Synthesis of a DOTA (Gd3+)-conjugate of proton-pump inhibitor pantoprazole for gastric wall imaging studies

Article abstract of DOI:10.1016/j.bmcl.2012.06.051

Magnetic resonance imaging (MRI) is used to evaluate gastrointestinal (GI) structure and functions in humans. Despite filling the viscus lumen with a contrast agent, visualization of the viscus wall is limited. To overcome this limitation, we de novo synthesized a conjugate that covalently combines a Gd-based MRI contrast agent, encaged with a chelating agent (DOTA), with pantoprazole, which is a widely used proton pump inhibitor that binds to proton pumps in the stomach and colon. The DOTA linkage was installed at a mechanism-based strategic location in the pantoprazole molecule to minimize a possible negative effect of the structural modification on the drug. It is anticipated that by defining the wall of the stomach and colon, this compound will facilitate functional MRI of the GI tract in humans.

Full text of DOI:10.1016/j.bmcl.2012.06.051

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2808–2811
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a DOTA (Gd3+)-conjugate of proton-pump inhibitor
pantoprazole for gastric wall imaging studies
a,
Ghulam M. Maharvi a, , Adil E. Bharucha b, , Abdul H. Fauq
⇑
⇑
a Chemical Synthesis Core Facility, Mayo Clinic Jacksonville, FL 32224, USA
b Gastroenterology and Hepatology, Mayo Clinic, 200 First Street, S.W., Rochester, MN 55905, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Magnetic resonance imaging (MRI) is used to evaluate gastrointestinal (GI) structure and functions in
humans. Despite filling the viscus lumen with a contrast agent, visualization of the viscus wall is limited.
To overcome this limitation, we de novo synthesized a conjugate that covalently combines a Gd-based
MRI contrast agent, encaged with a chelating agent (DOTA), with pantoprazole, which is a widely used
proton pump inhibitor that binds to proton pumps in the stomach and colon. The DOTA linkage was
installed at a mechanism-based strategic location in the pantoprazole molecule to minimize a possible
negative effect of the structural modification on the drug. It is anticipated that by defining the wall of
the stomach and colon, this compound will facilitate functional MRI of the GI tract in humans.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 26 April 2012
Revised 13 June 2012
Accepted 15 June 2012
Available online 23 June 2012
Keywords:
Pantoprazole
Imaging
Stomach wall
Contrast agent
DOTA
Conjugate
The role of membrane proteins as transporters and mediators of
cellular interaction with environment make them excellent targets
for drug targeting. These enzymes, located in the cellular lipid bi-
layer, exert their biological effects through active transport mech-
anisms that involve pumping specific cations through the cell.1–3
This cation flux is crucial for cellular control of electrochemical
gradients and cell homeostasis. Some of these proton pump inhib-
itors (PPIs), such as gastric H+,K+-ATPase inhibitors, exemplified by
pantoprazole, omeprazole, lansoprazole and rabeprazole—Fig. 1)
are used to treat peptic ulcer and gastroesophageal reflux disease.4
In conditions such as functional dyspepsia and gastroparesis,
disturbances of gastric motility can accelerate or delay gastric
emptying.5 Existing techniques to assess stomach functions are
limited by radiation exposure (e.g., scintigraphy), invasiveness
(e.g., gastroduodenal motility), and limited spatial resolution. Re-
search studies suggest that MRI can measure gastric volumes and
motility in humans.6,7,10
OMe
OMe
N
OMe
O
H
N
O
S
S
H
N
N
N
OCHF2
N
OMe
Pantoprazole
Omeprazole
CF3
O(CH2)3OMe
O
O
S
HOOCH2C
CH2COOH
H
Na
CH2COOH
COOH
N
S
N
N
N
N
N
N
N
N
HOOC
N
N
OCHF2
N
OCHF2
CH2COOH
Lansoprazole
HOOCH2C
Rabeprazole
CH2COOH
Figure 1. Prototypes of H+,K+-ATPase inhibitors.
(DOTA)
CH2COOH
⇑
N
Corresponding authors. Tel.: +1 507 284 6439 (A.E.B.); tel.: +1 904 953 2845;
fax: +1 904 953 7117 (A.H.F.).
HOOC
N
N
COOH
CH2COOH
CH2COOH
E-mail addresses: maharvi.ghulam@mayo.edu (G.M. Maharvi), Bharucha.adil@
mayo.edu (A.E. Bharucha), fauq.abdul@mayo.edu (A.H. Fauq).
Co-author. Tel.: +1 904 953 8034; fax: +1 904 953 7117.
Figure 2. Polyamino polycarboxylate ligands.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.051

G. M. Maharvi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2808–2811
2809
OMe
OMe
OMe
N
OMe
O
H+
H+
O
H
N
H
N
+
S
S
-
N
H
N
N
OCHF2
OCHF2
H+
H+
-
H+ -transfer
OMe
OMe
O
H+
H+
O
S
H
N
H
N
+
S
-
N
MeO
+
N
H
N
OCHF2
OCHF2
+
N
MeO
H
H
H+
O
S
H
N
MeO
MeO
MeO
MeO
S
N
OCHF2
N H
+
N
N
+
N
OCHF2
Sulfenamide
Enz-SH
-H2O
H
O
S
S
Enz
MeO
MeO
MeO
MeO
S
N
Enz-SH
-H2O
N
+
N
N
+
N
OCHF2
N
OCHF2
H
H
Figure 3. Proposed mechanism of pantoprazole-type PPI inhibition of ATPase enzyme.
Moreover, for the first time dynamic MRI identified increased
gastric motility in patients with rapid gastric emptying.7
mechanism is considered to involve reversible H+-transfers be-
tween the pyridine nitrogen and the two nitrogen of the benzimid-
azole ring (Fig. 3). Under physiological conditions, the nucleophilic
pyridine nitrogen atom attacks the neighboring highly electro-
philic protonated imine bond (C@N) of the benzimidazole ring
and open it to generate a sulfenate moiety that can either directly
establish a disulfide covalent bond with the enzyme through a cys-
teine residue, or it can bond with the same cysteine after ring clo-
sure to a 1,2,4-thiadiazinium ion. Thus, it appears that the DOTA
linker could not be linked through any of nitrogen atoms because
of their involvement in the enzyme inhibition mechanism. Since
other positions on the pantoprazole-type PPI molecules were al-
ready optimized for activity, we envisioned appending a DOPA-
linked ether at the 5-position of the benzimidazole ring of
pantoprazole.
However, segmenting the stomach from surrounding structures
on MR images is a manual, time-consuming, and rate-limiting pro-
cess. To overcome this limitation and facilitate visualization of the
gastric wall, we sought to develop a novel gadolinium conjugate,
tagged to an intravenous proton-pump inhibitor approved for hu-
man use. A plausible way to encage Gd cations is to use one of the
several polyamino polycarboxylate ligands that are commercially
available (Fig. 2). For our studies reported herein, we opted to
use
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid
(DOTA) as a Gd-entrapped linker on the pantoprazole molecule.
This linker which stably traps Gd-ion by chelation was designed
after Gadoteridol/ProHance model (Bracco Diagnostics, Inc.,
Princeton).
In designing a synthetic strategy for a pantoprazole–DOTA con-
jugate, determination of location of an appropriate point of linker
attachment in the drug molecule was needed. To this end, an
apparent and practically straightforward strategy would appear
to be to link a DOTA moiety through one of the nitrogen atoms
of the benzimidazole ring. However, a plausible approach was to
give due consideration to earlier speculations and observations
regarding mechanism of PPI inhibitory action. Nissen4a and Fujisa-
ki4b have described a mechanism of inhibition of P-type ATPase
inhibitors represented by pantoprazole structural types. This
The synthesis of the pantoprazole–DOTA conjugate (Scheme 1)
started with 5-amino-1-pentanol that was protected with a tert-
butyloxycarbonyl (Boc) group to give 2. After mesylation, the result-
ing mesylate 3 was converted to the iodide 4. A SN2 displacement of
the iodide by 3-nitro-4-aminophenol under basic conditions fur-
nished the phenolic ether 5 in an overall yield of 95% over three
steps. After the linker was installed, the nitroaniline 5 was con-
verted to a diamine 6 by catalytic reduction. A 2-mercaptobenzimi-
dazole ring 7 was then secured by reacting the diamine 6 with
carbon disulfide under basic conditions.8,9 An SN2-displacement of

2810
G. M. Maharvi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2808–2811
H
H
N
MsCl
(Boc)2O
N
OH
H2N
OMs
OH
Boc
Boc
CH2Cl2, Et3N,
CH2Cl2, r.t. 2 h,
100%
2
-10 oC, 30 min, 97%
3
NH2
NO2
NH2
NO2
H
H
N
KI, DMF
70 oC, 2 h, 92%
HO
O
N
I
Boc
Boc
NaH, DMF, 0 oC-r.t.
1.5 h, 92%
5
4
SH
N
NH2
O
HN
O
NH2
CS2
H2, 10% Pd-C
EtOH, KOH/H2O,
80 oC, 3 h, 92%
Boc
HN
MeOH, r.t.,
1h, 100%
Boc
HN
6
7
OCH3
H
H3CO
OCH3
OCH3
N
N
S
4N 1,4-Dioxane.HCl
r.t., 50 min, 100%
H
N
Cl
O
N
Boc
8
N
9
ACN, Et3N, r.t., 12 h, 97%
OH
O
HO
H
H3CO
S
OCH3
N
N
N
N
O
N
O
O
O
+
O
ClH.H2N
N
N
N
10
O
11
HO
OH
O
O
H3CO
N
HO
N
N
N
H3CO
S
O
N
O
GdCl3, H2O, r.t., 87%
DMSO, r.t., 12h, 98%
N
HN
OH
O
O
N
H
12
OMe
OMe
OH
OH
O
O
OMe
N
OMe
O
3+
HO
HO
N
N
N
N
Gd
N
N
N
3+
N
Gd
N
3+
S
S
O
O
N
O
N
O
HN
HN
OH
OH
O
O
O
N
H
O
N
H
1
1a
Scheme 1. Synthesis of pantoprazole–DOTA conjugate.
chloride from the benzylic chloride 8 with the thiol 6 in acetonitrile
provided the benzimidazole sulfide 9 in high yield. After removal of
the Boc from 9, the resulting free amine 10 was finally coupled with
the activated DOTA-succinimide (Macrocyclics, Dallas, TX) 11 in
DMSO to give the conjugate 12 in quantitative yield. Simple stirring
of an aqueous of 12 with GdCl3 furnished desoxy-pantoprazole–
DOTA conjugate 1 as a crude that was purified by HPLC on a RP Vy-
dac (150 Â 220 mm) column (gradient elution: 10% B–100% B in
40 min (B = 80% aq CH3CN containing 0.1% TFA, A = H2O containing
0.1% TFA; FR = 8 mL/min; kmax = 254 nm; rt = 19.0 min).
The pure desoxy-pantoprazole–DOTA (Gd3+) 1, obtained as a
yellow-brown solid in 87% yield, was fully characterized by 1H
and C-13 NMR, and ESI-based mass spectrometry which confirmed
a stable trapping of Gd3+ ion. This compound, when stored at
À20 °C was found to be stable for at least two months. The sulfide
1 was partially oxidized with m-chloroperoxybenzoic acid to fur-
nish the final contrast agent pantoprazole–DOTA (Gd3+) 1a. Purifi-
cation and spectral characterization of 1a was achieved in the same
way as for the sulfide 1. Unfortunately, this compound was found
to rapidly degrade to unidentified products as concluded by its

G. M. Maharvi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2808–2811
2811
purity by mass spectrometry and 1H NMR. The low temperature
References and notes
storage of 1a did not improve did not prevent its degradation. Since
the precursor 1 was found to have a long shelf life, this was used in
gastric wall imaging studies with the hope that the sulfide 1 itself,
or its possibly in vivo oxidized form might serve the purpose. The
successful imaging results using the PPI contrast conjugate agent 1
will be described in a separate communication (manuscript in
preparation).
In conclusion, this Letter reports a pump inhibitor pantoprazole
that has been linked to a Gd-encaging module DOTA to make a no-
vel conjugate. This conjugate was designed to be used as a stomach
wall contrast imaging agent. In order to minimize perturbation in
the normal function of the drug, a mechanism-based de novo
chemical synthesis was designed and successfully executed.
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Acknowledgments
This study was supported in part by USPHS NIH Grant R01
DK068055 and is gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.051.