Halogenated benzimidazole carboxamides target integrin α 4β1 on T-cell and B-cell lymphomas

Article abstract of DOI:10.1158/0008-5472.CAN-09-3736

Integrin α₄β₁ is an attractive but poorly understood target for selective diagnosis and treatment of T-cell and B-cell lymphomas. This report focuses on the rapid microwave preparation, structure-activity relationships, and biologica

Full text of DOI:10.1158/0008-5472.CAN-09-3736

Published OnlineFirst June 8, 2010; DOI: 10.1158/0008-5472.CAN-09-3736
Therapeutics, Targets, and Chemical Biology
Cancer
Research
Halogenated Benzimidazole Carboxamides Target Integrin
α4β1 on T-Cell and B-Cell Lymphomas
Richard D. Carpenter1, Arutselvan Natarajan2, Edmond Y. Lau4, Mirela Andrei3, Danielle M. Solano1,
Felice C. Lightstone4, Sally J. DeNardo2, Kit S. Lam3, and Mark J. Kurth1
Abstract
Integrin α4β1 is an attractive but poorly understood target for selective diagnosis and treatment of
T-cell and B-cell lymphomas. This report focuses on the rapid microwave preparation, structure-activity
relationships, and biological evaluation of medicinally pertinent benzimidazole heterocycles as integrin
α4β1 antagonists. We documented tumor uptake of derivatives labeled with 125I in xenograft murine mod-
els of B-cell lymphoma. Molecular homology models of integrin α4β1 predicted that docked halobenzimi-
dazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. The
high-affinity halogenated ligands identified offer attractive tools for medicinal and biological use,
including fluoro and iodo derivatives with potential radiodiagnostic (18F) or radiotherapeutic (131I) applica-
tions, or chloro and bromo analogues that could provide structural insights into integrin-ligand interactions
through photoaffinity, cross-linking/mass spectroscopy, and X-ray crystallographic studies. Cancer Res; 70(13);
5448–56. ©2010 AACR.
Introduction
nausea, vomiting, diarrhea, hair loss, anemia, and various or-
gan toxicities. Although significant effort has been directed
through lengthy syntheses of complex cytotoxic natural pro-
ducts, not until recently has attention been focused towards
target-selective chemotherapeutics that could reduce off-tar-
get binding and ensuing side effects (1).
Current cancer chemotherapeutic agents aim to annihilate
tumors through mechanisms such as DNA alkylation, unnat-
ural base pair recognition, inhibition of topoisomerases,
and microtubule stabilization mechanisms. However, these
agents have a narrow therapeutic index, are administered
near their maximum tolerated dose, and are largely nondis-
criminatory in recognizing either normal or cancerous cells.
As a consequence, patients suffer from serious side effects
including neutropenia, thrombocytopenia, neuropathy,
The resting or activated conformations of α4β1 integrin
allow for the application of target-selective agents for malig-
nant lymphoid cancers; activated α4β1 integrin is expressed
on leukemias, lymphomas, melanomas, and sarcomas (2, 3).
Integrins are heterodimeric transmembrane receptor pro-
teins crucial for cell-cell, cell-matrix, and cell-pathogen in-
teractions (4). Integrin α4β1 plays an important role in
autoimmune diseases and inflammation (5), as well as tu-
mor growth, angiogenesis, and metastasis (6–11). Indeed,
α4β1 integrin facilitates tumor cell extravasation (6), pre-
vents apoptosis of malignant B-chronic lymphocytic leuke-
mia cells (7), is key to drug resistance in both multiple
myeloma and acute myelogenous leukemia (8), and has been
selectively targeted with peptidomimetics (3, 12–16). None-
theless, α4β1 integrin has not garnered much attention as
either a therapeutic or a diagnostic cancer target due to
the lack of potent and specific agents.
In accord with our program to discover potent and se-
lective ligands that target various cancers, we have devel-
oped two in vivo imaging agents that have been successful
in murine models (3, 12–17). Figure 1A and B show the
structure of these agents; the bisaryl urea 1-Cy5.5 (LLP2A-
Cy5.5; Fig. 1A; refs. 3, 14) and the benzothiazole analogue
2-Cy5.5 (KLCA14-Cy5.5; Fig. 1B; ref. 15), both showed high
affinity and specificity for B- and T-cell lymphomas con-
taining activated α4β1 integrin, with the latter showing im-
proved tumor/kidney signal. This is likely due to the
Authors' Affiliations: 1Department of Chemistry, University of California,
Davis, Davis, California; 2Divisions of Hematology and Oncology, and
Radiodiagnosis and Therapy, Department of Internal Medicine, U.C.
Davis Cancer Center, University of California, Davis School of
Medicine, Sacramento, California; 3Division of Hematology and
Oncology, Department of Internal Medicine, U.C. Davis Cancer Center,
University of California, Davis, School of Medicine, Sacramento,
California; and 4Physical and Life Sciences Directorate, Lawrence
Livermore National Laboratory, Livermore, California
Note: Supplementary data for this article are available at Cancer
Research Online (http://cancerres.aacrjournals.org/).
K.S. Lam and M.J. Kurth share senior authorship.
Current address for R.D. Carpenter: Department of Biomedical Engineer-
ing, Genome and Biomedical Sciences Facility, University of California,
Davis, Davis, CA.
Current address for A. Natarajan: Molecular Imaging Program at
Stanford, Department of Radiology, Stanford University, Stanford, CA.
This research is dedicated in remembrance of our former colleague
Professor Aaron D. Mills (University of Idaho).
Corresponding Author: Mark J. Kurth, Department of Chemistry, Univer-
sity of California, Davis, Davis, CA 95616. Phone: 530-554-2145;
Fax: 530-752-8895; E-mail: mjkurth@ucdavis.edu.
doi: 10.1158/0008-5472.CAN-09-3736
©2010 American Association for Cancer Research.
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Halogenated Benzimidazole Antagonists of α4β1 Integrin
improved solubility of 2-arylamino azole heterocycle (13, 15)
Materials and Methods
over the bisaryl urea as these heterocycles have a lower log
P, a higher dipole moment, and an increased acidity of the
2-arylamino N–H (pKa = 5.5–6.5; ref. 18). However, both
1-Cy5.5 and 2-Cy5.5 have approximately one third of the mo-
lecular weight committed to the α4β1 integrin-targeting motif
with the rest of the molecular weight devoted to the linker and
the dye. Furthermore, due to the limited range of tissue pen-
etration, optical imaging is not practical for whole-body imag-
ing in humans. There is a need to develop a condensed α4β1
integrin radiotargeting agent that could not only be used for
positron emission tomography and single-photon emission
computed tomography imaging but also for radiotherapy.
With efforts integrating heterocyclic chemistry, cell adhesion
assays, molecular modeling, and radiochemistry, we report
herein the discovery of the bromobenzimidazole carboxamide
5 (IC50 = 115 pmol/L), the structure-activity relationship be-
tween halobenzimidazole carboxamides 3 to 6, and the biolog-
ical evaluation of radioiodinated derivates 7, 8, and 18
(structures shown in Fig. 1C and D).
Chemical synthesis
Compounds 3 to 9, 11 to 13, and 16 and 17 were synthe-
sized as outlined in Supplementary Schemes 1 to 4. Com-
pounds 1 and 2 (as well as 1-Cy5.5 and 2-Cy5.5), 9, 14, and
15a and b have been previously reported (3, 13, 15, 19, 20).
The synthesis of 3 to 6 was analogous to our previous work
(15); however, the analytical data for these compounds is
listed below. The synthesis of 7 and 8, 10 to 13, and 16 to
21 is described below.
(S)-6-(1-Carbamoylcyclohexyl-amino)-5-[(S)-2-{4-(5-
fluoro-1H-benzo[d]imidazol-2-yl)amino)-benzamido]-
6-[(E)-3-(pyridin-3-yl-acrylamido}hexanamido]-6-
oxo-hexanoic acid (3)
Following our procedure for benzimidazole analogues (15)
afforded 3 as a white solid (10 mg, 44%): ESI-MS (m/z) 798.8
(M + H)+, ESI-HRMS for C41H49FN9O7 (M + H)+: calcd,
798.3733; found, 798.4481 (m/z). Purity was determined to
Figure 1. Structures of: (A) 1-Cy5.5 (3), (B) 2-Cy5.5 (15), (C) halobenzimidazole analogues 3 to 7, and (D) the bishalo analogues 8 and 18, which incorporate
the bromobenzimidazole moiety and a distal radioiodide.
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Carpenter et al.
be 96% as determined by analytic high-performance liquid
chromatography (HPLC).
[
125I]-(S)-6-[1-{(S)-3-Amino-2-(4-hydroxy-3-
iodobenzyl)-3-oxopropanoyl-carbamoyl}
cyclohexylamino]-5-[(S)-2-{4-(5-bromo-1H-benzo[d]
imidazol-2-ylamino)benzamido]-6-[(E)-3-(pyridin-3-
yl)-acrylamido}hexanamido]-6-oxohexanoic acid (8)
A 2.5 mmol/L solution of compound 5 (200 μg, 50 μL) in
1 mol/L of sodium phosphate (adjusted to pH 7.0 with
H3PO4) was added to 50 μL of a 5 mmol/L solution of chlo-
ramine T in DMSO (200 μL) and mixed in a vial containing
Na125I (1 mCi, 10 μL), 10 mol% diethyldiamine and 10 mol
% ratio of CuI at 50°C for 20 minutes. After 20 minutes,
this mixture was quenched with 100 μL of a 5 mmol/L so-
lution of NaHSO3 in H2O for 15 minutes. Crude radio-
labeled 8 was eluted through a C-18 spin column with
250 μL of 1:1 acetonitrile/water to afford pure 8 in 90%
radiochemical yield, with specific activities ranging from
25.4 to 38.0 MBq (2 μCi)/μg, and >95% purity. The final pu-
rified products showed >95% monomeric compounds by
C-18 TLC, RP-HPLC, and CAE runs of 11 and 45 minutes;
the unbound radioiodine was <5%.
(S)-6-(1-Carbamoylcyclohexylamino)-5-[(S)-2-{4-(5-
chloro-1H-benzo[d]imidazol-2-ylamino)-benzamido]-
6-[(E)-3-(pyridin-3-yl)acrylamido}hexanamido]-6-
oxo-hexanoic acid (4)
Following our procedure for benzimidazole analogues (15)
afforded 4 as a white solid (22 mg, 57%): ESI-MS 814.3, 816.3
(m/z) (M + H)+, ESI-HRMS for C41H49ClN9O7 (M + H)+: calcd,
814.3438; found, 814.2894 (m/z). Purity (HPLC), 96%.
(S)-5-[(S)-2-{4-(5-Bromo-1H-benzo[d]imidazol-2-
ylamino}benzamido]-6-[(E)-3-(pyridin-3-yl)-
acrylamido}hexanamido]-6-
(1-carbamoylcyclohexylamino)-6-oxo-hexanoic acid (5)
Following our procedure for benzimidazole analogues
(15) afforded 5 as a white solid (28 mg, 39%): ESI-MS
(m/z) 858.4, 860.6 (M + H)+, ESI-HRMS for C41H49BrN9O7
(M + H)+: calcd, 906.2794; found, 906.2843 (m/z). Purity
(HPLC), 99%.
General procedure for halobenzimidazole acids:
4-(5-fluoro-1H-benzo[d]imidazol-2-ylamino)benzoic
acid (10)
(S)-6-(1-Carbamoylcyclohexylamino)-5-[(S)-2-{4-(5-
iodo-1H-benzo[d]imidazol-2-ylamino)benz-amido]-6-
[(E)-3-(pyridin-3-yl)acrylamido}hexanamido]-6-
oxohexanoic acid (6)
Following our procedure for benzimidazole analogues
(15) afforded 6 as a white solid (18 mg, 48%): ESI-MS
906.3 (m/z) (M + H)+, ESI-HRMS for C41H49IN9O7 (M +
H)+: calcd, 906.2794; found, 906.2843 (m/z). Purity
(HPLC), 96%.
Our previously reported methods (15) were used with
the following exceptions: a sealable microwave tube was
used as the reaction vessel and after the consumption of
9 as determined by TLC, 1,3-diisopropylcarbodiimide
(451 μL, 2.90 mmol) was added, the tube was sealed, and
the reaction mixture was microwave-heated to 80°C for
11 minutes. After our previously described workup, this
crude ester was then dissolved in DMF (8 mL), transferred
to a sealable microwave tube, treated with Ba(OH)2 (1.65 g,
9.65 mmol), and microwave-heated to 140°C for 21 minutes.
The mixture was filtered as previously described to afford
10 (225 mg, 86%) as a gray solid: ESI-MS (m/z) 272 (M + H)+.
Purity (HPLC), 98%.
[
125I]-(S)-6-(1-Carbamoylcyclohexylamino)-5-[(S)-2-{4-
(5-iodo-1H-benzo[d]imidazol-2-ylamino)-benzamido]-
6-[(E)-3-(pyridin-3-yl)acrylamido}hexanamido]-6-
oxo-hexanoic acid (7)
Radioiodination of the compound bromobenzimazole
carboxamide 5 using Na125I was evaluated by a number
of methods and conditions but the following procedure
provided the most consistent and best yields. Briefly,
50 μL of a 2.5 mmol/L solution of compound 5 in 1
mol/L of sodium phosphate (pH 7.0) was added to 50
μL of a 5 mol/L solution of chloramine T in distilled water
and mixed in a vial containing 185 MBq of Na125I and 10
mol% of CuI at 50°C for 20 minutes. This mixture was
then quenched with 100 μL of 5 mmol/L sodium bisulfite
for about 15 minutes. Radiolabeled 7 was eluted through a
C18 spin column with 250 μL of 1:1 acetonitrile/water.
Radiolabeling yields were in the range of 20% to 25%, with
specific activities ranging from 25.4 to 38.0 MBq (0.8–1.2
μCi)/μmol (specific activity was calculated and corrected
after final purification). Quality assurance for compound
7 was estimated by reverse-phase HPLC (RP-HPLC) with
radioactive and UV detectors and the labeled peptide
showed a single peak of >95% purity. The final purified
products showed >95% monomeric compounds by C18
TLC, RP-HPLC, and CAE runs of 11 and 45 minutes; the
unbound radioiodine was <5%.
4-(5-Chloro-1H-benzo[d]imidazol-2-ylamino)benzoic
acid (11)
Following the general procedure for halobenzimidazole
acids afforded 11 as a gray solid (249 mg, 89%): ESI-MS
(m/z) 288, 290 (M + H)+. Purity (HPLC), 99%.
4-(5-Bromo-1H-benzo[d]imidazol-2-ylamino)benzoic
acid (12)
Following the general procedure for halobenzimidazole
acids afforded 12 as a gray solid (205 mg, 64%): ESI-MS
(m/z) 332, 334 (M + H)+; found C, 50.70; H, 3.03; N, 12.63.
Purity (HPLC), 100%.
4-(5-Iodo-1H-benzo[d]imidazol-2-ylamino)benzoic
acid (13)
Following the general procedure for halobenzimidazole
acids afforded 13 as a brown solid (275 mg, 75%): ESI-MS
(m/z) 380.0 (M + H)+. Purity (HPLC), 97%.
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Halogenated Benzimidazole Antagonists of α4β1 Integrin
(S)-6-[1-{(S)-3-Amino-2-(4-hydroxybenzyl)-3-
oxopropanoylcarbamoyl}cyclohexyl-amino]-5-
[(S)-2-{4-(5-bromo-1H-benzo[d]imidazol-2-ylamino)
benzamido]-6-[(E)-3-(pyridin-3-yl)acrylamido}-
hexanamido]-6-oxohexanoic acid (17)
Our previously reported methods (15) afforded first 16 and
then 17 as a yellow solid (6 mg, 4.9%): ESI-MS 1023.69 (m/z)
(M + H)+. Purity (HPLC), 91%.
Animal biodistribution study for 7
Eleven female BALB/c nu/nu mice, 5 to 6 weeks old
(U.C. Davis Animal Care Facility), were xenografted s.c. in
the abdomen with 5 × 106 Raji cells. Three weeks after
inoculation, the tumor size of the mice was measured.
The mice were injected i.v. with 4 to 6 μCi of 125I-labeled
7 (5 μg, 6 nmol) with normal saline as the vehicle. The
mice were sacrificed at 4, 24, and 48 hours postinjection
and tissue samples were excised. The tissue samples were
weighed and radioactivity was measured in a gamma
counter. Uptake in harvested organs was expressed as %
ID/g of tissue.
[
125I]-(S)-6-[1-((S)-1-Amino-6-(4-iodobenzamido)-1-
oxohexan-2-ylcarbamoyl)-cyclohexyl-amino]-5-
[(S)-2-(4-(5-bromo-1H-benzo[d]imidazol-2-ylamino)
benz-amido]-6-[(E)-3-(pyridin-3-yl)acrylamido)
hexanamido]-6-oxohexanoic acid (18)
Animal biodistribution study for 18
Six female BALB/c nu/nu mice, 5 to 6 weeks old (U.C. Davis
Animal Care Facility), were xenografted s.c. in the abdomen
with 6 × 106 Raji cells. Three weeks after inoculation, the
tumor size of the mice was measured. The mice were
injected i.v. with 18 (5 μg, 4 nmol, 12 μCi specific activity)
with normal saline as the vehicle. Three mice were sacrificed
at 24 hours postinjection and the other three at 48 hours
postinjection, and tissue samples were excised. The tissue
samples were weighed and radioactivity was measured in a
gamma scintillation counter. Uptake in harvested organs was
expressed as % ID/g of tissue.
Amine 21 (50 μg, 41.2 μmol) and N-succinimidyl-4-[125I]io-
dobenzoate (14 μg, 41.2 μmol, 200 μCi; ref. 21) in alkaline
water (pH 8.0) was warmed to 60°C for 1 hour. Crude 18
was eluted through C18 spin column with 250 μL of 1:1
acetonitrile/water to afford pure 18 in 47% radiochemical
yield, with a specific activity of 1.7 μCi/g, and >95% purity.
Purity (C18 TLC and RP-HPLC), >90%.
(S)-5-((S)-2-(4-(5-Bromo-1H-benzo[d]imidazol-2-
ylamino)benzamido)-6-((E)-3-(pyridin-3-yl)acrylamido)
hexanamido)-6-(1-((S)-1,6-diamino-1-oxohexan-2-
ylcarbamoyl)cyclohexylamino)-6-oxohexanoic acid (21)
Our previously reported methods (15) afforded 21 (4.9 mg,
4.2%) as a white powder: ESI-MS 988.46 (m/z) (M + H)+. Pu-
rity (HPLC), 100%.
Results and Discussion
Chemistry
Both the azole carboxamide and bromoazole acetamide
series were previously known to be ineffective ligands for
α4β1 integrin. Indeed, previous structure-activity relationship
studies found that a methylene unit between the amide and
the phenyl ring (i.e., arylacetamide) was believed to be a crit-
ical motif for potency (3), whereas bromo substitution was
ineffective at increasing potency (13, 15). Nonetheless, molec-
ular modeling studies revealed a channel near the ligand
binding site where a halogen atom could potentially interact
(15). Halogenated ligands are all particularly attractive for
use in medicine and biology as either a radiodiagnostic
(18F), a radiotherapeutic (131I), or a molecular structure tool
(Cl or Br). In the latter example, either chloro or bromo li-
gands could be used to provide valuable molecular insight
into the integrin structure in either photoaffinity cross-
linking/mass spectroscopy experiments (Cl, 3:1 35Cl:37C;
Br, 1:1 79Br:81Br), as well as cocrystallization X-ray studies
(heavy atom effect). This provided the impetus behind the
synthesis of halobenzimidazole analogues 10 to 13
(Supplementary Scheme 1).
Although milder tandem reactions have been recently de-
veloped to afford m- and p-azole esters in good yields (30,
31), the reaction conditions shown in Supplementary
Scheme 1 quarter the amount of time to deliver halobenzi-
midazole acids 10 to 13 through microwave-mediated
chemistry (32). Briefly, commercially available aniline esters
were treated with thiophosgene to afford the aryl isothiocya-
nate ester 9 in 83% yield. Following purification by short
path column chromatography, this aryl isothiocyanate ester
Multiple sequence alignment and model building
The sequence alignment between α4β1 integrin and the
template (αIIbβ3 integrin; ref. 22; see Supplementary
Figs. S3–4) was performed using PSI-BLAST-ISS (23). Se-
quences having similarity to both α4β1 and αIIbβ3 integrins
were used as seeds to generate corresponding PSI-BLAST
profiles (24), which were extracted and compared. Protein
models were generated using MODELLER (25).
Docking simulations
Compounds were docked in the vicinity of Trp188 in (α4)
using Autodock (26). The partial atomic charges for the li-
gands were obtained using the AM1-BCC (27) method and
united atom charges were used for the integrin (28). An 80 ×
90 × 90 grid was used with a spacing of 0.375 Å and centered
above Trp188. A Lamarckian algorithm was used to generate
ligand conformations using previously reported parameters
whereas keeping the energy evaluations at a maximum of
150,000 (29). A total of 5,000 conformers were generated
for each ligand and clustered using a 2.0 Å root mean square
deviation.
Cell adhesion assay
The cell adhesion assay method is described elsewhere
(13, 15).
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Carpenter et al.
was reacted with 4-halo-o-phenylenediamine to yield an inter-
mediate bisaryl thiourea that, in the presence of 1,3-diisopro-
pylcarbodiimide, was cyclized to yield crude benzimidazole
esters and 1,3-diisopropylthiourea as a by-product. These es-
ters are rapidly saponified to afford halobenzimidazole acids
10 to 13 (64–89% yield) that are analytically pure following
acidification and filtration (33). This streamlined route, cou-
pled with previous reports focusing on milder reagents and
conditions that minimize purification (15, 30), significantly
improves the preparation of these medicinally pertinent azole
heterocycles which are present in nearly one-quarter of the
top 100 drugs (34).
With these halobenzimidazole acids in hand, effort was
then directed towards the synthesis of target molecules
3 to 6 (Supplementary Scheme 2). The tripeptide 14, pre-
pared previously from Rink amide resin (13), was N-acylated
with halobenzimidazole acid precursors 10 to 13, followed by
acid-mediated deprotection and resin cleavage to afford
Figure 2. A, potency (IC50),
estimated binding energies,
calculated interaction energies,
and amide-halogen geometries,
where the amide represents a
nearby primary amide of Asn161
(α4 subunit) side chain: a, see
ref. 13 for more on 15a, ref. 15
for more on 15b. b, energies are
expressed in units of kcal/mol.
c, estimated binding energies
(Ebind), in which Ebind = RT *
ln[IC50(X = halogen)/IC50(X = H)].
d, calculated interaction energy
for amide-halogen interaction
(gas phase) using MP2/6-311++G
(d,p)//B3LYP/6-311++G(d,p) level
of theory, second values are
the BSSE-corrected energies.
e, distances for the van der Waal's
(vdW) radii of each halogen
are expressed in angstroms.
f, distances (in angstroms)
represent the C(O)NRH⋯XAr
H-bond length. g, represents the
H-X-Ar angle in the C(O)NRH⋯XAr
H-bond. B, electrostatic potential
maps for 3 to 6 and 15a and 15b.
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Halogenated Benzimidazole Antagonists of α4β1 Integrin
Figure 3. Preparation of 18 from the tyrosine derivative 19, as described in ref. 15.
crude 3 to 6. Purification by RP-HPLC and lyophilization de-
livered pure halobenzimidazole carboxamide analogues 3 to
6 in 22% to 46% overall yield from Rink amide resin.
56-methylbenzimidazole (15b; ref. 15) were 1,000-fold less
potent than fluoro, chloro, and bromo analogues 3 to 5 as
well as 100-fold less potent than iodo analogue 6. These data
suggest that hydrophobic interactions were not responsible
for ultrapotency, as both 15a and 15b were equipotent. Ad-
ditionally, steric interactions were also not responsible, as
1,000-fold difference was seen between isosteres 15a and 3
as well as 15b and 4. Electrostatic interactions were also ex-
amined; however, these interactions were not important as
the electrostatic potential of 3 to 6 and 15a and 15b (see
Fig. 2B) did not correlate with the observed potency. There-
fore, attention turned towards aryl halide–derived interac-
tions which have occurred in prior systems through aryl
halide–H bonding (35) or halogen-carbonyl dipole-dipole
interactions (36, 37).
To further understand if the aryl halide–H-bond and/or di-
pole-dipole interactions were involved, the binding energy
(Ebind) was estimated for 3 to 6 (see Fig. 2A) using the equa-
tion Ebind = RT * ln[IC50(X = halogen)/IC50(X = H)], where R is
the gas constant and T is the temperature at 25°C. Quantum
mechanical calculations were performed where halobenzimi-
dazoles interacted with primary amide (i.e., Asn/Gln), ammo-
nium (Lys), or carboxylate (Asp/Glu) side chains.
Interestingly, only calculations involving the interaction
between the primary amide side chain of Asn or Gln with
halobenzimidazoles gave results consistent with the experi-
mental observations, thereby eliminating Lys, Asp, and Glu
residues as well as charge-transfer interactions. These data
suggest that the nature of either the H-bond donor or
carbonyl source is critical for ultrapotency.
In vitro biological evaluation
Halobenzimidazole carboxamide analogues 3 to 6 were
then subjected to cell adhesion competitive inhibition as-
says to determine in vitro activity and potency. The 25-mer
peptide CS-1 (DELPQLVTLPHPNLHGPEILDVPST), the
binding motif of fibronectin to α4β1 integrin, provides a
natural ligand to measure the binding affinities (IC50) of
the halobenzimidazole carboxamide analogues 3 to 6.
Briefly, 96-well plates were coated with neutravidin fol-
lowed by treatment of biotinylated CS-1 to immobilize the
natural ligand to the well of the plate. The remaining non–
neutravidin-bound sites were blocked with bovine serum
albumin and the wells were incubated with Molt-4 cells
(human T-cell leukemia containing α4β1 integrin). The plates
were then washed, fixed with formalin, stained with crystal
violet, and absorbance (570 nm) was measured using a UV/
Vis spectrophotometer equipped to read 96-well plates. Inhi-
bition was calculated as a percentage resulting from the con-
centration-dependent curve (see Supplementary Materials),
with the potency of 3 to 6 shown in Fig. 2A. Although all
compounds have an affinity for α4β1 integrin at <5 nmol/L,
these data suggest that the type of halogen atom plays a crit-
ical role for ultrapotency in this class of halobenzimidazole
analogues. The bromo, fluoro, and iodo derivatives are par-
ticularly promising; the bromo compound 5 is only 2-fold less
potent than previous leads (4, 19), whereas the fluoro (3) and
iodo (6) analogues could potentially be highly potent radio-
diagnostic (F-18) and radiotherapeutic (I-131) agents.
The nature of the amide-aryl halide interaction was elu-
cidated by investigating the geometries and interaction en-
ergies of both the carbonyl oxygen and the amide N–H
interacting with the halobenzimidazole. Having the hydro-
gen N–H interacting with the halogen gave a stabilizing in-
teraction for halobenzamidazoles 3 to 6. Although the
carbonyl oxygen interacting with heavier halo analogues 4
to 6 was stabilizing, it was unfavorable when interacting
Structure-activity relationship and theoretical
calculations
Interestingly, the structure-activity relationship for this
class of benzimidazole carboxamides (see Fig. 2A) revealed
that both the unsubstituted benzimidazole (15a; ref. 13) and
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Carpenter et al.
with fluoro analogue 3. The gas phase–calculated interaction
energies (IE) were then determined at the MP2/6-311++G(d,
p)//B3LYP/6-311++G(d,p) level and ranged from 3.5 to 4.0
kcal/mol as shown in the chart of Fig. 2A. These IE values were
primarily due to the H-bond between the primary amide of
Asn161 (α4 subunit) and the halobenzimidazole moiety. The
IE values predict that fluoro analogue 3 would be the most po-
tent; however, all IE values are very close (within 0.5 kcal/mol)
and were comparable with experimentally observed Ebind va-
lues. The variation in potency is likely explained by bromo an-
alogue 5 having the halogen with the requisite van der Waal's
atomic radii (2.00 Å for Br; ref. 38) and positioning of the hal-
ogen atom (105.8° angle of the C(O)NRH⋯X-Ar H-bond, where
X is the central atom of the angle and a halogen, is taken from
the minimized energy structures shown generically in Fig. 2A)
allowing for this key amide-halogen H-bond (bond length be-
tween H⋯X = 2.87 Å), whereas permitting other moieties to in-
teract with the α4 and β1 subunits (such as the butanoate-Mg2+
interaction at the MIDAS site of the β1 subunit; ref. 15). This
theory helps explain why the larger iodo analogue 6 (the poor-
est H-bond acceptor) is approximately 10-fold less potent than
the fluoro (3), chloro (4), and bromo (5) analogues and 100-
fold more potent than the unsubstituted (15a) and methyl
analogues (15b).
of 5 with several potential heteroatoms available for copper
chelation. However, treatment of 5 with [125I]NaI/chloramine
T successfully delivered 7 with a radiochemical yield of 20%
and a specific activity of 1.0 μCi/μg (125I T1/2 = ∼60 d).
Concurrently, Raji cells (human B-cell lymphoma), which
abundantly express α4β1 integrin (16), were cultured, centri-
fuged, and injected into 11 female BALB/c nude mice. These
xenograft tumors were allowed to grow to 50 to 200 mm. The I-
125 analogue 7 was then injected into the tail vein, with ani-
mals sacrificed and organs and tumors removed, weighed, and
counted after 24 and 48 hours. Although only one dose formu-
lation was given and dose injections were performed at the
same time in an identical manner for all mice, the blood clear-
ance and biodistribution data allowed us to separate the mice
into two groups: one with slow blood clearance and one with
fast blood clearance (Supplementary Figs. S1 and S2). Three
mice showed slow clearances (two mice at 24 h and one mouse
at 48 h). These mice had approximately 15% still circulating in
the blood thus providing high tumor uptakes of >6% ID/g. The
liver, spleen, and marrow uptakes were <8% ID/g at 48 hours.
However, five mice (three mice at 24 h and two mice at 48 h)
showed that 7 was cleared rapidly from the blood and body.
The major dose (25% ID/g) was mainly accumulated in the liv-
er, spleen, and marrow, whereas the tumor uptake of these
mice was very low (<1.5% ID/g).
Although these preliminary data have low statistical signif-
icance, we believe these results warrant further discussion and
might be due to one or more factors. The radioiodo analogue
7, having reduced in vitro affinity for α4β1 integrin by nearly
10-fold compared with bromo analogue 5, may have decreased
in vivo affinity and selectivity. The iodobenzamidyl moiety of 7
might also be metabolically degraded, as seen with p-radioio-
dobenzamide derivatives (42, 43). Although we did not see
in vitro peptide aggregation of 7, it has been reported that
the fast clearance patterns might be due to in vivo aggregation
resulting in ineffective tumor targeting (44).
Radioiodination via aromatic Finkelstein and initial
biodistribution studies
The condensed radioiodide derivatives 7 and 8 were attrac-
tive targets to potentially serve as therapeutic or diagnostic
agents for T- and B-cell lymphomas. Radioiodo derivative 7
was particularly attractive, as this could be synthesized from
the bromo analogue 5 in a copper-mediated radioiodination
(39, 40). Buchwald's copper(I)-mediated aromatic Finkelstein
reaction with cold sodium iodide (i.e., ArBr → ArI) that pro-
ceeds with average yields of 97% for many aryl and heteroaryl
systems seemed a highly promising route to deliver the I-125
enriched 7 from the aryl bromide 5 (Supplementary Scheme 3;
ref. 41). In our hands, this method for radiohalogenation was
unsuccessful, presumably due to the structural sophistication
Radioiodination via electrophilic aromatic substitution
In addition to these mixed in vivo results (Supplementary
Figs. S1 and S2), 7 was difficult to prepare in high radiochem-
ical yield and crude purity. This provided the impetus for the
synthesis of 3-radioiodotyrosine-derivative 8, as tyrosine
residues are rapidly radioiodinated with high regio- and
chemoselectively (21, 45). Moreover, previous optical conju-
gates 1-Cy5.5 and 2-Cy5.5 showed that the primary amide
was successfully modified to a secondary amide without af-
fecting activity, potency, or selectivity (3, 12, 14–16). With this
in mind, Rink amide resin was first swollen in DMF for 3 hours,
followed by Fmoc-deprotection and N-acylation with semi-
orthogonally protected tyrosine to deliver the tyrosylated resin
16 (Supplementary Scheme 2). This resin was further elaborat-
ed into the bromobenzimidazole tetrapeptide 17 through
analogous chemistry (Supplementary Scheme 2) and further
elaborated elsewhere (15). This tyrosine derivative 17 was then
rapidly radioiodinated using I-125–enriched sodium iodide
with iodogen as an oxidant to afford the 3-radioiodo tyrosine
derivative 8 in 90% radiochemical yield and with a specific ac-
tivity of 2 μCi/μg. Unfortunately, 8 performed poorly in in vitro
Figure 4. Ex vivo radio uptake data of 18 for various tumors and organs.
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Published OnlineFirst June 8, 2010; DOI: 10.1158/0008-5472.CAN-09-3736
Halogenated Benzimidazole Antagonists of α4β1 Integrin
binding studies (<4%) and thus in vivo studies were not pur-
sued. However, these in vitro results should not necessarily be
viewed detrimentally; the o-hydroxyl group has been known to
weaken the carbon-iodine bond and radioiodotyrosine deriva-
tives have been known to undergo in vivo degradation due to
their structural similarities to thyroid hormones (46).
synthesis of azole heterocycles, which previously required
harsh reaction conditions, long reaction times, and highly
toxic reagents, can now be rapidly prepared through micro-
wave-mediated synthesis using safer reagents with minimal
purification. This report also provides an excellent example
of how molecular models could be used as a guide to predict
analogues that will have high affinity and to understand key
weak force interactions. The potency of this unique class of
compounds is likely attributed to a key aryl halide-hydrogen
bond between the halobenzimidazole moiety and the prima-
ry amide side chain of Asn161 in the α4 subunit. In preli-
minary studies, the condensed radioiodobenzimidazole
analogue 18 has shown that a low tumor/kidney ratio may
be achievable with a covalently attached radiolabeled modal-
ity, while minimizing the size and cost of the payload-ligand
conjugate. These halobenzimidazoles are particularly attrac-
tive as this allows for the design of highly condensed
ligand-payload conjugates for radiotherapeutic (I-131) and
radiodiagnostic (F-18) agents for selectively detecting and
treating T- and B-cell lymphomas that express α4β1 integrin.
Additionally, the bromo analogue 5 could provide valuable
molecular insights into the binding site and integrin struc-
ture through photoaffinity cross-linking/mass spectroscopy
(1:1 79Br:81Br) as well as cocrystallization X-ray studies (heavy
atom effect).
Radioiodination via succinimidyl ester chemistry and
biodistribution studies
Attention was then turned towards the synthesis of 4-
radioiodobenzamidolysine-derivative 18, as the bulkiness
of the iodotyrosine may contribute to the poor in vitro
binding. Rink amide resin was first swollen in DMF for
3 hours, followed by Fmoc-deprotection and N-acylation
with Fmoc-Lys(Dde)-OH to deliver resin 19 (Fig. 3). Resin
19 was further elaborated into resin 20 through analogous
chemistry (Supplementary Scheme 2) and described else-
where (15). N-acylation of 20 with Fmoc-Lys(Alloc)-OH
was followed by Fmoc-deprotection, and then coupling
with bromobenzimazole acid 5. Alloc deprotection,
followed by N-acylation with trans-3-(3-pyridyl)acrylic acid,
Dde deprotection, and trifluoroacetic acid cleavage yielded
21. The lysinated bromobenzimadole 21 was radioiodinated
by coupling the free amine of the lysine with the NHS-ester
of I-125 enriched 4-iodobenzoic acid [prepared from p-
aminobenzoic acid as outlined by Khalaj and colleagues
(47)] to afford the 4-radioiodobenzamidolysine derivative
18 in 47% radiochemical yield and with a specific activity
of 1.7 μCi/μg.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
To study the preliminary biodistribution of 18, Raji cells
(human B-cell lymphoma) expressing α4β1 integrin were
cultured, centrifuged, and injected into six nude mice. These
xenograft tumors were grown to 50 to 200 mm. The I-125
analogue 18 was then injected into the tail vein, with half of
the animals being sacrificed after 24 and 48 hours. As shown
in Fig. 4, the organs and tumors were removed and counted
with 18 having good tumor uptake (12 1% ID/g at 24 h and
4.5 1% ID/g at 48 h) and minimal uptake in other organs.
In particular, the low kidney uptake [tumor/kidney(t = 24 h)
∼4:1; tumor/kidney(t = 48 h) ∼2.5:1] was encouraging as this,
in this initial assessment, has shown that radiolabeledazole
analogue 18 might be a promising payload-ligand conjugate
for targeting activated α4β1 integrin–expressed tumors.
Acknowledgments
Assistance with data compilation by Gary Mirick (U.C. Davis Cancer
Center) is duly acknowledged.
Grant Support
National Cancer Institute(U19CA113298), National Science Foundation (CHE-
0614756), National Institute for General Medical Sciences (RO1-GM076151),
American Chemical Society's Division of Medicinal Chemistry Predoctoral
Fellowship sponsored by Sanofi-Aventis (R.D. Carpenter), Howard Hughes
Medical Institute Med into Grad Fellowship (R.D. Carpenter), U.C. Davis
R.B. Miller Graduate Fellowship and Outstanding Dissertation Award (R.D.
Carpenter), Alfred P. Sloan Minority Ph.D. Program (D.M. Solano), and U.C. Davis
Alliance for Graduate Education and the Professoriate Advantage Program (D.M.
Solano). NMR spectrometers were funded in part by the National Science Foun-
dation (CHE-0443516 and CHE-9808183). A portion of this work was performed
under the auspices of the U.S. Department of Energy by Lawrence Livermore
National Laboratory under contract DE-AC52-07NA27344.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Conclusion
The results presented have shown the importance of ad-
vancing leads to target cancerous but not normal cells by ex-
ploiting the conformational differences of α4β1 integrin. The
Received 10/08/2009; revised 03/27/2010; accepted 04/29/2010; published
OnlineFirst 06/08/2010.
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Published OnlineFirst June 8, 2010; DOI: 10.1158/0008-5472.CAN-09-3736
Halogenated Benzimidazole Carboxamides Target Integrin α4β
1 on T-Cell and B-Cell Lymphomas
Richard D. Carpenter, Arutselvan Natarajan, Edmond Y. Lau, et al.
Cancer Res 2010;70:5448-5456. Published OnlineFirst June 8, 2010.
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