Application of encoded library technology (ELT) to a protein-protein interaction target: Discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists

Article abstract of DOI:10.1016/j.bmc.2014.01.050

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.

Full text of DOI:10.1016/j.bmc.2014.01.050

Bioorganic & Medicinal Chemistry 22 (2014) 2353–2365
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Application of encoded library technology (ELT) to a protein–protein
interaction target: Discovery of a potent class of integrin lymphocyte
function-associated antigen 1 (LFA-1) antagonists
Christopher S. Kollmann a, Xiaopeng Bai a, Ching-Hsuan Tsai a, Hongfang Yang a, Kenneth E. Lind a,
Steven R. Skinner a, Zhengrong Zhu a, David I. Israel a, John W. Cuozzo a, , Barry A. Morgan a, Koichi Yuki b,à
,
Can Xie b,§, Timothy A. Springer b, Motomu Shimaoka b,–, Ghotas Evindar a,
a GlaxoSmithKline, Platform Technology & Science, MDR Boston, 830 Winter Street, Waltham, MA 02451, USA
⇑
b Immune Disease Institute, Children’s Hospital Boston, Harvard Medical School, Program in Cellular and Molecular Medicine, Department of Biological Chemistry and
Molecular Pharmacology, 3 Blackfan Circle, Rm. 3100, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of protein–protein interactions remains a challenge for traditional small molecule drug
discovery. Here we describe the use of DNA-encoded library technology for the discovery of small
molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen
1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of
4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were
affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family
were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-
associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both
ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were
demonstrated to bind to cells expressing the target protein.
Received 25 November 2013
Revised 15 January 2014
Accepted 24 January 2014
Available online 7 February 2014
Keywords:
Encoded Library Technology
DNA-encoded libraries
Affinity-based selections
Lymphocyte Function-associated Antigen 1
Intercellular Adhesion Molecule 1
Protein-Protein Interactions
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
discovery efforts.1 Most protein–protein interactions (PPI) occur
over large, flat surface areas that have proved more difficult to
Targeting small molecule drugs to the interfaces between pro-
teins has great therapeutic potential, but remains a challenge for
traditional high-throughput screening drug discovery efforts.
Three quarters of marketed pharmaceuticals target either enzymes
or G-protein coupled receptors target classes known for containing
classical ligand pockets that are amenable to traditional drug
target than classical ligand binding pockets.2,3
Integrin LFA-1 (lymphocyte function-associated antigen-1) is a
major leukocyte cell adhesion molecule that binds to its major
ligand ICAM-1 (intercellular adhesion molecule-1) on endothelial
cells and dendritic cells.4 LFA-1 plays a pivotal role in regulating
leukocyte trafficking to sites of inflammation as well as in inducing
immune responses, thereby representing an established therapeutic
target for autoimmune and inflammatory diseases.5 A therapeutic
antibody to LFA-1, efalizumab (Raptiva™) has been demonstrated
to be effective for the treatment of patients with psoriasis, a T-cell
mediated autoimmune disease in the skin.6 In addition, LFA-1
inhibitors are currently being investigated in clinical trials for the
treatment of uveitis7 and other ocular inflammation.8
The ligand binding domain of LFA-1 termed inserted (I) domain
adopts a Rossmann fold that contains a metal ion-dependent adhe-
sion site (MIDAS) located on the top, whereas its C- and N-terminal
connections are located on the distal bottom face.9 The ability of
the I domain to bind ligand is regulated by conformational
changes, as the affinity for its ligand is dramatically enhanced by
a ‘piston-like’ downward axial displacement of its C-terminal helix.
Abbreviations: ELT, encoded library technology; LFA-1, lymphocyte function-
associated antigen 1; ICAM-1, intercellular adhesion molecule 1; PPI, protein–
protein interactions.
⇑
Corresponding author. Tel.: +1 781 795 4423; fax: +1 781 795 4496.
E-mail address: ghotas.x.evindar@gsk.com (G. Evindar).
Present address: X-Chem Inc, 100 Beaver Street Suite 101, Waltham, MA 02453,
USA.
à
Present address: Peking University, State Key Laboratory of Biomembrane and
Membrane Biotechnology, Laboratory of Receptor Biology, 5 Yiheyuan Road, Beijing
100871, China.
§
Present address: Department of Anesthesiology, Perioperative and Pain Medicine,
Children’s Hospital Boston, and Harvard Medical School, 300 Longwood Avenue,
Boston, MA 02115, USA.
–
Present address: Mie University Graduate School of Medicine, 2-174 Edobashi,
Tsu-City, Mie 514-8507, Japan.
http://dx.doi.org/10.1016/j.bmc.2014.01.050
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2354
C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
The C-terminal downward shift is conformationally linked to the
conversion of the MIDAS to the high-affinity configuration that
tightly binds to ligand. Since only the high-affinity form tightly
binds to ICAM-1, perturbation of the activation-dependent confor-
mational conversion of the LFA-1 I domain to the high-affinity form
has been an important target for small-molecule drugs.5 A class of
potent small-molecule antagonists to LFA-1, termed alpha I alloste-
ric antagonists, have been reported that bind to the cavity under-
neath the C-terminal helix of the I domain, thereby stabilizing
the low-affinity LFA-1 conformation.10–12 Thus far, the small-mol-
ecule antagonists that bind to the I domain were selected from a
conventional random screening based on the results of functional
assays that tested the ability of candidate compounds to block
LFA-1-ICAM-1 interaction. Despite the critical role of the I domain
in ligand binding, selection of antagonists by explicitly targeting
the I domain has limited investigation.13
We have previously described encoded library technology (ELT)
as a novel hit identification technology that includes the synthesis
of combinatorial DNA-encoded libraries (DELs) containing 106–1010
members, the interrogation of those libraries for ligands by affinity
selection, hit structure deconvolution through DNA sequencing
and finally the resynthesis of small molecule hits off-DNA for
activity confirmation.14,15 We have also previously used ELT to
identify inhibitors of Aurora A and p38 MAP kinases,15 ADAMTS-5
metalloprotease16 and sirtuins.17 While encoded libraries have been
reportedly used to discover inhibitors of protein–protein interac-
tions, to our knowledge none of these inhibitors have demonstrated
sub-micromolar potency.18–20 With the intention of expanding the
applications of ELT to PPIs, we launched a campaign to identify
specific small molecule inhibitors of the LFA-1/ICAM-1 interaction
by targeting the I domain, and we report here the first use of the
technology to discover a potent class of small molecules that block
the interaction of LFA-1 with its ligand ICAM-1.
ELT libraries. Most of the selected features were confirmed to be
active inhibitors after off-DNA synthesis. In this paper, we report
on one feature identified from a triazine library. Successful ELT
campaigns using DNA-encoded libraries (DELs) incorporating
1,3,5-triazine as a core structure have been disclosed previ-
ously.15,16 A schematic drawing of the desired triazine library is
shown as DEL-A in Figure 1. DEL-A utilizes 192 Fmoc protected
amino acids coupled with the amine moiety of the desired ELT
headpiece as cycle 1, followed by Fmoc deprotection and function-
alization of the amine group of the cycle 1 with cyanuric chloride.
The triazine core is decorated with 479 amines as cycle 2. Further
elaboration of the triazine moiety with 96 diamines as cycle 3 fol-
lowed by further decoration of the cycle 3 with 459 amine-capping
building blocks (carboxylic acids, aldehydes, sulfonyl chlorides and
isocyanates) as cycle 4 generated a library with a theoretical com-
plexity of 4.1 billion compounds. The synthesis details of this li-
brary have been previously disclosed.15
As previously described, the output of an ELT selection is conve-
niently visualized by a cubic scatter plot representation in which
each axis represents building blocks used in a individual cycle in
the library and the size of the data point represents the number
of times each unique DNA sequence was observed.15 Thus, after fil-
tering out members of the library that are lowly represented or
considered background because of presence in a no target selec-
tion, enriched chemotypes will appear as planes (one building
block in common) or lines (two building in common). For a four cy-
cle library such as DEL-A, two plots are required to visualize the
complete selection. Representative plots that were used to analyze
and visualize the selection outcome and the highly selected en-
riched families/features are shown in Figure 2. The selected feature
is demonstrated by a plane in the cube analysis. This feature’s
enrichment is driven specifically by a single building block from
cycle 1 (BB1), 2-amino-2,3-dihydro-1H-indene-2-carboxylic acid
(1), which was attached to the DNA through its carboxylic acid
functional group. The combination of the amino acid 1 and the tri-
azine core represent the selected pharmacophore 6. Within the se-
lected plane, there are four prominent horizontal lines; an
indication of preference for four different BB2s. There is a great
similarity within these selected BB2s (compounds 2 through 5) a
good indication of some preliminary internal SAR for selection at
cycle 2. Cycles 3 and 4 appear to be variable based on the lack of
selection for specific building blocks as indicated by the lack of
preference for spots within each line.
2. Results and discussion
2.1. ELT selection and data analysis
Affinity selections were performed against soluble LFA-1 I-do-
main after chemical biotinylation. The target protein was immobi-
lized on a streptavidin agarose resin packed column, and then
exposed to the on-DNA library prior to extensive washing to re-
move non-binders. Heat denaturation of the protein was used to
elute bound library molecules and the eluant was incubated with
fresh protein to start a new round of selection. A parallel selection
against a column lacking protein was done to allow for the identi-
fication of molecules enriched by non-target affinity. After three
rounds of selection the eluted population was amplified and se-
quenced, and the sequences translated to identify small molecule
binders to the LFA-1 I-domain. Only sequences that were unique
and constituted a complete translatable ELT encoding were used
for the analysis. The no target sample sequencing yielded 46,476
such sequences and the target sample gave 268,586. Data analysis
yielded a few attractive features (chemically related enriched com-
pounds that share one or more building blocks) across a number of
2.2. Chemistry
In preparation for off-DNA synthesis and activity confirmation a
few spots within the selected lines were chosen for follow up. The
synthetic strategy was designed to generate the pharmacophore
earlier in the synthesis and add on the variable components later
(Scheme 1). This allowed for testing the intermediate in the activ-
ity assay and therefore additional SAR exploration by testing the
possible fragments and intermediates. Off-DNA synthesis initiated
with ethylamine coupling with Boc-protected acid 7 and Boc re-
moval of the desired product to afford amine 8. We chose the most
Figure 1. Design of DEL-A.

C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
2355
Figure 2. (a) Selected cycle 1 building block (BB1); (b) Spotfire™ cube data views of selected LFA-1 features with selected cycle 2 building blocks (BB2s); (c) potential
chemotype pharmacophore analyzed from the feature analysis.
(a)
Cl
R
N
N
N
i, ii
iii, iv
H
N
N
H
N
N
HO
Boc
N
H
NH2
HN
O
O
v
O
7
8
14
BB2
N
HN
R
N
N
N
H
N
N
HN
O
15
O
(b)
O
N
CF3
12
13
vi, ii
HO
CF3
HN
HN
NH
10
O
S
O
OCF3
Boc
N
O
O
N
OCF3
S
Cl
vii, ii
9
11
Scheme 1. Off-DNA synthesis of LFA-1 selected hits. Reagents and conditions: (i) ethylamine hydrochloride (1.0 equiv), 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl
uronium hexafluorophosphate methanaminium (HATU, 1.2 equiv), N,N-diisopropylethylamine (DIPEA, 3.0 equiv), N,N-dimethylformamide (DMF); (ii) 20% trifluoroacetic acid
(TFA) in dichloromethane (DCM); (iii) cyanuric chloride (1.0 equiv), DIPEA (3.0 equiv), MeCN; (iv) desired amine, DIPEA (3.0 equiv), MeCN, room temperature; (v) desired
amine, DIPEA (3.0 equiv), MeCN; 80 °C; (vi) HATU (1.2 equiv), DIPEA (3.0 equiv), DMF; (vii) DIPEA (2.0 equiv), tetrahydrofuran (THF), rt.

2356
C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
prominently selected cycle 3 BB, 2,5-diazabicyclo[2.2.1]heptane 9,
and a couple of cycle 4 variable region BBs, compounds 10 and 11,
and pre-assembled the desired cycle 3 and cycle 4 precursors 12
and 13 for off-DNA synthesis. A one pot reaction of the amine 8
with cyanuric chloride followed by addition of the precursors 12
and 13 gave aryl halide 14. Displacement of the chloride on com-
pound 14 under heating conditions provided final compound 15.
2.3. Activity assay and SAR establishment
The synthesized compounds were tested for inhibition of the
binding of ICAM-1 to LFA-1 by an ELISA-type ligand binding assay.
Representative results from the dose dependent titration of the
first four synthesized compounds are shown in Figure 3. Given
the precursors 12 and 13 as two combinations of cycles 3 and 4,
we chose only two cycle 2 BBs in our explorations. The four poten-
tial combination compounds (Table 1, entries 1–4) plus a truncated
cycle three analog (entry 5) were the first compounds synthesized
off-DNA and tested for feature activity confirmation. Choosing the
cycle 2 BB as (S)-1-(4-bromophenyl)ethanamine (2), the corre-
sponding amide compounds 16a gave an IC50 potency of 16 nM,
7-fold more potent than the corresponding sulfonamide analog
16b. Replacing the (S)-1-(4-bromophenyl)ethanamine (2) with
the achiral 2-(3,4-dichlorophenyl)ethanamine (5), produce amide
16c with an IC50 potency of 23 nM while the corresponding sulfon-
amide 16d gave analogous activity of 22 nM as amide 16a. This
demonstrated the importance of both the cycle 2 groups and the
presence of an amide/sulfonamide moiety in the inhibitor
Figure 3. Dose dependent inhibition of ICAM-1 binding to LFA-1 by resynthesized
compounds 16a–d as measured by ELISA. Data points are the mean of two
replicates and error bars represent SEM.
Table 1
Activity of the first set of compounds made off-DNA
R1
N
N
N
H
N
R2
HN
O
16
a
Entry
1
Compounds
R1
R2
IC50
Br
Br
O
N
CF3
16a
16 nM
N
HN
O
O
N
OCF3
S
2
3
4
5
16b
16c
16d
16e
117 nM
23 nM
N
N
HN
Cl
Cl
O
N
CF3
HN
Cl
O
O
N
OCF3
S
Cl
Cl
22 nM
N
N
HN
Cl
12,900 nM
HN
a
Inhibition of ICAM-1 binding to LFA-1 was measured by ELISA.

C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
2357
Table 2
structure. Truncation of cycle3–cycle4 group to pyrrolidine dimin-
ished the inhibitor activity to double digit M, clear indication of
the major role played by the cycle3–cycle4 bridged diamine–
Structure–activity study (SAR) exploration around the BB1 and BB2 component of the
scaffold
l
R1
amide/sulfonamide moieties in the target inhibition.
X
N
Exploration of additional SAR around cycle 2 (R1) and cycle 1
(R3) was fruitful in defining the necessary moieties responsible
for the chemotype affinity. In this investigation we chose cycle3–
cycle-4 region constant as the corresponding amide. Maintaining
the cycle 1 as 2-amino-N-ethyl-2,3-dihydro-1H-indene-2-carbox-
amide (8), changing the cycle 2 residue from (S)-1-(4-bromophe-
nyl)ethanamine to the des-bromo analog reduced the activity by
N
N
CF3
R3
N
N
17
R3
a
Entry Compounds R1
X
IC50
10-fold. Inverting the
rimental to the compound activity, while elimination of the
-methyl group all together produce weak M activity. Further
SAR exploration by removal of the two chloro groups from com-
pound 16c gave compound 17d with a 20-fold diminished potency.
Further truncation of 17d by removal of the phenyl group
abolished the compounds activity (Table 2, entry 5). Replacement
of cycle3–cycle4 group to pyrrolidine diminished the inhibitor
a-methyl group chirality from S to R was det-
NH
NH
NH
NH
NH
1
2
3
4
5
6
17a
17b
17c
17d
17e
17f
–CO–
–CO–
–CO–
–CO–
–CO–
–CO–
180 nM
(S)
HN
a
l
HN
HN
HN
HN
HN
O
O
O
O
O
>10,000 nM
7900 nM
488 nM
(R)
HN
activity to double digit
lM a clear indication of the major role
played by the cycle3–cycle4 bridged diamine–amide/sulfonamide
moieties target inhibition. Additional SAR exploration around cycle
1 was observed to be rewarding. Replacement of the ethylamide
HN
group with
a carboxylate maintained compound potency at
37 nM while the des-carboxylate analog was observed to be
10-fold less potent. Further truncation of the cycle group by
removal of the phenyl group abolished the compound activity
(17h, entry 8). Given the excellent potency of the carboxylate
17f, the corresponding sulfonamide was synthesized and was
confirmed to have a potency of 65 nM (17i, entry 9).
HN
HN
Cl
>10,000 nM
37 nM
2.4. Cell adhesion activity
Given the great potency of the chemotype we examined a num-
ber of compounds in a cell adhesion assay. A human lymphocyte
Jurkat cell-line that expresses native wild-type (WT) LFA-1 was
used to test for compound inhibition of cell adhesion to ICAM-1.
Jurkat cells were allowed to interact with ICAM-1 substrates. Upon
PMA stimulation, Jurkat cells showed a good level of cell adhesion
to ICAM-1. Both the competitive antagonist antibody TS1/2221
used as a control and a number of our compounds potently inhib-
ited Jurkat cell adhesion to ICAM-1 (Fig. 4A). To study the mode of
action of the LFA-1 antagonists that we discovered, we employed
K562 cells stably transfected to express wild type LFA-1 (WT,
Fig. 4B–D) or mutant LFA-1 (HA, Fig. 4B–D) that contains an engi-
neered disulfide locked high-affinity I domain.22 The mutant LFA-1
is blocked only by competitive but not allosteric inhibitors. How-
ever, upon DTT treatment that disrupts the engineered disulfide,
the mutant LFA-1 is reverted to a functionally wild-type LFA-1,
thereby becoming inhibitable by either competitive or allosteric
antagonists In this way, this mutant LFA-1 allows for discrimina-
tion between competitive and allosteric modes of LFA-1 inhibi-
tion.23 TS1/22 inhibited not only wild-type LFA-1 (Fig. 4B), but
also mutant LFA-1 both in the absence (Fig. 4C) and presence
(Fig. 4D) of DTT treatment, thereby being confirmed to be a com-
petitive inhibitor. By contrast, an established allosteric antagonist
LFA70324 as well as our compounds were able to inhibit WT LFA-
1 (Fig. 4B) but unable to inhibit the mutant LFA-1 in the absence
of DTT treatment (Fig. 4C). However, they were able to inhibit
the mutant HA LFA-1 in the presence of DTT treatment (Fig. 4D).
These results suggest that our compounds function in the same
mode as LFA703; that is, in allosteric inhibition. As our compounds
bind to the I domain, their binding sites are likely to locate to the
allosteric pocket underneath the C-terminal helix, to which estab-
lished alpha I allosteric antagonists including LFA703 were found
to bind.
Cl
HO
NH
HN
Cl
O
Cl
Cl
Cl
NH
7
8
9
17g
17h
17i
–CO–
–CO–
358 nM
>10,000
HN
Cl
NH
HN
Cl
–SO2– 65 nM
HO
NH
HN
O
a
Inhibition of ICAM-1 binding to LFA-1 was measured by ELISA.
2.5. Synthesis and affinity of fluorescently labeled compounds
Compounds 16c and 16d were chosen as representative for fur-
ther investigation. We generated fluorescent compounds by di-
rectly conjugating the Cy3 dye to the DNA attachment point
(Scheme 2). Also, we synthesized compounds 16c and 16d with
DNA-headpiece attached to generate compounds 20a and 20b then
ligated them to biotinylated-DNA to generate compounds 21a and
21b (Scheme 3). The addition of fluorescently labeled streptavidin
phycoerythrin (PE) allowed visualization of binding by a warhead

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C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
Figure 4. Inhibition of LFA-1-mediated cell adhesion to ICAM-1. (A) Adhesion to ICAM-1 of Jurkat cells expressing native WT LFA-1 in the presence or absence of PMA
stimulation. (B and C) Adhesion to ICAM-1 of K562 transfectants expressing WT (B) or mutant high-affinity (C and D) LFA-1 in resting (Mg/Ca) or stimulating (Mn) conditions.
(A–D) Data are one of two representative experiments with mean S.D. of quadruplicates. Compounds 16a–d at 50
lM, a reference allosteric antagonist LFA703 at 50 lM, and
a reference competitive antagonist TS1/22 at 10
lg/mL were used.
Cl
Cl
HN
X
N
N
N
CF3
HO
N
H
N
N
O
17f, X = -CO-
17i
, X = -SO2-
i, ii, iii
HO3S
Cl
Cl
N
HN
X
N
O
N
N
CF3
H
N
N
N
H
N
N
N
H
O
HO3S
18a, X = -CO-
18b
, X = -SO2-
Scheme 2. Fluorophore labeling of selected LFA-1 hits. Reagents and conditions: (i) tert-butyl (4-aminobutyl)carbamate (1.5 equiv), HATU (1.5 equiv), DIPEA (5.0 equiv),
DMF; (ii) 20% TFA in DCM; (iii) Cy3-NHS ester (1.0 equiv), DIPEA (3.0 equiv), DCM/DMSO (3:1).

C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
2359
Cl
Cl
Cl
Cl
HN
HN
X
N
N
N
N
N
CF3
i, ii, iii
H
N
iv
H
N
NH2
N
H
N
Cl
N
H
N
N
O
O
19
20a
, X = -CO-
20b, X = -SO2-
Cl
Cl
HN
X
N
N
N
CF3
v
H
Biotin
N
N
H
N
N
O
21a
, X = -CO-
21b, X = -SO2-
Scheme 3. On-DNA synthesis of biotinylated selected LFA-1 hits. Reagents and conditions: (i) 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,3-dihydro-1H-indene-2-
carboxylic acid (40.0 equiv), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 40.0 equiv), sodium borate buffer (pH 9.4), MeCN/H2O (1:1), 0
°C, 2 h; (ii) 10% piperidine in sodium borate buffer (pH 9.4); (iii) cyanuric chloride (30 equiv), MeCN/H2O (1:1), 0 °C, 2 h then added 2-(3,4-dichlorophenyl)ethanamine
(50 equiv) in DMA, 0 °C, 12 h; (iv) Compound 12 or 13 (50.0 equiv), sodium borate buffer (pH 9.4), MeCN, 80 °C, 1 h; (v) enzymatic ligation of a biotinylated-DNA.
attached to DNA as in the library. The Cy3 labeling initiated with a
peptide coupling of compounds 17f and 17i to mono-Boc deco-
rated 1,4-diamino butane. After removal of the Boc protecting
group the corresponding amine was coupled with Cy3-NHS ester
to afford final compounds 18a and 18b. The corresponding on-
DNA biotinylated approach was initiated in a library mimetic fash-
ion. The synthesis proceeded analogously to our previous reports
using ELT headpiece (HP).15 Coupling of the free amine functional
group of the HP with Fmoc-protected cycle 1 amino acid was fol-
lowed by the Fmoc removal to produce cycle1 as free amine. Reac-
tion of the cycle amine with cyanuric chloride followed by addition
of the second amine afforded on-DNA precursor 19. Reaction of
precursor 19 with amine 12 and 13 provided final compounds
20a and 20b which were further ligated with a biotinylated piece
of DNA to produce 21a and 21b. We consider the immediate
knowledge of a usable attachment point to be a major benefit of
the ligands discovered through ELT, as tool compounds can be rap-
idly synthesized. By making the DNA conjugated molecules we
were able to examine the affinity of the small molecule warhead
as in the original library. In addition to determining the potency
of the conjugated compounds in the LFA-1 ELISA, we also investi-
gated the compound’s binding to LFA-1 in its natural state on the
cell surface using fluorescent flow cytometry.
produce comparable data to the ethylamine 16c and 16b while
the on-DNA compounds gave 10-fold lower potency but retained
sufficient activity for further cell surface LFA-1 investigation.
We then used the fluorescently labeled compounds 18a and
18b and the on-DNA compounds (20a and 20b) to examine the
binding of these compounds to native LFA-1. Flow cytometry was
used to measure the fluorescence increase due to compound bind-
ing to either parent K562 cells (naïve) that lack LFA-1 expression or
19385WT (K562 cells stably transfected to express LFA-125). After
incubation of the cells with biotinylated on-DNA compounds
(21a and 21b) streptavidin PE was added to detect binding. Fluo-
rescence increase due to compound binding was compared to phy-
coerythrin labeled mouse anti-human CD11a antibody binding as a
positive control for LFA-1 expression (Fig. 5). We observed a higher
background fluorescence signal from the Cy3 labeled compounds
on the naïve cells, but fluorescence signal increased 2–3 fold when
LFA-1 expressing cells were used (Table 4). The on-DNA com-
pounds had lower levels of background signal and an LFA-1 depen-
dent fluorescent signal increase of 45 and 13 fold (Table 4). This
data indicates the requirement of LFA-1 expression for binding
and shows that the original on-DNA library member as selected
by affinity to soluble LFA-1 I-domain retains affinity for the native
protein as expressed on a cell membrane.
The Cy3 and DNA conjugated compounds were both inhibitors
of the LFA-1/ICAM-1 interaction (Table 3). These data were com-
pared to one another as well as the corresponding ethylamide
16c (23 nM) and 16d (22 nM). The Cy3 conjugated compounds
3. Conclusion
Our strategy of selecting for binders to a soluble recombinant
protein domain from a complex chemical library was validated
by our discovery of potent small molecules with novel SAR that in-
hibit the LFA-1/ICAM-1 PPI in both ELISA and cell adhesion assays.
This potency was retained after modification for use as tool com-
pounds and the compounds proved to retain affinity for native pro-
tein. The latter phenomena provided an opportunity for ELT
selections against a desired target in its natural state on cell sur-
face. We are currently exploring this technology development
opportunity.
Table 3
LFA-1 activity of modified inhibitors
a
Entry
Compounds
Modification
IC50
1
2
3
4
18a
18b
20a
20b
Cy3
Cy3
DNA
DNA
100 nM
28 nM
330 nM
295 nM
a
Inhibition of ICAM-1 binding to LFA-1 was measured by ELISA.

2360
C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
Figure 5. Fluorescent cell cytometry of the binding of on-DNA, fluorescently labeled compounds to LFA-1 expressing cell. The x and y axes show number of cells per channel
and fluorescence intensity in each channel, with intensity on a log scale.
ng/mL. Cells were labeled with calcein acetoxymethyl ester
Table 4
(calcein-AM) (Invitrogen). 50
mock (DMSO), LFA-1 competitive antagonist TS1/22 (final concen-
tration 10 g/mL) were given to wells. DMSO concentration was
lL of cells containing compounds,
Fluorophore signal increase due to inhibitor binding to LFA-1
Mean signal
K562 naïve
cells
K562 LFA-1
expressing cells
Fold signal
increase
l
held constant at 0.5%. Cells were incubated at 37 °C for 45 min in
the dark, and centrifuged at 200g for 5 min. After centrifugation,
nonadherent cells that accumulated in the center of the V bottom
wells were quantified using the Fluoroskan Ascent microplate fluo-
rometer (Thermo Scientific) with the filter sets of excitation at 485
nm and emission at 535 nm. ICAM-1 binding % was defined in the
following formula as follows.
1
2
3
4
5
PE
a
CD11a
93
12,072
699
1360
1833
652
129
3
2
45
13
18a
18b
21a
21b
215
610
41
51
4. Experimental procedures
4.1. Protein expression
½1 ꢀ ðFIICAMꢀ1=FIBSAÞꢁ ꢂ 100 ¼ % of adhesive cells
FIICAM-1: the fluorescence signal when cells bind to ICAM-1; FIBSA
the fluorescence signal in absence of ICAM-1 and presence of BSA.
In some experiments, K562 transfectants expressing wild-type
or mutant disulfide-locked high-affinity LFA-1 K287C/K294C (Ref.
22) were used. K562 transfectants were cultured in RPMI1640/
:
The LFA-1 I domain was expressed in Escherichia coli as an
inclusion body, solubilized in guanidine, refolded by dilution, and
purified to homogeneity by
a
gel filtration as previously
Lb2 heterodi-
described.26 Soluble extracellular portion of LFA-1 (
a
10% FBS, 4
coated with ICAM-1-Fc (1
Following wash with TBST, wells were blocked with HBS/2% BSA
for 2 h at room temperature. After wash, wells were given 50
l
g/mL puromycin at 37 °C. V-bottom 96 wells were
l
g/mL) or BSA (1 g/mL) overnight.
l
mer) containing the C-terminal coiled-coil artificial transmem-
brane domains and Hexahistidine and Strep-II tags was expressed
in CHO Lec 3.2.8.1 cells and purified to homogeneity by a sequential
affinity chromatography as previously described.27
l
L
each of HBS/2 mM MgCl2/CaCl2 or HBS/2 mM MnCl2. Cells were
labeled with 20,70-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein
acetoxymethyl ester (BCECF-AM)(Invitrogen). An aliquot (50
lL) of
4.2. Cell adhesion assays
cells containing compounds, mock (DMSO)(final concentration 10
l
l
M), LFA-1 allosteric antagonist LFA703 (final concentration 50
M), or competitive antagonist TS1/22 (final concentration
Cell adhesion assays using a V-bottom plate were performed as
previously described28 with minor modifications.29 Briefly, Jurkat
cells were cultured in RPMI1640/10% FBS at 37 °C. V-bottom 96
10 lg/mL) were given to wells. Cells were incubated at room tem-
perature for 30 min in the dark, and centrifuged at 400g for 5 min.
In some experiments, K562 cells expressing high-affinity LFA-1
(K287C/K294C) were pretreated with 20 mM dithothreitol (DTT)
for 10 min and then underwent continuous treatment with 5 mM
DTT during a 30-min incubation with ICAM-1. After that, the
procedures were same as those with Jurkat cells.
wells (Corning) were coated with ICAM-1-Fc (1
lg/mL) (R&D
Systems) or BSA (1 g/mL) overnight. Following wash with Tris-
l
buffered saline/0.05% Tween (TBST), wells were blocked with
HEPES-buffered saline (HBS)/2% BSA for 2 h at room temperature.
After wash, wells were given 50
lL each of HBS/2 mM MgCl2/CaCl2
or HBS/2 mM MgCl2/CaCl2 + phorbol myristate acetate (PMA) 200

C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
2361
4.3. Affinity screening of encoded library
collected by centrifugation, counted, and washed twice with PBS.
1 lM compound concentration in PBS buffer was incubated with
Sulfo-NHS-LC-biotin (Pierce Chemical, USA) was used to bioti-
2 ꢂ 105 cells in the presence of 0.5 mg/mL of sheared salmon
nylate LFA-1 I-domain protein by incubating 150
lg of protein
sperm DNA to block non-specific DNA interaction at 4 degrees
with a 10:1 biotin/protein ratio for 30 min at room temperature.
Biotinylated protein was then desalted into phosphate buffered
for 1 h. Cells were then washed with 100
For biotinylated on-DNA compounds, cells were then incubated
with Streptavidin-PE (Invitrogen) at 1:100 ratio in 50 L of PBS
at 4 degrees for 30 min. After staining, cells were washed and fluo-
lL of PBS buffer 3 times.
saline and used for selection. For affinity selection, 100
0.1 mg/mL protein solution was pipeted up and down in a 200
Phynexus column packed with 5 L agarose streptavidin resin for
20 min to immobilize 10 g of protein. 5 nmol of library in selec-
l
L of
l
lL
l
rescence was measured by FACS analysis on
cytometer.
a LSR II flow
l
tion buffer (25 mM Hepes, pH 7.4; 150 mM NaCl; 1 mM CHAPS;
1 mM MgCl2; 1 mg/mL sheared salmon sperm DNA (Ambion))
was incubated on the column for 1 h, the column was then washed
4.7. Compound synthesis
10 times with 100 lL of selection buffer to remove non-binders.
4.7.1. General method
Bound molecules were eluted by heat denaturing the protein at
80 °C for 10 min. Eluant was then incubated with fresh immobi-
lized protein to start a second round of affinity selection. Three
rounds of selection were performed. To exclude molecules bound
to the resin matrix a parallel selection was done in identical fash-
ion with no protein target as a no target control (NTC). For DNA
sequencing, the final eluant containing 5e7 molecules was ampli-
fied by PCR for 25 cycles, then sequenced using Illumina high
throughput sequencing technology.
1H NMR spectra were recorded on a Varian Mercury 400 plus.
Chemical shifts are expressed in parts per million (ppm, d units).
Coupling constants (J) are in units of hertz (Hz). Splitting patterns
describe apparent multiplicities and are designated as s (singlet), d
(doublet), t (triplet), q (quartet), dd (double doublet), dt (double
triplet), m (multiplet). Purification of final compounds for biologi-
cal testing was performed on a Gilson GX-281 system with a Phe-
nomenex Luna 5
l
C8(2) 100 ꢂ 21.20 mm 100A column running
gradient of 10–80% MeCN/H2O (+0.1% TFA) over 20 min with flow
rate of 22 mL/min. The purity of final compounds was checked
using an Agilent 1100 HPLC system coupled with a Thermo Finni-
4.4. ELISA testing for LFA-1 antagonism
gan LCQ Mass Spectrometer–Phenomenex Luna 3
l C8(2) 100A
An ELISA-type assay to study LFA-1-ICAM-1 interaction was
performed as previously described with minor modification.
50 ꢂ 3.00 mm column running gradient of 10–95% MeCN/H2O
(+0.1% formic acid) over 15 min with flow rate 0.5 mL/min. High-
resolution mass measurement was performed on Bruker MicroTOF
electrospray mass spectrometer coupled with an Agilent 1100
HPLC system. All mass spectra were performed by electrospray
ionization (ESI) positive mode (Scheme 4).
Briefly, LFA-1 protein was diluted to 2.5
ine (TBS), pH 8.0 plus 1 mM MgCl2 and 1 mM CaCl2. Protein was di-
rectly immobilized by adding 50 L/well to an ELISA plate and
lg/mL in Tris buffered sal-
l
incubated overnight at 4 °C. All subsequent steps were carried
out at room temperature. Wells were washed 3 times with
300
with 150
l
L/well with TBS plus 0.05% Tween-20 (TBST), then blocked
L/well TBS+0.1% gelatin for 1 h. Compounds were seri-
4.7.1.1. 2-Amino-N-ethyl-2,3-dihydro-1H-indene-2-carboxam-
ide (8). To a mixture of 2-((tert-butoxycarbonyl)amino)-2,3-
dihydro-1H-indene-2-carboxylic acid (7) (500 mg, 1.803 mmol)
and HATU (823 mg, 2.164 mmol) in 10 mL DMF was added DIPEA
(0.945 mL, 5.41 mmol) and ethylamine hydrochloride (221 mg,
2.70 mmol), and the reaction was allowed to stir at rt overnight.
The reaction mixture was diluted with EtOAc (100 mL), washed
with 10% NH4Cl (2 ꢂ 100 mL), saturated NaHCO3 (1 ꢂ 100 mL), sat-
urated NaCl (1 ꢂ 100 mL), dried with MgSO4, and evaporated to
dryness. 500 mg (1.643 mmol) of the residue was dissolved in
6 mL DCM and added TFA (3.16 mL, 41.1 mmol). The reaction mix-
ture was stirred at room temp for 2 h, then azeotroped with DCM
(2 ꢂ 20 mL). The crude TFA salt was put on vacuum pump over-
night, and the residue was re-dissolved in EtOAc (50 mL) and
washed with 1 N NaOH (1 ꢂ 50 mL), and saturated NaCl
(1 ꢂ 50 mL), dried with MgSO4, filtered, and evaporated to dryness
to obtain the desired free amine 2-amino-N-ethyl-2,3-dihydro-1H-
indene-2-carboxamide (8) (350 mg, 1.628 mmol, 99% yield). MS
(ESI) m/z [M+1]+ = 205.04.
l
ally diluted in 100% DMSO at 200ꢂ the final assay concentration
prior to being diluted 1:100 to 2ꢂ final concentration in Hepes buf-
fered saline (HBS). Wells were again washed 3 times with 300 lL/
well TBST prior to 25
added. Compounds were incubated with LFA-1 for 30 min, then
25 L/well of 20 g/mL ICAM-1-Fc in HBS+2 mM MnCl2 was added
and plates were incubated for 60 min. Wells were washed 5 times
with 300 L/well TBST+1 mM MnCl2. Horseradish peroxidase con-
lL of serially diluted compounds being
l
l
l
jugated goat anti-human IgG (Pierce) was added to allow the
detection of bound ICAM-1-Fc. Antibody was diluted 1:5000 into
HBS+1 mM MnCl2 and 50
for 30 min. Wells were washed 10 times with 300
TBST+1 mM MnCl2. 100 L/well ABTS substrate (Invitrogen) was
added and reaction allowed to proceed for 20 min before being
stopped by the addition of 100 L/well 0.01% sodium azide in citric
acid. Absorbance at 415nM was then measured. Wells containing
no LFA-1 were used for background signal subtraction and percent
inhibition was calculated from wells containing no compound.
l
L/well was added prior to incubation
lL/well
l
l
4.7.1.2. 2,5-Diazabicyclo[2.2.1]heptan-2-yl(3-(trifluoromethyl)
phenyl)methanone (12). To a mixture of 3-(trifluoromethyl)
benzoic acid (10) (450 mg, 2.37 mmol) and HATU (912 mg,
2.4 mmol) in DMF (6 mL) was added DIPEA (2 mL, 11.5 mmol). To
the reaction mixture was then added tert-butyl 2,5-diazabicy-
clo[2.2.1]heptane-2-carboxylate (9) (469 mg,2.37 mmol) and the
reaction was allowed to stir at rt for 1 h. The reaction mixture
was diluted with EtOAc (100 mL), washed with 10% NH4Cl
(2 ꢂ 100 mL), saturated NaHCO3 (1 ꢂ 100 mL), saturated NaCl
(1 ꢂ 100 mL), dried with MgSO4, and evaporated to dryness to give
the Boc-protected material. The Boc-protected material was re-
dissolved in 6 mL DCM and to the solution was added TFA (3 mL,
39 mmol). The reaction was stirred at rt for 2 h. The solvent was
4.5. DNA ligation
Compounds 20a and 20b with library DNA headpiece attached
were ligated to biotinylated double stranded DNA by incubating
with 20 lM T4 DNA ligase (NEB) in ligation buffer at 16 °C for
18 h. Ligation reactions were then ethanol precipitated prior to fur-
ther experiments.
4.6. Cell-binding analysis via FACS
K562 cells 19385 cells were cultured in RPMI media, supplied
with 10% FBS in a 5% CO2 incubator with shaking. Cells were

2362
C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
O
Cl
N
Cl
N
N
N
N
N
N
CF3
N
H
N
N
H
N
HN
HN
O
O
14b
14a
Cl
Cl
Cl
O
O
N
OCF3
S
O
HN
N
N
N
N
N
CF3
N
H
N
N
HN
O
Cl
N
N
14c
Intermediate (I) for 17g
Scheme 4. Structures of intermediates.
evaporated to dryness. The residue was azeotroped with DCM
(2 ꢂ 20 mL) to yield 2,5-diazabicyclo[2.2.1]heptan-2-yl(3-(trifluo-
romethyl)phenyl)methanone (12) as a TFA salt (911 mg, >99%
yield). MS (ESI) m/z [M+1]+ = 271.14.
(81 mg, 0.222 mmol, 81% yield), MS (ESI) m/z [M+1]+ = 387.13; 14b
(84 mg, 0.135 mmol, 51 % yield). MS (ESI) m/z [M+1]+ = 586.22; and
14c (75 mg, 0.081 mmol, 30% yield), MS (ESI) m/z [M+1]+ = 638.08.
4.7.2. General method for synthesis of 16 and 17(a–e)
To a suspension of one of compound 14 (0.090 mmol) in NMP
(2 mL) at rt was added the desired amine (0.135 mmol). To the
reaction mixture was then added DIPEA (0.047 mL, 0.270 mmol).
The reaction mixture was then heated at 100 °C overnight. The de-
sired product was purified by prep Gilson HPLC.
4.7.1.3. 2-((2-(Trifluoromethoxy)phenyl)sulfonyl)-2,5-diazabi-
cyclo[2.2.1]heptane (13). To a solution of tert-butyl 2,5-diazabi-
cyclo[2.2.1]heptane-2-carboxylate (9) (469 mg, 2.37 mmol) and
DIPEA (2 mL, 11.5 mmol) in DMF (6 mL) was added 2-[(trifluoro-
methyl)oxy]benzenesulfonyl chloride (617 mg, 2.37 mmol) slowly.
The reaction mixture was allowed to stir at rt for 2 h. The reaction
mixture was diluted with EtOAc (100 mL), washed with saturated
NaHCO3 (1 ꢂ 100 mL), saturated NaCl (1 ꢂ 100 mL), dried with
MgSO4, and evaporated to dryness to give the Boc-protected
material. The Boc-protected material was dissolved in DCM
(6 mL) and to the solution was added TFA (3 mL, 39 mmol). The
reaction was stirred at rt for 2 h. The solvent was evaporated to
dryness and the residue was azeotroped with DCM (2 ꢂ 5 mL)
to yield 2-((2-(trifluoromethoxy)phenyl)sulfonyl)-2,5-diazabicyclo
[2.2.1]heptane (13) as a TFA salt (1.0 g, >99% yield). MS (ESI) m/z
[M+1]+ = 323.17.
4.7.2.1. 2-((4-(((S)-1-(4-Bromophenyl)ethyl)amino)-6-(5-(3-(tri-
fluoromethyl)benzoyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,3,
5-triazin-2-yl)amino)-N-ethyl-2,3-dihydro-1H-indene-2-carbox-
amide (16a). Compound 14b and (S)-1-(4-bromophenyl)ethan-
amine were used for the reaction to give 16a (40 mg, 0.048 mmol,
53% yield). 1H NMR (400 MHz, CDCl3) d 8.66–8.26 (m, 2H), 7.80–
7.39 (m, 6H), 7.27–7.06 (m, 6H), 6.00 (s, 1H), 5.07–4.38 (m, 3H),
3.79–3.15 (m, 10H), 2.06–1.87 (m, 2H), 1.53 and 1.51 (d, J = 8 Hz,
3H), 1.11–0.92 (m, 3H). 13C NMR (100 MHz, CDCl3) d 172.1,
168.6, 167.8, 160.3, 155.0, 154.4, 142.2, 144.8, 139.6, 131.8,
131.7, 131.7, 129.3, 128.2, 128.0, 127.3, 127.2, 127.1, 124.5,
124.4, 120.6, 117.7, 114.8, 59.4, 56.7, 56.6, 50.6, 43.3, 35.0,
34.9, 34.7, 22.3, 19.0, 14.7, 14.6. HRMS (M+H)+ calcd for
[C36H36BrF3N8O2 + H] 749.2170; found 749.2139.
4.7.1.4. 2-((4-Chloro-6-(pyrrolidin-1-yl)-1,3,5-triazin-2-yl)
amino)-N-ethyl-2,3-dihydro-1H-indene-2-carboxamide (14a);
2-((4-chloro-6-(5-(3-(trifluoromethyl)benzoyl)-2,5-diazabicyclo
[2.2.1]heptan-2-yl)-1,3,5-triazin-2-yl)amino)-N-ethyl-2,3-dihy
dro-1H-indene-2-carboxamide (14b); 2-((4-chloro-6-(5-((2-(tri
fluoromethoxy)phenyl)sulfonyl)-2,5-diazabicyclo[2.2.1]heptan-
2-yl)-1,3,5-triazin-2-yl)amino)-N-ethyl-2,3-dihydro-1H-indene-
2-carboxamide (14c). In three separate vials, to a suspension of
2,4,6-trichloro-1,3,5-triazine (50 mg, 0.271 mmol) in MeCN (3 mL)
at 0 °C was added a solution of the 2-amino-N-ethyl-2,3-dihydro-
1H-indene-2-carboxamide (8, 55.4 mg, 0.271 mmol) in MeCN
(1 mL) dropwise. To the reaction mixture was then added DIPEA
(0.142 mL, 0.813 mmol) dropwise. The reaction mixtures were
stirred at 0 °C for 15 min. To each vial was added the desired
secondary amine (pyrrolidine for 14a, 2,5-diazabicyclo[2.2.1]
heptan-2-yl(3-(trifluoromethyl)phenyl) methanone (12) for 14b,
and 2-((2-(trifluoromethoxy)phenyl) sulfonyl)-2,5-diazabicyclo
[2.2.1]heptane (13) for 14c) in MeCN (1 mL) dropwise at 0 °C.
The reaction mixtures were allowed to warm up to room temp.
The reactions’ completion was confirmed by LCMS after 2 h of stir-
ring. The mixtures were then purified with prep Gilson HPLC. 14a
4.7.2.2. 2-((4-(((S)-1-(4-Bromophenyl)ethyl)amino)-6-(5-((2-
trifluoromethoxy)phenyl)sulfonyl)-2,5-diazabicyclo[2.2.1]hep
tan-2-yl)-1,3,5-triazin-2-yl)amino)-N-ethyl-2,3-dihydro-1H-
indene-2-carboxamide (16b). Compound 14c and (S)-1-(4-
bromophenyl)ethanamine were used for the reaction to give 16b
(25 mg, 0.028 mmol, 31% yield). 1H NMR (400 MHz, CDCl3) d
8.48–8.38 (broad s, 1H), 8.26–8.01 (m, 2H), 7.68–7.64 (t, 1H),
7.47–7.4 (m, 4H), 7.20–7.06 (m, 6H) 6.01–5.87 (d, 1H), 5.04–4.97
(m, 1H), 4.73–4.59 (m, 2H), 3.72–3.3 (m, 10H), 1.86–1.67 (m,
2H), 1.53–1.47 (m, 3H), 1.08–1.02(m, 3H). HRMS (M+H)+ calcd
for [C35H36BrF3N8O4S + H] 801.1789; found 801.1769.
4.7.2.3.
2-((4-((3,4-Dichlorophenethyl)amino)-6-(5-(3-(trifluo
romethyl)benzoyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,3,5-tria-
zin-2-yl)amino)-N-ethyl-2,3-dihydro-1H-indene-2-carboxamide
(16c). Compound 14b and 2-(3,4-dichlorophenyl)ethanamine
were used for the reaction to give 16c (20 mg, 0.024 mmol, 27%

C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
2363
yield). 1H NMR (400 MHz, CDCl3) d 8.47–8.00 (m, 2H), 7.61–7.56
(m, 4H), 7.36–7.02 (m, 7H), 6.12–6.02 (d, 1H), 5.09–4.95 (m, 1H),
4.76–4.43 (dd, 1H), 3.82–3.13 (m, 12H), 2.87–2.76 (m, 2H), 2.09–
1.89 (m, 2H), 1.12–0.91 (m, 3H). 13C NMR (100 MHz, CDCl3) d
172.1, 160.3, 154.3, 139.7, 138.5, 138.4, 135.8, 132.4, 130.9,
130.7, 130.5, 129.2, 128.4, 127.5, 127.1, 124.5, 124.5, 124.4,
120.6, 117.7, 115.8, 67.3, 59.6, 57.7, 56.7, 54.2, 43.7, 43.1, 36.2,
34.8, 34.5, 14.7, 14.6. HRMS (M+H)+ calcd for [C36H35Cl2F3N8O2 + -
H] 739.2285; found 739.2276.
4.7.2.9. N-Ethyl-2-(4-(phenethylamino)-6-(5-(3-(trifluo
romethyl)benzoyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,3,
5-triazin-2-ylamino)-2,3-dihydro-1H-indene-2-carboxamide
(17d). Compound 14b and phenylethylamine were used for the
reaction to give 17d (54.5 mg, 0.07 mmol, 81% yield). 1H NMR
(400 MHz, CDCl3) d 8.60–8.54 (m, 1H), 7.82–7.52 (m, 5H),
7.31–7.14 (m, 9H), 6.06 (br s, 1H), 5.10–4.89 (d, 1H), 4.82–4.46
(m, 1H), 3.84–3.15 (m, 12H), 2.89 (br s, 2H), 2.10–1.89 (m, 2H),
1.13–1.04 (dt, 2H), 0.94–0.91 (t, 1H). HRMS (M+H)+ calcd for
[C36H37F3N8O2 + H] 671.3065; found 671.3067.
4.7.2.4. 2-((4-((3,4-Dichlorophenethyl)amino)-6-(5-((2-(trifluoro
methoxy)phenyl)sulfonyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,
3,5-triazin-2-yl)amino)-N-ethyl-2,3-dihydro-1H-indene-2-carbox-
amide (16d). Compound 14c and 2-(3,4-dichlorophenyl)ethan-
amine were used for the reaction to give 16d (20 mg, 0.023 mmol,
4.7.2.10. N-Ethyl-2-((4-(ethylamino)-6-(5-(3-(trifluoro-
methyl)benzoyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,3,5-tria-
zin-2-yl)amino)-2,3-dihydro-1H-indene-2-carboxamide
(17e). Compound 14b and ethylamine hydrochloride were used
for the reaction to give 17e (43.9 mg, 0.056 mmol, 63% yield). 1H
NMR (400 MHz, CDCl3) d 8.69–8.60 (m, 1H), 7.82–7.56 (m, 4H),
7.32–7.14 (m, 4H), 6.11 (s, 1H), 5.13–5.05 (t, 1H), 4.84 and 4.46
(d, 1H), 3.86–2.89 (m, 13H), 2.10–1.94 (m, 2H), 1.26–0.90 (m,
6H). HRMS (M+H)+ calcd for [C30H33F3N8O2 + H] 595.2752; found
595.2748.
25% yield). 1H NMR (400 MHz, CDCl3)
d 8.41–8.29 (d, 1H),
8.05–7.91 (m, 2H), 7.67–7.63 (t, 1H), 7.45–7.01 (m, 9H), 6.02–
5.90 (d, 1H), 4.72–4.63(m, 2H), 3.82–3.15(m, 12H), 2.86–2.82 (t,
2H), 1.91–1.69 (m, 2H), 1.10–1.02 (m, 3H). 13C NMR (100 MHz,
CDCl3) d 172.2, 154.3, 146.0, 139.6, 138.4, 134.8, 132.5, 131.7,
130.8, 130.7, 130.6, 128.3, 127.3, 127.2, 127.1, 126.7, 124.5,
124.4, 120.4, 67.3, 60.2, 59.8, 57.9, 55.5, 54.3, 54.0, 43.5, 43.4,
41.8, 37.2, 34.9, 34.4, 14.7. HRMS (M+H)+ calcd for [C35H35Cl2F3N8-
O4S + H] 791.1904; found 791.1891.
4.7.2.11.
2-((4-((3,4-Dichlorophenethyl)amino)-6-(5-(3-(tri
fluoromethyl)benzoyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,3,
5-triazin-2-yl)amino)-2,3-dihydro-1H-indene-2-carboxylic acid
(17f). To a suspension of 2,4,6-trichloro-1,3,5-triazine (200 mg,
1.085 mmol) in MeCN (3 mL) at 0 °C was added a solution of the
4.7.2.5. 2-((4-((3,4-Dichlorophenethyl)amino)-6-(pyrrolidin-1-
yl)-1,3,5-triazin-2-yl)amino)-N-ethyl-2,3-dihydro-1H-indene-2-
carboxamide (16e). Compound 14a and 2-(3,4-dichlorophenyl)
ethanamine were used for the reaction to give 16e (15 mg,
0.025 mmol, 28% yield). 1H NMR (400 MHz, CDCl3) d 8.18 (s, 1H),
7.63 (s, 1H), 7.36–7.02 (m, 7H), 6.10 (s, 1H), 3.75–3.25 (m, 12H),
2.87–2.83 (t, 2H), 2.00–1.94 (m, 4H), 1.08–1.05 (t, 3H). HRMS
(M+H)+ calcd for [C27H31Cl2N7O+ H] 540.2040; found 540.2020.
2-amino-2,3-dihydro-1H-indene-2-carboxylic
acid
(316 mg,
1.085 mmol) in MeCN (2 mL) dropwise. To the reaction mixture
was then added DIPEA (0.568 mL, 3.25 mmol) dropwise. The
reaction mixture was stirred at 0 °C for 30 min. To the reaction vial
was then added amine 12 (292 mg, 1.085 mmol) in MeCN (2 mL)
dropwise at 0 °C. The reaction mixture was allowed to warm up
to room temp. The reaction’s completion was confirmed by LCMS
after stirring for 2 h. To the vial was then added 2-(3,4-dichloro-
phenyl)ethanamine (412 mg, 2.169 mmol). The reaction mixture
was then heated at 100 °C overnight and purified by prep Gilson
HPLC to yield 17f (120 mg, 0.16 mmol, 14.8% yield). 1H NMR
(400 MHz, CDCl3) d 8.50–8.32 (m, 1H), 8.11 (br s, 1H), 7.80–7.53
(m, 4H), 7.32–6.97 (m, 7H), 5.05–4.35 (m, 2H), 3.91–3.24 (m,
11H), 2.85–2.75 (m, 2H), 2.06–1.92 (m, 2H). HRMS (M+H)+ calcd
for [C34H30Cl2F3N7O3 + H] 712.1812; found 712.1782.
4.7.2.6.
N-Ethyl-2-(4-((S)-1-phenylethylamino)-6-(5-(3-(tri-
fluoromethyl)benzoyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,
3,5-triazin-2-ylamino)-2,3-dihydro-1H-indene-2-carboxamide
(17a). Compound 14b and (S)-1-phenylethanamine were used
for the reaction to give 17a (34.5 mg, 0.046 mmol, 51% yield). 1H
NMR (400 MHz, CDCl3) d 8.41–8.05 (m, 2H), 7.81–7.54 (m, 4H),
7.37–7.10 (m, 9H), 6.00 (s, 1H), 5.11–4.39 (m, 2H), 3.77–2.91 (m,
11H), 2.04–1.88 (m, 2H), 1.56 (d, 3H), 1.12–1.05 (m, 2H),
0.94–0.92 (t, 1H). HRMS (M+H)+ calcd for [C36H37F3N8O2 + H]
671.3065; found 671.3059.
4.7.2.12.
(5-(4-((3,4-Dichlorophenethyl)amino)-6-((2,3-dihy
dro-1H-inden-2-yl)amino)-1,3,5-triazin-2-yl)-2,5-diazabicy-
clo[2.2.1]heptan-2-yl)(3-(trifluoromethyl)phenyl)methanone
(17g). To a suspension of 2,4,6-trichloro-1,3,5-triazine (191 mg,
1.036 mmol) in MeCN (5 mL) at 0 °C was added a solution of the
3,4-dichlorophenylehthylamine (196 mg, 1.036 mmol) in MeCN
(2 mL) dropwise. To the reaction mixture was then added DIPEA
(1.086 mL, 6.22 mmol) dropwise and then stirred at 0 °C for
15 min. To the vial was added the secondary amine 12 (279 mg,
1.036 mmol) in MeCN (2 mL) dropwise at 0 °C. The reaction mix-
ture was allowed to warm up to room temperature and allowed
to stir for additional 1 h, then diluted with EtOAc (100 mL),
washed with 10% NH4Cl (2 ꢂ 100 mL), saturated NaHCO3
(1 ꢂ 100 mL), saturated NaCl (1 ꢂ 100 mL), dried with MgSO4,
and evaporated to dryness. The residue was purified with ISCO
flash chromatography (40 g silica gel column, 10–80% EA in
Hexanes in 40 min with retention time at 23 min) to give the
intermediate (5-(4-chloro-6-((3,4-dichlorophenethyl)amino)-1,
3,5-triazin-2-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)(3-(trifluoro-
methyl)phenyl)methanone (I): (510 mg, 0.89 mmol, 86% yield).
MS (ESI) m/z [M+1]+ = 571.07. To a suspension of the intermedi-
ate I (100 mg, 0.175 mmol) in NMP (1 mL) at rt was added a
4.7.2.7.
N-Ethyl-2-(4-((R)-1-phenylethylamino)-6-(5-(3-(tri-
fluoromethyl)benzoyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,
3,5-triazin-2-ylamino)-2,3-dihydro-1H-indene-2-carboxamide
(17b). Compound 14b and (R)-1-phenylethanamine were used
for the reaction to give 17b (37 mg, 0.05 mmol, 55% yield). 1H
NMR (400 MHz, CDCl3) d 8.21–8.07 (m, 2H), 7.83–7.59 (m, 4H),
7.32–7.05 (m, 9H), 6.03 (s, 1H), 5.10–4.39 (m, 2H), 3.86–2.77 (m,
11H), 2.06–1.86 (m, 2H), 1.57 (d, 3H), 1.12–1.04 (m, 2H), 0.93–
0.90 (t, 1H). HRMS (M+H)+ calcd for [C36H37F3N8O2 + H]
671.3065; found 671.3049.
4.7.2.8.
2-((4-(Benzylamino)-6-(5-(3-(trifluoromethyl)
benzoyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-1,3,5-triazin-
2-yl)amino)-N-ethyl-2,3-dihydro-1H-indene-2-carboxamide
(17c). Compound 14b and benzylamine were used for the reac-
tion to give 17c (38.7 mg, 0.045 mmol, 51% yield). 1H NMR
(400 MHz, CDCl3) d 8.76–8.70 (m, 1H), 8.14–7.57 (m, 5H), 7.33–
7.14 (m, 9H), 6.05 (broad s, 1H), 5.08–4.44 (m, 4H), 3.84–3.12
(m, 10H), 2.09–1.90 (m, 2H), 1.13–0.90 (m, 3H). HRMS (M+H)+
calcd for [C35H35F3N8O2 + H] 657.2908; found 657.2922.

2364
C. S. Kollmann et al. / Bioorg. Med. Chem. 22 (2014) 2353–2365
solution of 2,3-dihydro-1H-inden-2-amine (46.5 mg, 0.35 mmol)
and DIPEA (0.122 mL, 0.7 mmol). The reaction mixture was then
heated at 90 °C overnight and purified by prep Gilson HPLC to
give 17g (40 mg, 0.046 mmol, 26.3% yield). 1H NMR (400 MHz,
CDCl3) d 7.96–7.55 (m, 6H), 7.37–6.99 (m, 7H), 5.12–4.48 (m,
3H), 3.83–3.54 (6H), 3.38–3.22 (m, 2H), 3.04–2.81 (m, 4H),
2.12–2.00 (m, 2H). HRMS (M+H)+ calcd for [C33H30Cl2F3N7O +
H] 668.1914; found 668.1892.
showed almost complete conversion. To the solution was added
a solution of 2-(3,4-dichlorophenyl)ethanamine in DMA (50 equiv,
20 lmol, 100 lL, 200 mM), and the mixture was shaken gently at
4 °C overnight. LCMS showed complete conversion to 19. To the
solution was then added 10% volume of 5 M NaCl solution and
3X volume of cold anhydrous ethanol, cooled at ꢀ78 °C for 1 h
and then centrifuged at 3000r/min for 10 min. The supernatant
was discarded and the residue was re-dissolved in 400 lL of so-
dium borate buffer (pH = 9.4) as product 19 for the next step. MS
4.7.2.13. (5-(4-(Cyclopentylamino)-6-((3,4-dichlorophenethyl)
amino)-1,3,5-triazin-2-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)
(3-(trifluoromethyl)phenyl)methanone (17h). The procedure
for the synthesis of 17g was followed with the use of cyclopentan-
amine (30 mg, 0.35 mmol) to yield 17h (39 mg, 0.048 mmol, 27.3%
yield). 1H NMR (400 MHz, CDCl3) d 7.89–7.56 (m, 5H), 7.44–7.26
(m, 3H), 7.07–6.99 (m, 1H), 5.17–4.50 (m, 2H), 4.27–4.16 (m,
1H), 3.83–3.36 (m, 6H), 2.87–2.80 (m, 2H), 2.14–1.98 (m, 4H),
1.76–1.57 (m, 6H). HRMS (M+H)+ calcd for [C29H3 °Cl2F3N7O + H]
620.1914; found 620.1909.
(ESI) m/z [Mꢀ3]/3ꢀ = 1880.03 (calcd 1880.0).
4.7.3.4. Synthesis of compound 20a. To a solution of 19 (0.25
l
mol, 250
l
L, 1 mM) in sodium borate buffer (pH = 9.4) was added
mol, 62.5 L, 200 mM) and
a solution of 12 in MeCN (50 equiv, 12.5
l
l
the mixture was shaken gently at 80 °C for 1 h. LCMS showed com-
plete conversion to 20a. To the solution was then added 10% volume
of 5 M NaCl solution and 3ꢂ volume of cold anhydrous ethanol,
cooled at ꢀ78 °C for 1 h and then centrifuged at 3000r/min for
10 min. The supernatant was discarded and the residue was re-dis-
solved in 400 lL of water and purified with Prep HPLC to yield 20a
4.7.2.14. 2-((4-((3,4-Dichlorophenethyl)amino)-6-(5-((2-(triflu-
oromethoxy)phenyl)sulfonyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-
1,3,5-triazin-2-yl)amino)-2,3-dihydro-1H-indene-2-carboxylic
acid (17i). The procedure for the synthesis of 17f was followed
with the use of amine 13 to yield 17i (120 mg, 0.15 mmol, 13.8%
yield). 1H NMR (400 MHz, CDCl3) d 8.46–7.98 (m, 3H), 7.68–7.60
(m, 1H), 7.43–6.97 (m, 9H), 4.87–4.23 (m, 2H), 3.77–3.09 (m,
11H), 2.79–2.76 (t, 2H), 1.88–1.57 (m, 2H). HRMS (M+H)+ calcd
for [C33H30Cl2F3N7O5S + H] 764.1431; found 764.1411.
(0.06
1958.7).
l
mol, 24% yield). MS (ESI) m/z [Mꢀ3]/3ꢀ = 1957.96 (calcd
4.7.3.5. Synthesis of compound 20b. The procedure for the syn-
thesis of 20a was used with 19 (0.18
starting materials to yield 20b (0.09
lmol) and 13 (9.0
lmol) as
l
mol, 52% yield). MS (ESI)
m/z [Mꢀ3]/3ꢀ = 1975.38 (calcd 1976.0).
Conflict of interest
4.7.3. Synthesis of fluorescence polarization (FP) ligands
The authors have declared no conflict of interest.
4.7.3.1. Synthesis of compound 18a. To a mixture of 17f (50 mg,
0.07 mmol) and HATU (40 mg, 0.105 mmol) in DMF (3 mL) was
added DIPEA (0.061 mL, 0.351 mmol) and 1,1-dimethylethyl
(4-aminobutyl)carbamate (19.8 mg, 0.105 mmol), and the reaction
was allowed to stir at rt overnight. LCMS analysis showed complete
conversion. The reaction mixture was diluted with EtOAc (100 mL),
washed with 10% NH4Cl (2 ꢂ 100 mL), saturated NaHCO3
(1 ꢂ 100 mL), saturated NaCl (1 ꢂ 100 mL), dried with MgSO4,
and evaporated to dryness. The residue was re-dissolved in DCM
(3 mL) and added TFA (0.25 mL, 3.23 mmol) and stirred at rt for
2 h. The solvent was removed under vacuum and azeotroped with
DCM (2 ꢂ 20 mL) to yield the free amine intermediate (54.7 mg,
0.07 mmol). A fraction of the free amine intermediate (2.4 mg,
0.003 mmol) was then re-dissolved in DCM (3 mL) and added DI-
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l
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