Selectively targeting T- and B-cell lymphomas: A benzothiazole antagonist of α4β1integrin

Article abstract of DOI:10.1021/jm800313f

Current cancer chemotherapeutic agents clinically deployed today are designed to be indiscriminately cytotoxic, however, achieving selective targeting of cancer malignancies would allow for improved diagnostic and chemotherapeutic tools. Integrin α₄β₁, a heterodimeric cell surface receptor, is believed to have a low- affinity conformation in resting normal lymphocytes and an activated high-affinity conformation in cancerous cells, specifically T- and B-cell lymphomas. This highly attractive yet poorly understood receptor has been selectively targeted with the bisaryl urea peptidomimetic antagonist i. However, concerns regarding its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzothiazole moiety, resulting in an analogue with improved physicochemical properties, solubility, and kidney:tumor ratio while maintaining potency (6;IC₅₀ = 53 pM). The results presented herein utilized heterocyclic and solid-phase chemistry, cell adhesion assay, and in vivo optical imaging using the cyanine dye Cy5.5 conjugate.

Full text of DOI:10.1021/jm800313f

14
J. Med. Chem. 2009, 52, 14–19
Articles
Selectively Targeting T- and B-Cell Lymphomas: A Benzothiazole Antagonist of r4ꢀ1 Integrin
Richard D. Carpenter,† Mirela Andrei,‡ Olulanu H. Aina,‡ Edmond Y. Lau,§ Felice C. Lightstone,§ Ruiwu Liu,‡ Kit S. Lam,*,‡
and Mark J. Kurth*,†
Department of Chemistry, UniVersity of California DaVis, One Shields AVenue, DaVis, California 95616, Department of Internal Medicine,
DiVision of Hematology/Oncology, UC DaVis Cancer Center, 4501 X Street, Sacramento, California 95817, Chemistry, Materials, Earth, and
Life Sciences, Lawrence LiVermore National Laboratory, 7000 East Street, LiVermore, California 94550
ReceiVed March 19, 2008
Current cancer chemotherapeutic agents clinically deployed today are designed to be indiscriminately
cytotoxic, however, achieving selective targeting of cancer malignancies would allow for improved diagnostic
and chemotherapeutic tools. Integrin R4ꢀ1, a heterodimeric cell surface receptor, is believed to have a low-
affinity conformation in resting normal lymphocytes and an activated high-affinity conformation in cancerous
cells, specifically T- and B-cell lymphomas. This highly attractive yet poorly understood receptor has been
selectively targeted with the bisaryl urea peptidomimetic antagonist 1. However, concerns regarding its
preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl
urea to a 2-arylaminobenzothiazole moiety, resulting in an analogue with improved physicochemical
properties, solubility, and kidney:tumor ratio while maintaining potency (6; IC50 ) 53 pM). The results
presented herein utilized heterocyclic and solid-phase chemistry, cell adhesion assay, and in vivo optical
imaging using the cyanine dye Cy5.5 conjugate.
Introduction
Many of the current cancer chemotherapeutic agents clinically
deployed today are designed to be indiscriminately cytotoxic
through DNA alkylation, unnatural base-pair incorporation,
inhibition of topoisomerases, and microtubule stabilization
mechanisms. Cancer chemotherapy, often administered near its
maximum tolerated dose (MTDa), aims to annihilate tumors with
tolerable toxicity. Several of these agents exemplified in Figure
1 possess a narrow therapeutic index that limits effectiveness.
As a consequence, underdosing at the tumor site is problematic,
with patients suffering from significant side effects including
nausea, vomiting, diarrhea, malnutrition, hair and memory loss,
anemia, immunosuppression, hemorrhaging, chronic pain, and
various organ toxicities. Tremendous success has been achieved
Figure 1. Structures of commonly administered indiscriminating
cytotoxic chemotherapeutic agents.
* To whom correspondence should be addressed. For K.S.L.: phone,
(916) 734-8012; fax, 916-734-7946; E-mail: kit.lam@ucdmc.ucdavis.edu.
For M.J.K.: phone, (530) 752-8895; fax, 530-752-8895; E-mail:
mjkurth@ucdavis.edu.
†
through lengthy syntheses of ornate cytotoxic natural products
with significantly less attention being granted toward selectiVe
chemotherapeutics that would result in decreased off-target
binding and ensuing side effects.
Department of Chemistry, University of California Davis.
Department of Internal Medicine, Division of Hematology/Oncology,
‡
UC Davis Cancer Center.
§
Chemistry, Materials, Earth, and Life Sciences, Lawrence Livermore
National Laboratory.
a Abbreviations: PK, pharmacokinetic; MTD, maximum tolerated dose;
MIDAS, metal ion-dependent adhestion site; PEG, polyethylene glycol;
FOS, function-oriented synthesis; Fmoc-Ach-OH, 1-[((9H-fluoren-9-yl)me-
thoxy)carbonylamino]cyclohexanecarboxylic acid; HOBt, hydroxybenzot-
riazole; DIC, 1,3-diisopropylcarbodiimide; DMF, N,N-dimethylformamide;
Fmoc-Aad(tBu)-OH, (S)-2-[((9H-fluoren-9-yl)methoxy)carbonylamino]-6-
tert-butoxy-6-oxohexanoic acid; DdeK(Fmoc)OH, (S)-6-[((9H-fluoren-9-yl)-
methoxy)carbonylamino]-2-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethy-
lamino]hexanoic acid; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate; DIEA, diisopropylethylamine; TFA, triflu-
oroacetic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline;
FmocK(Dde)OH, (S)-2-[((9H-fluoren-9-yl)methoxy)carbonylamino]-6-[1-
(4,4-dimethyl-2,6-dioxocyclohexylidene)ethylamino]hexanoic acid; FmocK-
(Alloc)OH, (S)-1-(9H-fluoren-9-yl)-3,11-dioxo-2,12-dioxa-4,10-diazapentadec-
14-ene-5-carboxylic acid.
To achieve target selectivity, therapeutic compounds must
be able to differentiate cancer cells from normal cells. In T-
and B-cell lymphomas, targeting the activated form of cell
surface receptors expressed on cancer cells allows for dif-
ferentiation, as normal or inactivated versions remain untargeted.
Specifically, the cell surface receptor R4ꢀ1 integrin regulates
lymphocyte trafficking1 and homing in normal adult cells.2,3
A
ꢀ-subunit conformational change4 activates R4ꢀ1, which regu-
lates tumor growth, metastasis, and angiogenesis, in addition
to promoting the dissemination of tumor cells to distal organs.5
The ligand LLP2A (1; see Figure 2) recognizes this change and
shows potential as a noninvasive imaging and therapeutic agent
10.1021/jm800313f CCC: $40.75
2009 American Chemical Society
Published on Web 12/15/2008

SelectiVely Targeting T- and B-Cell Lymphomas
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 15
Scheme 1. Synthesis of Analogues 3-11 (See Supporting
Information for Structures)a
a (a) (Cl)2CdS, Et3N, EtOAc; (b) 5-R2-4-R3-2-XH-anilines, CH2Cl2, 16 h,
followed by N-cylcohexylcarbodiimide, N’-methyl polystyrene resin, then
LiOH, H2O/dioxane, ∆; (c) swell/DMF, 3 h; (d) 20% piperidine/DMF; (e)
Fmoc-Ach-OH, DIC, HOBt, DMF; f (d); (f). Fmoc-Aad(tBu)-OH, DIC,
HOBt, DMF; f (d); (g) Dde-K(Fmoc)-OH, DIC, HOBt, DMF; f (d); (h)
(E)-3-(pyridin-3-yl)acrylic acid, DIC, HOBt, DMF; (i) 2% H2NNH2/DMF;
(j) 12-20, HBTU, EtN(iPr)2, DMF; (k) TFA, (iPr)3SiH, H2O.
The removal of the polystyrene-bound thiourea by filtration
simplifies purification and this technology allows for the
extension of a previously reported benzimidazole preparation11
to benzoxazole and benzothiazole systems as well. The resulting
heterocyclic esters, when subjected to saponification conditions,
deliver the m- and p-heterocyclic acid precursors 12-20 in
68-84% overall yield from the aryl isothiocyanate. Diversifica-
tion at the o-position was precluded due to a 6-exo-trig
cyclization under saponification conditions affording benzimi-
dazoquinazolinones.12
Effort was next forwarded on preparing the Rink resin-bound
tripeptide 22 and analogue targets 3-11. Tripeptide 22 was
prepared by first Fmoc-deprotecting Rink resin followed by
activating an appropriately protected amino acid via treatment
with DIC/HOBt in DMF and addition of this solution to the
resulting amino resin. This process was repeated twice more
with the respective precursors 12-20, each dissolved in a
solution of HBTU/DIEA and DMF, were coupled to the
polymer-tripeptide 22. No detectable polymerization occurred
with the 2-arylaminoheterocyclic acids 12-20, enabling this
reaction to occur without additional protecting groups. The
heterocyclic tripeptides were cleaved from the polymer under
acidic conditions to give heterocyclic analogues 3-11 (see Table
1 for yields).
The potencies of these heterocyclic analogues were deter-
mined using a Molt-4 cell adhesion assay by inhibiting R4ꢀ1-
mediated cell adhesion to the known ligand CS-1. A neutravidin-
coated 96-well plate was incubated with biotinylated CS-1. The
wells were then blocked, followed by incubation with Molt-4
cells expressing activated R4ꢀ1 integrin (1.3 × 105 cells/well).
Serially diluted ligands were incubated and washed; the remain-
ing bound cells were then fixed and stained. Inhibition was
quantified by measuring absorbance at 570 nm and IC50 data
were extrapolated from the concentration-dependent inhibition
curves.
Figure 2. Evolution of ligand analogues using function-oriented
synthesis (FOS): (a) Structure of 1 (LLP2A), which can be optically
or radioconjugated and shows potential as an imaging or therapeutic
agent for lypmphoma;6 (b) structure of water soluble benzimidazole
analogue 2 (IC50 ) 305 pM);7 (c) heterocyclic analogues 3-11 and
requisite precursor heterocyclic acids 12-21 and aryl isothiocyanates
22a-d.
despite kidney uptake observed in xenograft models.6 This
prompted creation of a water soluble benzimidazole analogue
KLCA4 (2)7 that would be dianionic8 at physiological pH
(bisarylamino NH + CO2H), thereby improving solubility and
decreasing kidney uptake based on electronic factors.7,9,10 While
2 has picomolar potency, it is still 10-fold less potent than the
bisaryl urea 1. Herein, we report the design of an equipotent
(to 1), comparably soluble (to 2) benzothiazole analogue 6 that,
when optically conjugated using Cy5.5, demonstrates excellent
tumor uptake with preliminary evidence showing an improved
kidney:tumor ratio in xenograft models. Key to this approach
is the heterocyclic design, which, in a condensed fashion,
improves the ligand’s physicochemical properties without
PEGylation or a poly charged tail.
Results and Discussion
Our previous benzimidazole ligand showed excellent binding
to human R4ꢀ1 integrin; however, it still did not bind as
efficiently as LLP2A (1). The design of improved analogues,
was centered around systematic modifications to the heterocycle,
its substituents, and its side chain to regain the binding affinity
while improving the pharmacokinetics over previous leads. As
delineated in Scheme 1, m- and p-aniline esters were thiophos-
genated, delivering the aryl isothiocyanate esters 21a-d in
81-93% yield. These aryl isothiocyanate esters were then
reacted with 4,5-diversified o-nucleophillic anilines, yielding
thiourea intermediates that undergo a tandem reaction with a
carbodiimide-resin to deliver thiocondensed heterocyclic esters.
The SAR data for this class of compounds is summarized in
Table 1 and Figure 3, with p-orientation being critical as

16 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Carpenter et al.
Scheme 2a
Table 1. Overall Yield, Purity, Molecular Ion, and IC50 of Heterocyclic
Analogues
compound
yield, %
purity, %
[M + H]+
IC50
1
2
3
4
5
6
7
8
37 pM
305 pM
30 nM
379 nM
490 nM
53 pM
4 nM
50
54
44
62
74
69
61
64
58
97
98
95
100
99
100
99
100
100
858, 860
886, 888
794
811
809
794
795
781
797
2 nM
1 µM
1 µM
9
10
11
347 nM
a Reagents and conditions: (a) swell, DMF, 24 h; (b) 20% piperidine/
DMF; (c) Fmoc-K(Dde)-OH, DIC, HOBt, DMF f (b); (d) Fmoc-NH-linker-
CO2H, DIC, HOBt, DMF f (b) f (d) f (b); (e) Fmoc-Ach-OH, DIC,
HOBt, DMF f (b); (f) Fmoc-Aad(OtBu)-OH, DIC, HOBt, DMF f (b);
(g) Fmoc-K(Alloc)-OH, DIC, HOBt, DMF f (b); (h) (i) 15, HBTU, DIEA,
DMF; (ii) Pd(PPh3)4, PhSiH3, DMF; (i) (E)-3-(pyridin-3-yl)acrylic acid, DIC,
HOBt, DMF; (j) 2% H2NNH2/DMF; (k) Cy5.5-NHS, DIEA, DMF; (l) TFA,
H2O, (iPr)3SiH.
Pd0 and phenylsilane (Caution: Pressure buildup!) followed by
N-acylation of the resulting amine with 3-pyridylacrylic acid
affords an intermediate with the complete framework of 6.
Deprotection of the Dde group, N-acylation with Cy5.5-NHS,
and acid hydrolysis liberates 6-Cy5.5 from the solid support
with concomitant deprotection of the t-butyl ester.
Figure 3. (a) Original SAR findings of 1 juxtaposed to that of the
heterocyclic analogues. (b) Structure of the equipotent (to 1) benzothia-
zole acetamide 6.
m-substitution was impotent. Moreover, carboxamides (where
n ) 0) were largely ineffective in achieving an equipotent
analogue. The benzoxazole 9 and 4-methylbenzoxazole 10
showed complete loss of potency, while benzothiazole 11, and
56-methylbenzimidazole 5 showed mid nM potency. The 4-me-
thylbenzoxazole acetamide 7 exhibited low nM potency. While
holding constant the acetamide (n ) 1), removal of the methyl
group results in no appreciable change for benzoxazole 8, but
replacement of X ) O, NH with X ) S in benzothiazole 6 further
increases the potency to the low pM range (IC50 ) 53 pM).
From nine heterocyclic analogues, the benzothiazole acetamide
6 was discovered to be equipotent to 1. The sulfur atom is
probably an isostere for the NH moiety of 2 as both heteroaryl
rings are likely antiperiplanar to the phenyl ring. The benzox-
azole 8, however, adopts a near-planar orientation likely making
the smaller oxygen atom a less successful isostere.
Near-infrared optical imaging preliminary studies were then
performed to measure tumor and organ uptake in lymphoma
xenograft bearing murine models using the fluorophore Cy5.5-
labeled benzothiazole acetamide. While this conjugate is not
ideal in terms of size and sophistication (triples the molecular
weight; 5-fold cost increase), this allows for safe, nonradioactive
studies to initially assess in vivo biological activity before radio
studies. Scheme 2 delineates the synthesis of 6-Cy5.5 starting
with Rink amide resin, followed by a series of Fmoc-depro-
tection and N-acylation iterations with appropriately protected
amino acids and linkers delivering resin 23. Key to this synthesis
is the use of two orthogonally protected lysines that will be
selectively deprotected as well as a hydrophilic linker to
minimize dye interference with integrin binding. The R-car-
bamate of lysine was Fmoc-deprotected and N-acylated with
the benzothiazole phenylacetic acid 15 under HBTU/DIEA
conditions. Chemoselective deprotection of the Alloc group with
Keeping in mind that a safe in vivo evaluation was desired
prior to radio studies, activated R4ꢀ1 expressed Molt-4 (T-cell
lymphoma) or Raji tumors (B-cell lymphoma) were subcutane-
ously implanted into the right shoulder of the nude mice. As a
negative control, A549 nonsmall cell lung cancer (not expressing
R4ꢀ1) was implanted into the left shoulder. To establish adequate
doses, varying amounts of 6-Cy5.5 were administered via the
tail vein into nude mice bearing subcutaneously Molt-4 or Raji
and A549 xenografts. As depicted in Figure 4, some mice were
serially imaged ranging from 5 min to 120 h post injection.
Other mice were sacrificed at various time points, with ex vivo
specificity and uptake measurements of pertinent organs and
tumors for both agents at similar doses. Tumor uptake was
observed as early as 5 min post injection and persisted for up
to 120 h. To determine organ:tumor ratios, regions of interest
were drawn around the tumor and each organ in the ex vivo
images and mean signal intensity was obtained by subtracting
the lowest intensity background signal (heart) from each
intensity value. Preliminary evidence indicates 6-Cy5.5 is
comparable to 1-Cy5.5 in terms of tumor specificity and uptake
while showing improvement in the kidney:tumor ratio. Regard-
less of the imaging agent, an appropriate decrease in signal is
observed as the dose decreases. Furthermore, both agents
showed low liver, muscle, lung, and spleen signal while
consistently showing skin, lymph node, and negative tumor
signal. Optical probe uptake by the R4ꢀ1 integrin negative tumors
may in part be explained by the expression of activated R4ꢀ1
integrin in tumor vasculature.13 These findings strengthen the
hypothesis that the improved kidney:tumor ratio may be
attributed to the presence of an additional negative charge from
the arylaminobenzothiazole N-H8 at physiological pH. This

SelectiVely Targeting T- and B-Cell Lymphomas
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 17
NH), preliminary evidence shows the 6-Cy5.5 conjugate has
improved solubility and kidney:tumor ratio while retaining
excellent tumor uptake and comparable organ specificity. This
work further enables lymphoma targeting with a highly potent
ligand and highlights the importance of physicochemical factors
in the rational design of analogues to maintain potency while
preventing toxicity. Although 6-Cy5.5 is not an ideal agent, these
nonradioactive preliminary in vivo optical studies show en-
couraging results, with future efforts pointing toward the
incorporation of radioisotopes for more rigorous pharmacoki-
netic and radio chemotherapeutic and/or imaging purposes.
Experimental Section
General Synthetic Procedures. All chemicals were purchased
from commercial suppliers and used without further purification.
Rink amide resin (0.5 mmol/g loading, 100-200 mesh) was
purchased from Tianjin Nankai Hecheng Sci & Tech. Co., Ltd.,
(batch number GRM-0406-J). N-cylcohexylcarbodiimide, N′-methyl
polystyrene resin (1.3 mmol/g loading, 200-400 mesh) was
purchased from Novabiochem. Analytical TLC was carried out on
precoated plates (silica gel 60, F254) and visualized with UV light.
NMR spectra (1H at 300 MHz, 400 MHz, 600 MHz; 13C at 75
MHz, 100 MHz) were recorded in DMSO-d6, methanol-d4, and
acetone-d6 as solvents and chemical shifts are expressed in parts
per million relative to residual undeuterated solvent. The specifica-
tions of the LC/MS are as follows: electrospray (+) ionization, mass
range 100-900 Da, 20 V cone voltage, and Xterra MS C18 column
(2.1 mm × 50 mm × 3.5 µm). CC refers to normal-phase silica-
gel column chromatography. Concentration refers to rotary evapora-
tion under reduced pressure. After each solid-phase step, the resin
was washed by sequential treatment with the following solvents:
DMF (2 × 5 mL), H2O (2 × 5 mL), CH3OH, (3 × 5 mL), and
CH2Cl2 (5 × 5 mL).
General Procedure for Heterocyclic Acids: 4-(56-Methyl-1H-
benzo[d]imidazol-2-ylamino)benzoic Acid (14). Following our
previously reported procedure,7 to a solution of o-phenylenediamine
(1.90 g, 17.6 mmol) in CH2Cl2 (75 mL) was added a solution of
the aryl isothiocyanate ester (For 2a, 3.0 g, 16.8 mmol) in CH2Cl2
(75 mL) dropwise over 30 min, followed by stirring for 16 h at
room temperature. After TLC showed that the aryl isothiocyanate
was consumed, polystyrene-bound DCC resin was added [for 14,
(39 mg, 50.4 mmol)] and the reaction proceeded at room temper-
ature until TLC showed the intermediate thiourea was consumed.
In most instances, this was between 4-8 h, but in some cases this
took as long as 16 h (14, 4.5 h). The resin was filtered, followed
by concentration of the filtrate. The resulting residue was taken up
in ethyl acetate/H2O, followed by washing (H2O, brine), drying
(MgSO4), and concentrated to give the benzimidazole ester (4.08
g) that was used without further purification. A solution of this
benzimidazole ester (4.08 g, 15.3 mmol) in dioxane/H2O (125 mL/
80 mL) was treated with LiOH (1.83 g, 76.4 mmol) and the solution
was refluxed for 16 h. The reaction mixture was concentrated, and
the residue was taken up in aqueous 2 M NaOH. This basic water
layer (pH ∼10) was washed twice with ether before being acidified
with concentrated HCl to pH ∼2-3, at which point 14 precipitated
as a light-gray solid (3.43 g, 74%): mp 368-370 °C. IR (neat)
3542 (st, br), 3284 (sh), 3050 (sh), 2984, 1699 (st). 1H (300 MHz,
DMSO-d6): δ 7.97 (d, J ) 6.6 Hz, 2H), 7.58 (d, J ) 7.2 Hz, 2H),
7.35 (d, J ) 7.8 Hz, 1H), 7.28 (s, 1H), 7.05 (d, J ) 8.1 Hz), 2.35
(s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 166.9, 146.6, 141.3,
133.1, 131.1, 130.6, 128.4, 126.6, 124.6, 120.1, 112.3, 112.1, 21.2.
ESI MS (m/z) 268 (M + H)+. Anal. Calcd for C15H13N3O: C, 67.40;
H, 4.90; N, 15.72. Found: C, 67.63; H, 4.91; N, 15.78. Purity was
determined to be 99% by HPLC analysis on the basis of absorption
at 220 nm.
Figure 4. In vivo near-infrared light (NIRF) images using 100 µL of
6-Cy5.5 with R4ꢀ1-bearing tumors grown on the upper right side (Molt-
4), lower right side (Raji), and non-R4ꢀ1-bearing tumor grown on the
left side as a negative control (A549) at the following doses and times:
(a) 10 nmol, t ) 4 h; (b) 10 nmol, t ) 24 h; (c) 10 nmol, t ) 48 h; (d)
10 nmol, t ) 120 h. Ex vivo NIRF image mean intensity values 24 h
postinjection of pertinent organs and tumors at the following doses:
(e) 1-Cy5.5 (0.25 nmol, 24 h postinjection); and (f) 6-Cy5.5 (1 nmol,
24 h postinjection). Ex vivo NIRF image graphs (g) Side-by-side organ:
tumor [mean-lowest organ] ratio at comparable doses for 1-Cy5.5 and
6-Cy5.5. Note that lung images were not available for 0.25 nmol
1-Cy5.5.
additional negative charge aids in solubility and may promote
excretion by preventing kidney uptake.
Conclusion
Using synthesis, cell adhesion assays, and optical imaging
with xenograft murine models, the bisaryl urea moiety (1) has
been transformed from a bisaryl urea to the benzothiazole
analogue 6 without sacrificing low picomolar potency to
activated R4ꢀ1 integrin. Because of the presence of an additional
acidic hydrogen from a relatively unusual acid (bisarylamino
H2N-K[(E)-3-(Pyridin-3-yl)acrylamide]-Aad(OtBu)-Ach-Rink
Polystyrene (22). Rink amide resin (2.35 g, 1.19 mmol) was swollen
in DMF (30 mL) for 3 h, followed by treatment with 20% piperidine
in DMF (20 mL). After washing, the resin was then treated with a

18 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Carpenter et al.
premixed solution of Fmoc-Ach-OH (Fmoc-Ach-OH; 1.30 g, 3.57
mmol), 1,3-diisopropylcarbodiimide (DIC; 3.57 mmol, 553 µL),
and hydroxybenzotriazole (HOBt; 482 mg, 3.57 mmol) in DMF
(20 mL) followed by shaking for 6 h. After a negative Kaiser test14
washing, this sequence of deprotection/coupling was repeated thrice
more with Fmoc-Aad(tBu)-OH (Fmoc-Aad(tBu)-OH; 1.57 g, 3.57
mmol), Dde-K(Fmoc)-OH (Dde-K(Fmoc)-OH; 1.85 g, 3.57 mmol),
and (E)-3-(pyridin-3-yl)acrylic acid (532 mg, 3.57 mmol). After
washing, the Dde-tripeptide resin was washed and treated with 2%
H2NNH2 in DMF (20 mL) for 20 min, followed by washing to
afford the free amino-tripeptide resin 22: IR (neat) 3430 (sh), 3370
process was repeated once more, and the combined filtrates were
concentrated under a gentle stream of nitrogen, precipitated with
ether, centrifuged, and decanted. The precipitate was then purified
by preparatory HPLC, and the combined fractions were lyophilized
to afford 6-Cy5.5 (13 mg, 34%) as a blue powder: ESI MS (m/z)
2221 (M + H)+. ESI HRMS (m/z) for C106H131N16NaO25S5: Calcd
2242.7882 (M + Na)+. Found: 2242.7871. Purity was determined
to be 100% by HPLC analysis.
General Procedure for Aryl Isothiocyanate Esters: Ethyl
4-Isothiocyanatobenzoate (21a). Following our previously reported
procedure,21 a solution of an appropriate aniline ester (4.5 g, 27.3
mmol) and triethylamine (60.1 mmol, 8.37 mL) in ethyl acetate
(160 mL) was treated with thiophosgene (30.0 mmol, 2.30 mL) in
ethyl acetate (130 mL) dropwise over 30 min at 0 °C. After addition,
the cooling bath was removed and the reaction mixture was allowed
to gradually warm up to room temperature over 12 h. The workup
consisted of diluting with ethyl acetate, followed by washing
sequentially with water (200 mL × 2) and brine (200 mL). The
organic layer was dried (MgSO4), concentrated, and the crude
product was purified via short path CC (hexanes/ethyl acetate, 9:1)
to give 2a (5.03 g, 89%). The analytical data are in accord with
literature values.15
Ethyl 2-(4-Isothiocyanatophenyl)acetate (21b). Following the
general procedure for aryl isothiocyanateesters yielded 2b (5.20 g,
93%). The analytical data are in accord with literature values.16
Methyl 3-(4-Isothiocyanatophenyl)propanoate (21c). Following
the general procedure for aryl isothiocyanate esters yielded 21c (5.41
g, 90%). The analytical data are in accord with literature values.7,17
Methyl 3-Isothiocyanatobenzoate (21d). Following the general
procedure for aryl isothiocyanate esters yielded 21d (7.06 g, 92%
yield). The analytical data are in accord with literature values.18
(sh), 3084, 1740 (st), 1684 (st), 1680 (st), 1662 (st), 1654 (st) cm-1
.
General Procedures for Heterocyclic Analogues: (R)-5-(R)-[2-
(2-[4-(Benzothiazol-2-ylamino)-phenyl]acetylamino)-6-(E)-(3-pyri-
din-3-yl-acryloylamino)hexanoylamino]-5-(1-carbamoylcyclo-hex-
ylcarbamoyl)pentanoic Acid (6). Heterocyclic acid 15 (53 mg, 0.180
mmol), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (HBTU; 67 mg, 0.180 mmol), and DIEA (54.7 mL,
0.360 mmol) were dissolved in DMF (3 mL) and the homogeneous
solution was allowed to stand for 10 min. This solution was then
added to the free amino tripeptide resin 22 (120 mg, 0.06 mmol)
and was shaken for 4 h. After washing, the resin was then cleaved
with 3 mL of a 95:2.5:2.5 cleavage solution of TFA:H2O:TIPS for
2 h, followed by draining and washing with the cleavage solution.
This cleavage process was repeated once more, and the combined
filtrates were concentrated under a gentle stream of nitrogen,
precipitated with ether, centrifuged, and decanted. The precipitate
was then purified by preparatory HPLC, and the combined fractions
were lyophillized to afford 6 (27 mg, 62% from Rink Amide resin)
as a white powder: ESI MS (m/z) 810 (M + H)+. EI HRMS (m/z)
for C42H50N8O7S: Calcd 810.3523 (M + H)+. Found: 811.3549
Purity was determined to be 99% by HPLC analysis. Tabulated
analytical data for 3-11 as well as 6-Cy5.5 are shown in Table 1
of the Supporting Information.
3-(56-Bromo-1H-benzo[d]imidazol-2-ylamino)benzoic Acid (12).
Following the general procedure for heterocyclic acids yielded 12
(Yield 1.27 g, 78%). The analytical data are in accord with our
previously reported values.7
2-(1E,3E,5E)-5-3-(R)-1-(1-(R)-2-(R)-2-[2-(4-(Benzo[d]thiazol-2-
ylamino)phenyl)\acetamido]-6-(E)-3-(pyridin-3-yl)acrylamido)hex-
anamido)-5-carboxypentanamido)cyclohexyl)-27-carbamoyl-
1,9,13,21,25,33-hexaoxo-5,17-dioxa-2,8,14,20,26,32-hexa-
azaheptatriacontan-37-yl)-1,1-dimethyl-6,8-disulfonato-1H-
benzo[e]indol-2(3H)-ylid-ene)penta-1,3-dienyl)-3-ethyl-1,1-dimethyl-
1H-ben-zo[e]indolium-6,8-disulfonate (6-Cy5.5). Rink amide resin
(120 mg, 0.06 mmol) was swollen in DMF (3 mL) for 3 h, followed
by treatment with 20% piperidine in DMF (20 mL). After washing,
the resin was then treated with a premixed solution of Fmoc-
K(Dde)-OH (93 mg, 0.180 mmol), 1,3-diisopropylcarbodiimide
(DIC; 0.180 mmol, 27.9 µL), and hydroxybenzotriazole (HOBt;
24.3 mg, 0.180 mmol) in DMF (3 mL), followed by shaking for
6 h. After a negative Kaiser test14 washing, this sequence of
deprotection/coupling was repeated with N-(Fmoc-8-amino-3,6-
dioxa-octyl)succinamic acid (85 mg, 0.180 mmol), then Fmoc-Ach-
OH (66 mg, 0.180 mmol), Fmoc-Aad(OtBu)-OH (79 mg, 0.180
mmol), and Fmoc-K(Alloc)-OH (82 mg, 0.180 mmol). The free
R-amino resin was then treated with a premixed solution of 15 (51
mg, 0.180 mmol), HBTU (68 mg, 0.180 mmol), and DIEA (35.8
µL, 0.180 mmol) in DMF (3 mL) for 4 h. After washing, the ε-Alloc
protected amine was deprotected by treatment with a solution of
Pd(PPh3)4 (11 mg, 9 × 10-3 mmol) and phenylsilane (1.2 mmol,
149 µL) in DMF (3 mL) for 30 min. Note: Care should be taken
(i.e., Venting), as this reaction builds up heat and pressure. After
draining and washing, this deprotection was repeated once more.
This free ε-amino resin was then treated with a solution of (E)-3-
(pyridin-3-yl)acrylic acid (27 mg, 0.180 mmol), DIC (0.180 mmol,
27.9 µL), and hydroxybenzotriazole (HOBt; 24.3 mg, 0.180 mmol)
in DMF (3 mL). The ε-Dde protected amine was removed upon
treatment with 2% H2NNH2 in DMF (3 mL) for 5 min, then washed
with DMF and repeated for 15 min. After washing, to this free
ε-amino resin was added Cy5.5-NHS (90 mg, 0.090 mmol) and
DIEA (0.090 mmol, 15.8 µL) in DMF (3 mL) and allowed to shake
for 8 h. After washing, the resin was then cleaved with 3 mL of a
95:2.5:2.5 cleavage solution of TFA:H2O:TIPS for 2 h, followed
by draining and washing with the cleavage solution. This cleavage
3-(4-(56-Bromo-1H-benzo[d]imidazol-2-ylamino)phenyl)propano-
ic Acid (13). Following the general procedure for heterocyclic acids
yielded 13 (Yield 1.82 g, 83%). The analytical data are in accord
with our previously reported values.7
2-(4-(Benzo[d]thiazol-2-ylamino)phenyl)acetic Acid (15). Fol-
lowing the general procedure for heterocyclic acids yielded 15 (2.81
g, 70%). The analytical data are in accord with literature values.19
2-(4-(4-Methylbenzo[d]oxazol-2-ylamino)phenyl)acetic Acid (16).
Following the general procedure for heterocyclic acids yielded 16
(2.49 g, 75%) as a gray solid: mp 337-339 °C. IR (neat) 3539 (st,
1
br), 3268 (sh), 3049 (sh), 2984, 1724 (st). H (400 MHz, DMSO-
d6): δ 10.56 (br s, 1H), 7.69 (d, J ) 6.8 Hz, 2H), 7.24 (apparent t,
3H), 7.02-6.95 (m, 2H), 3.50 (s, 2H), 2.47 (s, 3H). 13C NMR (100
MHz, DMSO-d6): δ 172.9, 157.5, 146.6, 141.3, 137.5, 129.9, 128.6,
126.3, 124.7, 121.3, 117.5, 106.4, 22.5, 16.2. ESI MS (m/z) 283
(M + H)+. Anal. Calcd for C16H14N2O3: C, 68.07; H, 5.00; N, 9.92.
Found: C, 68.18; H, 5.00; N, 9.95. Purity was determined to be
99% by HPLC analysis on the basis of absorption at 220 nm.
2-(4-(Benzo[d]oxazol-2-ylamino)phenyl)acetic Acid (17). Fol-
lowing the general procedure for heterocyclic acids yielded 17 (1.89
g, 79%). The analytical data are in accord with literature values.20
4-(4-Methylbenzo[d]oxazol-2-ylamino)benzoic Acid (18). Fol-
lowing the general procedure for heterocyclic acids yielded 18 (1.67
g, 81%) as a light tan solid: mp 327-329 °C. IR (neat) 3560 (st,
1
br), 3248 (sh), 3032 (sh), 2994, 1696 (st). H (300 MHz, DMSO-
d6): δ 11.11 (s, 1H), 7.96 (d, J ) 9 Hz, 2H), 7.91 (d, J ) 9 Hz,
2H), 7.26 (apparent t, 1H), 7.01-6.97 (m, 2H), 2.45 (s, 3H). 13C
NMR (75 MHz, DMSO-d6): δ 167.2, 156.9, 146.7, 143.1, 141.0,
130.8, 126.8, 124.8, 123.9, 121.9, 116.8, 106.6, 16.3. ESI MS (m/
z) 269 (M + H)+. Anal. Calcd for C15H12N2O3: C, 67.16; H, 4.51;
N, 10.44. Found: C, 66.93; H, 5.34; N, 10.09. Purity was determined
to be 99% by HPLC analysis on the basis of absorption at 220 nm.
4-(Benzo[d]oxazol-2-ylamino)benzoic Acid (19). Following the
general procedure for heterocyclic acids yielded 19 (2.04 g, 76%).
The analytical data are in accord with literature values.21

SelectiVely Targeting T- and B-Cell Lymphomas
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 19
4-(Benzo[d]thiazol-2-ylamino)benzoic Acid (20). Following the
general procedure for heterocyclic acids yielded 20 (1.19 g, 85%).
The analytical data are in accord with literature values.22
Cell Adhesion Assay Background. R4ꢀ1 integrins are cell surface
heterodimeric glycoproteins that mediate cell adhesion to vascular
cell adhesion molecule-1 (VCAM-1 or CD 106) as well as to
extracellular matrix (ECM) protein fibronectin (FN).4 Integrin
expression and function depend on cell activation. The dynamic
changes in integrin affinity, avidity, or activation state are implicated
in cell migration, survival and apoptosis, cancer development, and
metastasis.23 Binding affinities (IC50s) of the ligands were studied
in a Molt4 T-cell leukemia adhesion assay by inhibiting the R4ꢀ1-
mediated cell adhesion to CS-1 peptide (DELPQLVTLPHPNLH-
GPEILDVPST), which is the binding motif of fibronectin to R4ꢀ1
receptor.
General Procedure for Cell Adhesion Assay. We prepared 96-
well plates by coating them with 1 µg/mL neutravidin for a one-
hour period, followed by adding 2 µM biotin-conjugated CS-1
peptide. The wells were then blocked with 1% bovine serine
albumin in phosphate buffer saline (PBS) solution, followed by
adding a volume of 80 µL consisted of 1.3 × 105 Molt4 cells, and
finally different dilutions of tested ligands in binding buffer (1 mM
Mn2+ TBS) were added to each well. To allow binding, the plates
were incubated for 30 min at 37 °C, followed by washing of
unbound cells with PBS. Bound cells were fixed with 10% formalin
buffered in phosphate for 30 min and stained with 0.1% crystal
violet. After washing and drying at room temperature, the dye was
dissolved in 1% SDS, and absorbance at 570 nm was measured
using a 96-well TECAN OD UV/vis spectrophotometer. Inhibition
was calculated as a percentage resulting from the concentration-
dependent curve.
Supporting Information Available: Analytical data for all new
compounds and bioassay data for 3-11. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Lin, K. C.; Castro, A. C. Very late antigen 4 (VLA4) antagonists as
anti-inflammatory agents. Curr. Opin. Chem. Biol. 1998, 2, 453–457.
(2) Yusut-Makagiansar, H.; Anderson, M. E.; Yakovleva, T. V.; Murray,
J. S.; Siahaan, T. J. Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1
as a therapeutic approach to inflammation and autoimmune diseases.
Med Res. ReV. 2002, 22, 146–167.
(3) Holzman, B.; Gosslar, U.; Bittner, M. R4 integrins and tumor
metastasis. Curr. Top. Microbiol. Immunol., 1998, 231, 125–141.
(4) Vincent, A. M.; Cawley, J. C.; Burthem, J. Integrin function in chronic
lymphocytic leukemia. Blood 1996, 87, 4780–4788.
(5) Marco, R. A.; Diaz-Montero, C. M.; Wygant, J. N.; Kleinerman, E. S.;
McIntyre, B. W. a4 integrin increases anoikas of human osteosarcoma
cells. J. Cell. Biochem. 2003, 88, 1038–1047.
(6) Peng, L.; Liu, R.; Marik, J.; Wang, X.; Takada, Y.; Lam, K. S.
Combinatorial chemistry identifies high-affinity peptidomimetics
against a4b1 integrin for in vivo tumor imaging. Nat. Chem. Biol 2006,
2, 381–387.
(7) Carpenter, R. D.; Andrei, M.; Lau, E. Y.; Lightsone, F. C.; Liu, R.;
Lam, K. S.; Kurth, M. J. Highly potent, water soluble benzimidazole
antagonist for activated R4ꢀ1 integrin. J. Med. Chem. 2007, 50, 5863–
5867.
(8) Perkins, J. J.; Zartman, A. E.; Meissner, R. S. Synthesis of 2-(alky-
lamino)benzimidazoles. Tetrahedron Lett. 1999, 40, 1103–1106.
(9) Oh, Y.-H.; Han, H.-K. Pharmacokinetic interaction of tetracycline with
nonsteroidal anti-inflammatory drugs via organic anion transporters
in rats. Pharm. Res. 2005, 53, 75–79.
(10) Babu, E.; Takeda, M.; Shinichi, N.; Yasuna, K.; Toshinori, Y.; Ho,
C. S.; Takashi, S.; Dhanapal, S.; Hitoshi, E. Human organic anion
transporters mediate the transport of tetracycline. Jpn. J. Pharmacol.
2002, 88, 69–76.
Xenograft Methods. Four-six week old male and female
athymic nude (Nu/Nu) mice were purchased from Harlan Sprague-
Dawley (Indianapolis, IN). Mice were housed in the animal facility
and fed ad lib with rodent pellets and water. Tumor xenografts
were created by injecting Molt-4 (T-cell lymphoma) or Raji (B-
cell lymphoma; 1 × 107 cells in 200 µL incomplete RPMI media)
subcutaneously on the right upper back and A549 (1 × 106 cells in
200 µL incomplete DMEM media) subcutaneously on the left upper
back under mild inhalation anesthesia (halothane). Studies com-
menced when xenografts reached between 10-15 mm. All proce-
dures were conducted under an approved protocol according to
guidelines specified by the National Institute of Health Guide for
Animal Use and Care. Animals were anesthetized intraperitoneally
with (1 µL/g bwt) of 6 mg/mL pentobarbitol sodium. The tails were
dilated with warm water and animal were injected iv. Animals were
imaged serially for tumor uptake and washout. The duration of each
scan was 30 s for each acquired exposure.
(11) Carpenter, R. D.; DeBerdt, P. B.; Lam, K. S.; Kurth, M. J. A
carbodiimide-based library method. J. Comb. Chem. 2006, 8, 907–
914.
(12) Carpenter, R. D.; Lam, K. S.; Kurth, M. J. Microwave-mediated
heterocyclization to benzimidazo[2,1-b]quinazolin-12(5H)-ones. J.
Org. Chem. 2007, 72, 284–287.
(13) Grazioli, A.; Alves, C. S.; Konstantopoulos, K.; Yang, J. T. Defective
blood vessel development and pericyte/pvSMC distribution in R4
integrin-deficient mouse embryos. DeV. Biol. 2006, 293, 165–177.
(14) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test
for detection of free terminal amino groups in the solid-phase synthesis
of pepetides. Anal. Biochem. 1970, 34, 595–598.
(15) Sayigh, A. A. R.; Ulrich, H.; Potts, J. S. Reaction of arylamines with
diethylthiocarbamoyl chloride. New synthesis of aryl isothiocyanates.
J. Org. Chem. 1965, 30, 2465–2466.
(16) Bowne, D. W.; Dyson, G. W. J. Chem. Soc. 1935, 178.
(17) Jakobsen, C. M.; Denmeade, S. R.; Isaacs, J. T.; Gady, A.; Olsen,
C. E.; Christensen, S. B. Design, synthesis, and pharmacological
evaluation of thapsigargin analogues for targeting apoptosis to prostatic
cancer cells. J. Med. Chem. 2001, 44, 4696–4703.
(18) Budesinsky, M.; Exner, O. Correlation of carbon-13 substituent-
induced chemical shifts: meta- and para-substituted methyl benzoates.
Magn. Reson. Chem. 1989, 27, 585–591.
(19) Sawhney, S. N.; Arora, S. K.; Singh, J. V.; Bansal, O. P.; Singh, S. P.
Synthesis and anti-inflammatory activity of 2-amino- and 2-alkylami-
no-6-benzothiazoleacetic acids, 4-(2′-benzothiazolylamino)-,4-(4′-sub-
situted-2-thiazolyl-amino)- and 4-(4′-substituted-3′-alkyl-4′-thiazoline-
2′-imino)phenylacetic acids. Indian J. Chem., Sect. B: Org. Chem.
Incl. Med. Chem 1978, 16, 605–609.
(20) Nakayama, A.; Machinaga, N.; Yoneda, Y.; Sugimoto, Y.; Chiba, J.;
Watanabe, T.; Imura, S. VLA-4 inhibitorInt. Patent JP11641, 2002.
(21) Varma, R. Synthesis of substituted 2-anilinobenzoxazoles. Curr. Sci.
1976, 45, 53–54.
(22) Tsubuki, T.; Nomura, M.; Sawada, T.; Kono, Y.; Sakoe, Y. Patent
258842, Jpn. Kokai Tokkyo Koho, 2000.
Acknowledgment. This research is dedicated to the legacy
of Dorothy Schoening who lost her lymphoma battle De-
cember 4, 2005. R.D.C. thanks the American Chemical
Society’s Division of Medicinal Chemistry for their Predoc-
toral Fellowship, the Howard Hughes Medical Institute for
their Med into Grad Fellowship, and UC Davis for their R.B.
Miller and Dissertation Awards. The authors also thank the
National Cancer Institute (U19CA113298), the National
Institute for General Medical Sciences (RO1-GM076151),
and the National Science Foundation (NMR spectrometers;
CHE-0443516 and CHE-9808183). This work was in part
performed under the auspices of the United States Department
of Energy by Lawrence Livermore National Laboratory under
contract number DE-AC52-07NA27344.
(23) Luscinskas, F.; Lawler, J. Integrins as dynamic regulators of vascular
function. FASEB J. 1994, 8, 929–938.
JM800313F