found: 976.5155.
2ꢀ[4,10ꢀBis(carboxymethyl)ꢀ7ꢀ[[[2ꢀ[2ꢀ(2ꢀ[2ꢀ[4ꢀ(4ꢀsulfamoylphenyl)ꢀ1
H
ꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl]ethoxy]ethoxy)ethoxy]ethyl]carbamoyl]methyl]1,4,7,10
yl]acetic acid (14).
tetraazacyclododecanꢀ1ꢀ
Compound 13 (0.219 mg, 0.23 mmol) was dissolved in formic acid (10 mL) or TFA/DCM
(1:1) and stirred at 60 °C. The reaction was monitored by HPLC. On completion, water (10
mL) was added and then the solvent removed in vacuo and then coꢀevaporated with water
(×3) followed by lyophilization. The sample was purified by reverse phase column
chromatography using a gradient of 100% H O → 95:5 MeOH:H O and the product isolated
2
2
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2
2
2
2
2
2
2
3
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4
4
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5
5
5
5
5
5
5
5
5
6
2
as a hygroscopic white gum (144 mg, 80%). Rf 0.53 (50:50 MeOH:H O RPꢀ18 TLC).
Samples were further purified via HPLC prior to radiolabelling (Isocratic 10:90 MeCN: H
2
O
+0.1% Formic acid – Waters Atlantis® T3 C18, 19 × 150 mm, 10 µm, at a flowrate of 12
1
mL/min). Product fractions were collected and solvent removed in vacuo (19%). H NMR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(500 MHz, DMSOꢀd
ArCH), 7.90 (m, 2H, 2
t,
6
)
δ
H
8.67 (s, 1H, CH triazole), 8.17 (br, 1H, NH), 8.02 (m, 2H, 2 ×
×
ArCH), 7.38 (s, 2H, SO NH ), 4.60 (t,
J
= 5.0 Hz, 2H, ηCH ), 3.88
2
2
2
(
J
= 5.3 Hz, 2H, ζCH
2
), the remaining signals were masked by broad H
2
O peak.
1
1
+
Correlations were observed in the Hꢀ H gCOSY, and HSQC spectra. LRMS (ESI ): m/z 786
+
+
[M+H] . HRMS (ESI) Calcd for C H N NaO S : 808.3281. Found: 808.3278.
3
2
51
9
12
2,2',2''ꢀ(10ꢀ(2ꢀOxoꢀ2ꢀ((3ꢀ(4ꢀ(4ꢀsulfamoylphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl)propyl)amino)ethyl)ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriyl)triacetic acid Gallium
nat
complex ( Gaꢀ1)
Compound 9 (48 mg, 0.071 mmol) was treated as per general procedure 2 to give the title
ꢀ
ꢀ
compound as a hygroscopic white solid. LRMS (ESI ): m/z = 734 [MꢀH] . HRMS (ESI) Calcd
+
for C27
H
38GaN
9
NaO
9
S :756.1661. Found: 756.1660.
2ꢀ[4,10ꢀBis(carboxymethyl)ꢀ7ꢀ[([2ꢀ[2ꢀ(2ꢀ[2ꢀ[4ꢀ(4ꢀsulfamoylphenyl)ꢀ1
H
ꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl]ethoxy]ethoxy)ethoxy]ethyl]carbamoyl]methyl]1,4,7,10
tetraazacyclododecanꢀ1ꢀ
nat
yl]acetic acid Gallium complex ( Gaꢀ2).
Compound 14 (50 mg, 0.064 mmol) was treated as per general procedure 2 to give the title
compound as a hygroscopic white solid. Rf 0.65 (RPꢀ18TLC 60:40 H O/MeOH). LRMS
2
+
+
+
(ESI ): m/z 852 [M+H] . HRMS (ESI) Calcd for C H GaN O S : 852.2472. Found:
32
49
9
12
852.2460.
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Page 27 of 43
Journal of Medicinal Chemistry
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Cell culture
®
Human MDAꢀMBꢀ231 breast cancer cells (ATCC ꢀ26™) for toxicity studies were cultured in
Roswell Park Memorial Institute medium (RPMI)ꢀ1640 media supplemented with 10% Fetal
Bovine Serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C and 5%
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4
CO . Cells were seeded at 3×10 cells/well (200 µL, RPMIꢀ1640) and allowed to grow for 24
2
h before being exposed to the compound of interest. Untreated cells were used as a control.
®
HCT116 human colorectal carcinoma cells (ATCC CCLꢀ247™) stably expressing a CA IXꢀ
targeting shRNA (shCA IX) or nonꢀtargeting shRNA (shNT) construct established as
6
1
described previously , were routinely cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% FBS at 37 °C.
Cell proliferation assay
Compounds were dissolved in RPMI media to a final concentration of 1 mM and linear
dilutions ranging from 1 mM – 1 µM were carried out in culture medium. After 24 h of
treatment at 37 °C, 5% CO , MTT (20 µL, 5 mg/mL) was added. After 3 hours, SDS (50 µL,
2
2
0% (w/v) SDS in 0.01M HCl) was added and the plates left overnight before being read by a
SpectraMax fluorescence plate reader (Molecular Devices) at 570 nm. Experiments were
performed in triplicate and where possible, were repeated three times. Values were
determined using the Graphpad prism software.
Radiolabelling
67
Radiolabelling with Ga
67
67
[ Ga]GaCl
3
in 0.1 M HCl was prepared from commercial [ Ga]citrate for injection
67
(
Lantheus) via standard conversion methods. To an acid washed microcentrifuge tube, the
DOTA complex (25 µL, 1 mM H O) and sodium acetate buffer (100 µL, 0.1 M, pH 4.3) were
2
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Journal of Medicinal Chemistry
Page 28 of 43
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3
combined and the solution agitated. GaCl (18ꢀ21 MBq in 0.1M HCl) was added and the
solution heated at 95 °C for 10 mins using a solid state heating block. The sample was stirred
via convection. Once complete, a sample (20 µL, ~3 MBq) was analysed by HPLC and the
radiochemical purity noted. Solutions were purified by HPLC (Atlantis® T3, 10 × 250 mm, 5
µM, flow rate 3 ml/min) (or rapid reverse phase Cꢀ18 solid phase extraction (SPE, washed
0
1
2
3
4
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8
9
0
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9
0
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0
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8
9
0
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7
8
9
0
2 2
with copious amounts of H O and compounds eluted in 50:50 EtOH:H O).
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[
Ga]ꢀ1 and [ Ga]ꢀ2 Stability versus Human serum
Human serum (150 µL), was placed in a previously acid washed 0.5 mL microcentrifuge tube.
To this was added 10× PBS concentrate (15 µL), water (60 µL) and the radioꢀcomplex (75 µL
in water, 0.24 ꢀ0.28 MBq). The mixture was agitated via a bench vortex mixer and then
incubated at 37.5 °C for the course of the study. Aliquots (5 ꢀ15 µL) were injected onto a
Phenomenex Biosep SECꢀS 3000 column (300mm × 7.8 mm ID, 5 µm, pore size 290 Å), with
a mobile phase of 50 mM sodium phosphate, 300 mM NaCl, pH 7.0, 0.2 µm filtered) at 1
mL/min on a HPLC system. Serum uptake was assessed as a percentage of the total activity in
chromatogram.
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7
67
[
Ga]ꢀ1 and [ Ga]ꢀ2 Stability in Phosphate buffered saline
10× PBS concentrate (20 µL), water (160 µL) and the radioꢀcomplex (20 µL in water, 0.2 ꢀ
.23 MBq) were added together in a previously acid washed 0.5 mL microcentrifuge tube.
The solution was agitated and then incubated at 37.5 °C for the course of the study. Aliquots
5 ꢀ50 µL) were injected onto a Waters Atlantis® T3 C18 column (150 mm × 4.6mm I.D., 3
0
(
µm), with a mobile phase of MeCN/ammonium formate buffer (120 mM, pH 4.43, 0.2 µm
filtered) (16:84) at 0.6 mL/min on a HPLC system. Stability was assessed as a percentage of
parent radioꢀcomplex.
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Page 29 of 43
Journal of Medicinal Chemistry
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Radiolabelling with Ga
Reaction progress was monitored by radioꢀHPLC equipped with an Inertsil ODS C18, 5 µM,
.6 × 250 mm column using a gradient method (100% H O → 100% MeCN over 17 mins, 1
ml/min). Sodium acetate or ammonium acetate buffer (3 M, 400 µL, pH 4.3ꢀ4.5) combined
4
2
10
11
12
13
14
15
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6
8
with ~800 MBq Ga in aqueous HCl (0.6M, ~1.2 mL) which was eluted from an iThemba
68
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1
2
480 MBq Ge/ Ga generator. Compound 14 in H O (40 µL, 1 mM) was added and the
solution was mixed by Eppendorf pipette (pH ~4.5) then heated at 99 °C for 10 mins.
Reaction mixtures were analysed by radioꢀHPLC and radiochemical purity was consistently
68
>95%. The specific activity of [ Ga]ꢀ2 at the time of radiolabelling was ~20 MBq/nmol (800
6
8
MBq/40nmole). Small animal imaging PET studies with [ Ga]ꢀ2 were carried out between 1ꢀ
68
2
.5 h post radiolabelling of 14 →[ Ga]ꢀ2. No further purification was carried out prior to
imaging. Samples (~3.7 MBq) were diluted in 0.9% saline solution prior to injection.
CA Inhibition Assay
An Applied Photophysics stoppedꢀflow instrument was used for assaying the CAꢀcatalyzed
58
CO
seven different concentrations of test compound by fitting the curves using PRISM
www.graphpad.com) and nonꢀlinear least squares methods; values represent the mean of at
2
hydration activity. IC50 values were obtained from dose response curves working at
(
3
9
least three different determinations as described previously. The inhibition constants (
were then derived by using the Cheng−Prusoff equation as follows:
= IC50/(1 + [S]/
where [S] represents the CO concentration at which the measurement was carried out and
the concentration of substrate at which the enzyme activity is at half maximal. All enzymes
K
i
)
K
i
K
m
),
2
K
m
68, 69
used were recombinant, produced in Escherichia coli as reported earlier.
The
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