A threonine turnstile defines a dynamic amphiphilic binding motif in the AAA ATPase p97 allosteric binding site

Article abstract of DOI:10.1039/c7ob00526a

The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cr

Full text of DOI:10.1039/c7ob00526a

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McGrath, M. Arkin, R. Gussio and D. Huryn, Org. Biomol. Chem., 2017, DOI: 10.1039/C7OB00526A.
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DOI: 10.1039/C7OB00526A
A Threonine Turnstile Defines a Dynamic Amphiphilic
Binding Motif in the AAA ATPase p97 Allosteric Binding Site
James C. Burnett,§ Chaemin Lim,‡ Brian D. Peyser,& Lalith P. Samankumara,‡ Marina Kovaliov,‡
Raffaele Colombo,‡ Stacie L. Bulfer,∥ Matthew G. LaPorte,‡ Ann R. Hermone,§ Connor F.
McGrath,§ Michelle R. Arkin,∥ Rick Gussio,*,& Donna M. Huryn,*,‡,† and Peter Wipf*,‡,†
§Leidos Biomedical Research, Inc., P.O. Box B, Frederick, MD 21702, United States
‡University of Pittsburgh Chemical Diversity Center, University of Pittsburgh, Pittsburgh, PA 15260,
United States
&Frederick National Laboratory for Cancer Research, Developmental Therapeutics Program, P.O. Box B,
Frederick, MD 21702, United States
∥Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, University of California,
San Francisco, CA 94158, United States
†Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, United States
Keywords. Protein homeostasis, ERAD, UPR, ubiquitin pathway inhibitor, autophagy, threonine turnstile,
allosteric binding site, dynamics, docking.
ABSTRACT. The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay
that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A
computational model based on SAR data and both Xꢀray and cryoꢀEM structures of the AAA ATPase p97
was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain
hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand
substituents. Conversely, diametric rotation of the χ1 torsion by 150ꢀ180o orients the side chain βꢀmethyl
groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar
substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set
of inhibitors of p97 covering a >2,000ꢀfold variation in potencies, with a preference for either smallꢀhighly
polar or smallꢀapolar groups. The threonine turnstile motif was further validated by a PDB search that
identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR
structures demonstrating additional gearꢀlike interactions between adjacent Thr pairs. Combined, these data
suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets.
TOC Text (20 words max): The turnstile motion of two neighboring threonines accommodates both polar
and apolar ligands in an allosteric binding site.

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accommodate both polar and apolar moieties in the
allosteric binding pocket.
Introduction
The hexameric AAA+ (ATPases Associated with
diverse cellular Activities) p97 serves as a major
regulator of protein homeostasis. p97 assists in
chromatinꢀ and mitochondriaꢀassociated degradation,
endoplasmic reticulum (ER)ꢀassociated degradation
(ERAD), unfolded protein response (UPR),
proteasome
degradation,
Golgi
reassembly,
endosomal tracking, protein aggregate processing,
1
and autophagy. Key to p97’s function is a
remarkable conformational mobility in its 6×3 multiꢀ
domain subunits. Binding to nucleotides during the
ATPase cycle repositions these domains around the
ringꢀshaped core, transducing phosphate hydrolysis
into mechanical force exerted on protein substrates.2
The specific elements that control the coordinated
ratchet motion within the D1, D2, and N domains in
the 550 kDa hexameric complex are still under
Fig. 1. Structures of inhibitors used for comparing
substituent effects at the 5ꢀposition of the indole or
analogous heterocyclic scaffolds. R1 see Table 1; R2 = H
or Me. Additional indole replacements include 5ꢀ and 7ꢀ
azaindoles, naphthalene, benzimidazole, and benzofuran.
Table 1. Biochemical Activities of p97 Inhibitorsa
3
R1 Indole
ADPGlo ADPGlo
IC50 [µM] Std. Dev.
investigation. p97 has been recognized as a
Entry Compound ID
Substitution (R2)
5ꢀCN (H)
potential target for treating cancer as well as
neurodegeneration,4,5 thus triggering drug discovery
efforts in both industry and academia.6,7,8,9,10
1
UPCDC30283 (1)
UPCDC30287 (2)
UPCDC30346 (3)
UPCDC30245 (4)
UPCDC30361 (5)
UPCDC30310 (6)
UPCDC30256 (7)
UPCDC30341 (8)
UPCDC30083 (9)
UPCDC30317 (10)
UPCDC30288 (11)
UPCDC30318 (12)
UPCDC30206 (13)
UPCDC30367 (14)
UPCDC30238 (15)
UPCDC30257 (16)
UPCDC30345 (17)
UPCDC30297 (18)
UPCDC30381 (19)
UPCDC30222 (20)
UPCDC30221 (21)
UPCDC30277 (22)
UPCDC30368 (23)
UPCDC30250 (24)
UPCDC30201 (25)
0.044
0.047
0.050
0.055
0.087
0.10
0.12
0.13
0.16
0.19
0.23
0.24
0.39
0.45
0.71
3.8
0.045
0.040
0.044
0.087
0.047
0.032
0.073
0.076
0.10
0.14
0.18
0.11
0.069
0.12
0.22
0.8
2
3
4
5
6
7
8
9
5ꢀNO2 (H)
5ꢀF (Me)
5ꢀF (H)
5ꢀCN (Me)
5ꢀCONH2 (H)
5ꢀOH (H)
5ꢀCO2Me (H)
5ꢀH (H)
5ꢀCl (H)
5ꢀN3 (H)
5ꢀCH3 (H)
benzo[α]carbazole
5ꢀCONHMe (H)
5ꢀOCH3 (H)
5ꢀOCF3 (H)
5ꢀazaindole (H)
5ꢀCF3 (H)
Results and discussion
In a recent series of indoleꢀbased allosteric
inhibitors of p97, we were unable to completely
rationalize the structureꢀactivity relationship (SAR)
of CF3ꢀbioisosteres at the indole Cꢀ5 position (R1) of
our lead structure (Figure 1). 11 Specifically, the
analysis of substituent effects revealed a remarkably
divergent electronic trend since both the 5ꢀnitro and
the 5ꢀmethyl substituent showed superior binding
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
4.0
1.7
4.67
2.0
affinities
versus
the
corresponding
5ꢀF,7ꢀazaindole (H) 4.74
1.3
naphthalene (H)
benzofuran (H)
5ꢀSF5 (H)
5ꢀCONMe2 (H)
benzimidazole (H)
NꢀMe indole (H)
5.2
1.1
pentafluorosulfanylꢀ, methoxyꢀ, trifluoromethoxyꢀ,
and trifluoromethylꢀsubstituted analogs.11 Even after
the subsequent elucidation of a cryoꢀEM structure
with a potent (IC50 = 55 nM) 5ꢀfluoroꢀsubstituted
inhibitor of the same structural series bound to p97
(PDB entry 5FTJ),3 the inhibitor binding data could
not be fully explained by their steric, polar, or
electronic properties. Therefore, in the present study,
we computationally refined a model of this p97
allosteric site based on the SAR of additional 5ꢀ
substituted indoles and select arene and heteroarene
analogs, which together span a range of more than 3
orders of magnitude in a biochemical assay (Figure
1 and Table 1). Examination of these small molecule
probes revealed an unprecedented turnstile motion
of two neighboring but discontinuous threonine
(Thr) residues, setting up a dynamic side chain ꢀ
20.1
21.5
31.4
38.5
>50
4.60
0.40
1.60
6.60
ꢀ
aAssay conditions: ADPGlo with 20 nM p97 ATPase WT in the
presence of 100 ꢀΜ ATP.12 Assays were repeated multiple times
(see Table S1 for an extended overview).
Central to developing a protein binding model
capable of rationalizing our p97 SAR was that all
inhibitors considered for the refinement contained an
identical tetramine side chain, thereby creating a
structurally homologous set of compounds differing
only in the substitution pattern in the indole region.
Semiempirical calculations initially suggested a
correlation between the assay data (Table 1) and
surface area calculations of the 5ꢀsubstituted
indoles.13 Specifically, van der Waals surface areas
of the phenyl indole moiety of compounds 1, 2, 4, 6ꢀ
heterocyclic
ligand
interaction
that
can

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12, 14ꢀ16, 18, 22 and 23 formed a leastꢀsquares Furthermore, we observed that at least one of the
linear fit with the log IC50 data (R=0.72, df = 14, p side chains of the Thr pair could form van der Waals
<0.01). Upon selecting only examples with nonpolar contacts with substituents at the 5ꢀposition of the
5ꢀsubstitutions (9ꢀ12, 15, 16, 18 and 22), the linear indole in some conformations of the canonical
fit slightly improved (R=0.91, df = 6, p <0.01). ensemble. Given the combined steric restriction and
Conversely, the correlation for analogs with polar divergent SAR of the 5ꢀposition substituents, along
substitutions (1, 2, 4, 6ꢀ8, 14, and 23) dropped to with the conformational flexibility of these loops,
R=0.81 (df = 6, p >0.01) (Figures S1ꢀS3). Among we hypothesized that, upon inhibitor binding and
several possible correlations of physicochemical
parameters with activity, only the van der Waals
surface areas provided R > 0.5. This suggested that
for inhibitors with nonpolar indole 5ꢀsubstituents,
the main factor determining potent activity was the
size of the substituent, with a preference for smaller
groups. Polar substituents formed an independent
cohort, and hydrogen bonding capabilities
influenced the biological activity in addition to
substituent size. Taken together, these data indicated
that the protein binding subsite at the indole 5ꢀ
position was sterically constrained and amphiphilic,
accommodating hydrogen bond donors and
acceptors, as well as small hydrophobic groups.
Fig. 2. Variable orientations of loops 507ꢀ511 and 611ꢀ
616 near the allosteric binding site of UPCDC30245 (4).
Shown is the distance range between the side chain βꢀ
carbons of loop residues Thr 509 and Thr 613 observed in
The cryoꢀEM coꢀstructure of 5ꢀF derivative 4 ADPꢀbound Xꢀray crystal structure 3CF3 (cyan) and
ADPꢀbound cryoꢀEM structures 5FTJ (magenta, with
inhibitor complex) and 5FTK (green), and ATPγSꢀbound
cryoꢀEM structures 5FTL (yellow) and 5FTM (orange).
(UPCDC30245) bound to p97 showed how this
specific inhibitor molecule engaged in multiple
favorable contacts in the protein’s allosteric binding
site, including a multipolar bond between the
fluorine and a backbone carbonyl carbon (see Figure
2D in ref 3). However, the docking of other
derivatives possessing polar and apolar 5ꢀposition
substituents in the structure failed to provide a
rationale for why substituents of divergent polarity
should be equally active (Table 1). For example, the
structure provided no explanation why a 5ꢀNO2
substitution would result in greater affinity than a 5ꢀ
amide substituent, or why a hydrophobic 5ꢀCH3
substituent would be significantly more potent than
a 5ꢀOCF3 derivative (Figure S4). Therefore, to gain
a better understanding of potential conformational
variabilities in amino acid backbone and side chain
arrangements surrounding this allosteric binding site
in the highly dynamic protein, we examined all
available hexameric p97 structures in the protein
data bank (PDB), including both cryoꢀEM and Xꢀray
structures (PDB entries 3CF1, 3CF2, 3CF3, 5C1A,
5C18, 5C19, 5FTJ, 5FTK, 5FTL, 5FTM, and
5FTN).3, 14 , 15 Interestingly, we found that two
separate loops (composed of residues 507ꢀ511 and
611ꢀ616), which border the site where the inhibitor
indole 5ꢀposition is localized in the cryoꢀEM coꢀ
structure, displayed a high degree of conformational
flexibility. In these different structures, the distances
between the βꢀcarbons of the amphiphilic secꢀ
propanol side chains of residues Thr 509 and Thr
613 ranged from approx. 3.2 Å to 7.8 Å (Figure 2).
concomitant hydrophobic collapse with the binding
site loops, the amphiphilic Thr side chain pair could
form a subsite in agreement with the sterically
restricted, amphiphilic SAR of the indole series.
Using cryoꢀEM coꢀstructure 5FTJ as a starting
point, we employed constrained molecular
mechanics and dynamics protocols to anneal the bisꢀ
Thr side chain pair to form a subsite for the 5ꢀpolar
substitutions (e.g., the 5ꢀOH of UPCDC30256, 7).
This subsite is characterized by a waterꢀcoordinated,
stabilized hydrogen bonding network with the polar
inhibitors (Figure 3a). When the Thr side chain χ1
torsions are rotated in a turnstile mode (150–180°), a
subsite formed by the Thr side chain methyl groups
accommodates nonpolar 5ꢀsubstitutions (e.g., 5ꢀ
methyl indole inhibitor UPCDC30318, 12, Figure
3b). Moreover, χ rotations of the Thr pair are
facilitated by the ATP binding cavity of the D2
domain, which is located on the opposite side of the
loops bordering the allosteric site. Specifically,
when the γꢀhydroxyl groups rotate out of the binding
pocket, the water molecule shown in Figure 3a is
extruded into the ATP binding cavity (as shown in
Figure 3b), and is accommodated, along with the γꢀ
hydroxyls, by bulk solvent in the cavity (Figure 3b).
Conversely, in the polar conformation, the γꢀmethyls
of the Thr pair, which have very low solvent
accessible surface areas, are oriented toward the

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ATP binding cavity, and the bulk solvent in the hydrophobic collapse with the side chains of Trp
cavity is not significantly affected.
476 and Ile 531 (Figure 4).
Molecular docking studies employing hydropathic
These alternate binding modes incorporate key scoring (Table S2 and Figure S5)16,17 with all of the
inhibitor contacts observed in the cryoꢀEM coꢀ inhibitors listed in Table 1 were used to further
structure (Figure 4), including the critical hydrogen refine the allosteric site on p97. Several key SAR
bond between the backbone amide carbonyl of Val aspects were investigated. Binding modes for high
493 and the inhibitor indole NH, πꢀstacking between affinity (IC50 <150 nM) polar 5ꢀsubstitutions (entries
the Phe 618 side chain phenyl and the inhibitor 1ꢀ8; 5ꢀCN, 5ꢀNO2, 5ꢀF, 5ꢀCN, 5ꢀCONH2, 5ꢀOH, and
indole, favorable hydrophobic collapse between the 5ꢀCO2Me) were distinguished by favorable
inhibitor phenylꢀindole and the side chains of hydrogen bonding networks similar to those shown
residues Pro 510, Ala 537, Pro 571, and Lys 614 in Figures 3a and 5a. For these derivatives, the polar
(i.e., the residue’s side chain methylenes), a 5ꢀmoieties had good complementary fits for the bisꢀ
favorable dipoleꢀdipole interaction between the Cys Thr polar subsite that were characterized by either
535 thiol and the inhibitor piperidine N, and a the formation of direct hydrogen bonds with a Thr
hydrogen bond between the carboxylate of Glu 534 side chain hydroxyl group, or via water mediation,
and the piperidineꢀ4ꢀamino substituent. In total, or both. Similarly, good complementarity was
these interactions result in a tightly bound phenyl achieved when docking potent (IC50 <500 nM) 5ꢀ
indole scaffold that facilitates inhibitor tetramine substituted apolar moieties (entries 9ꢀ13; 5ꢀH, 5ꢀCl,
side chain interactions with residues of helices 16 5ꢀN3, 5ꢀCH3, and benzo[α]carbazole). These binding
and 17 (Figure 4). Specifically, piperazine N1 is
predicted to hydrogen bond with the side chain
amide of Gln 494, while the substituent’s N4
hydrogen bonds with the carboxylate of Glu 534,
Fig. 4. Schematic of polar (top) and apolar (or
hydrophobic) (bottom) inhibitor binding modes
exemplified by 5ꢀhydroxyl analog UPCDC30256 (7, top)
and 5ꢀmethyl analog UPCDC30318 (12, bottom). In both
binding modes, the yellow shaded region represents the
amphiphilic bisꢀThr subsite. Blue dashes indicate
Fig. 3. Upon inhibitor binding, the loops containing the
hydrogen bonds/dipoleꢀdipole interactions, green dashes
Thr 509ꢀThr 613 pair collapse to form an amphiphilic
indicate hydrophobic contacts, and green lines depict the
binding site at the indole 5ꢀposition. (a) Polar subsite: the
hydrophobic and steric continuity of the binding site. Pink
Thr γꢀhydroxyls engage in a network of hydrogen bonds
shading indicates a critical hydrogen bond for optimal
with polar inhibitor substituents. (b) Hydrophobic subsite:
inhibitor activity, while green circles indicate πꢀstacking.
rotation of the Thr χ1 torsions by 150ꢀ180° results in γꢀ
methyl group interactions with small hydrophobic
moieties on the indole 5ꢀposition. In this conformation,
the bridging water molecule has been displaced from the
binding pocket.
modes were characterized by favorable hydrophobic
interactions with the side chain methyl groups of the
bisꢀThr, as depicted in Figure 3b.
and the isopropyl moiety desolvates via favorable

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Fig. 5. Comparison of the binding modes of the 5ꢀsubstituted amide series comprised of 6, 14, and 23, and for contrast,
ester derivative 8. Yellow dashes = hydrogen bonds; red dashes = unfavorable contacts. (a) The potent binding affinity
of UPCDC30310 (6, IC50 = 100 nM) is typical of the polar binding mode, with the two Thr side chain hydroxyl groups
and the indole 5ꢀposition amide nitrogen coordinating a water molecule in a hydrogen bonding network. Additionally,
the 5ꢀposition amide nitrogen forms a hydrogen bond with the backbone carbonyl carbon of Ser 511. (b) For
UPCDC30367 (14), substitution of a single methyl group on the amide nitrogen necessitates the engagement of the Thr
613 methyl group to accommodate the hydrophobic addition, while the polar amide carbonyl group hydrogen bonds
with the Thr 509 hydroxyl group. In this suboptimal binding mode (IC50 = 450 nM), the waterꢀmediated hydrogen
bond observed for 6 is lost, and the amide methyl moiety assumes an unfavorable, partially solventꢀexposed orientation
(red sphere). (c) Dimethylation of the amine in the C(5)ꢀamide (UPCDC30368, 23) is sterically prohibited (red sphere),
resulting in a near loss of all potency. (d) For comparison with methyl amide 14, the binding model of the more potent
methyl ester UPCDC30341 (8, IC50 = 130 nM) indicates that this substituent’s methyl group, unlike the
conformationally restricted Nꢀmethyl of 14, can avoid solvent exposure by orienting toward the bottom edge of the
binding pocket, while the methoxy oxygen engages in a weak hydrogen bond with the coordinated water molecule.
Disruption of the indole NH to Val 493 hydrogen other derivatives, such as 5ꢀfluoroꢀ7ꢀazaindole
bond (Figure 4) by Nꢀmethylation in UPCDC30201 UPCDC30381 (19) introduce a destabilizing polar
(25) or by replacement of the indole with a mismatch between the 7ꢀposition
naphthalene isostere in UPCDC30222 (20) ablates backbone carbonyl group of Leu 492.
or greatly diminishes activity, respectively.
N and the
Introduction of a nitrogen atom at the indole 3ꢀ
Finally, some polar 5ꢀsubstituents (14 and 23
position in benzimidazole UPCDC30250 (24) (Figure 5b and c, respectively)) and hydrophobic
reduces binding affinity by >2 orders of magnitude. substituents (15, 16, 18, and 22) are either sterically
The lone pair of the imine N of the benzimidazole less favorable (e.g., 14) or simply too large to fit into
presents an incompatible negative electrostatic either binding mode. For example, the increased
steric bulk of dimethylamide 23 results in the loss of
a waterꢀmediated hydrogen bond, solvent exposure,
and unfavorable steric contacts (i.e., versus potent
amide analog 6) (Figure 5aꢀc). Interestingly, unlike
methylamide 14 (IC50 = 450 nM), which is sterically
less favorable than amide 6, methyl ester 8 is
significantly more potent (IC50 = 130 nM), as the
more flexible methoxy group can rotate slightly to
avoid solvent exposure. This orientation also directs
the methoxy oxygen of the ester to form a hydrogen
bond with a water molecule (Figure 5d), thereby
rationalizing its similar potency to C(5)ꢀamide 6.
potential near the hydrophobic α−, β−, and aromatic
carbons of Phe 618. Furthermore, if this N atom is
complexed to a water molecule (via hydrogen
bonding) during the binding event, an even greater
hydrophobicꢀpolar incompatibility ensues.
The 5ꢀazaindole nitrogen of UPCDC30345 (17)
has an electrostatic potential similar to the
benzimidazole N, and is also suboptimal for this
binding pocket. Its negative electrostatic potential or
coordination with water places it too close to the
hydrophobic side chain components of Pro 510 and
the hydrocarbon component of Lys 614. Similarly,

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Figure 6. (a) Schematic of the criteria used to search the PDB for bisꢀThr pairs in the vicinity of ligands. (b) Protein
kinase B inhibitor with a 9Hꢀpurine component near the bisꢀThr pair in the polar conformation. In this structure, the
purine N9 engages in a hydrogen bond (yellow dash) with one of the Thr side chain hydroxyls. (c) Protein kinase B
inhibitor with a 7Hꢀpyrrolo[2,3ꢀd]pyrimidine moiety near the bisꢀThr pair in the apolar conformation. In this inhibitor,
N9 is replaced with a carbon, which consequently engages in favorable hydrophobic contacts (orange dash) with a Thr
methyl group.
Based on the importance of the bisꢀThr pair in
To further broaden the scope of the PDB analysis,
rationalizing the amphiphilic SAR of our allosteric the initial search criteria were relaxed to allow for
inhibitors of p97, we conducted a search of the PDB the identification of bisꢀThr pairs (within 7 Å of a
to examine the generality of this motif at small ligand) occurring in any secondary structural feature
molecule ligand binding sites. All PDB entries with (i.e., helices, sheets, and loops). From this search we
ligands having >200 residues in the longest chain found additional Xꢀray coꢀcrystal examples of
and a resolution ≤2.5 Å were evaluated. Figure 6a ligands interacting with at least one Thr of a bisꢀThr
illustrates the search criteria used for this study. pair. Examples include heat shock protein 90
Among the 19,881 entries meeting the criteria, 840 (HSP90), βꢀlactamases (CTXꢀMꢀ9 and BELꢀ1), and
(4.2%) were found to possess discontinuous (>5 AMPA glutamate receptor 2 (GluR2) (Figure S7).
residues apart in loop sequence) Thr pairs for which The HSP90 threonines both occur in β sheets, the βꢀ
lactamase threonines occur on a β sheet and a loop,
and the GluR2 threonines occur on a β sheet and an
α helix. While in these additional examples the bisꢀ
Thr pairs did not display the χ rotations observed in
PKB inhibitor coꢀcrystal structures, it is interesting
that they support a consistently polar binding mode
in the solid state (i.e., Xꢀray structures), as the
interacting ligand moieties in these examples were
always polar and interacted with the γꢀhydroxyl of
the Thr side chain. Consequently, we considered the
possibility that the bisꢀThr turnstile movement may
not be readily observable in solid state structures.
Therefore, we further expanded our search to
examine solution NMR structures for paired Thr χ
rotations, under the assumption that the cooperative
motion between paired Thr residues’ side chains
would be more readily detected in solution.
Accordingly, 8,687 nonꢀredundant NMR entries
meeting the search criteria described above were
identified. Of these, a subset of 271 structures
contained Thr pairs where both side chain χ torsions
rotated simultaneously by >50o. Although this subset
did not contain ligands interacting with bisꢀThr pairs,
the finding that 271 structures did display full and
simultaneous χ rotations occurring in Thr pairs
signifies that any subsite created by these residues
can be both polar and hydrophobic. It is noteworthy,
yet not surprising, that such bisꢀThr turnstile motion
the distance between the residues’ βꢀcarbons was ≤5
Å (similar to our model), and where the Thr βꢀ
carbons were in the vicinity of a ligand (Figure 6a).
Interestingly, we found an active site with a bisꢀThr
pair occurring on separate loops in protein kinase B
(ATK1) (see Supporting Information and Figure S6
for additional details). Similar to our model, the Thr
pair could also adopt conformations to accommodate
either a polar or a hydrophobic contact in the same
binding pose. In the coꢀcrystal structure (PDB entry
2UVY) depicted in Figure 6b,18 the bisꢀThr pair in
the polar binding mode is positioned for hydrogen
bonding directly to the 9Hꢀnitrogen of the inhibitor,
and when the 9Hꢀnitrogen is replaced with a carbon
(PDB entry 2X39, Figure 6c), 19 the bisꢀThr χ1
torsion changes to form an apolar conformation,
with the Thr βꢀmethyl groups closing off the
hydrophobic pocket. The specific compounds from
the indicated PDB entries possess different side
chain substituents on the respective positions of their
purine and pyrroloꢀpyrimidine cores, and thus a
comparison of their inhibitory activity is not
straightforward. However, similar potencies are
observed for analogous pairs with identical
substitutions (see core pairs 14 and 15, 17 and 18, 20
and 21, 22 and 23, and 24 and 25 in Table 2 in
reference 20).

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is more prominent in solution structures versus Xꢀ speculate that the function of such Thr pairs is to
ray structures.
position water molecules in protein allosteric sites,
as well as other locations that are either functionally
For example, Figure 7a shows the 15 conformers important, and/or structurally significant. For
deposited with NMR PDB entry 2FIN. In Figures 7b example, we suggest that, in the absence of an
and 7c, the Thr pair in the polar subsite inhibitor, the function of the bisꢀThr pair in the p97
conformation (conformer 12) and the hydrophobic allosteric site identified in the cryoꢀEM coꢀstructure3
subsite conformation (conformer 1) are shown, is to shuttle water molecules out of the structurally
respectively. Movie S1, which was created directly dynamic interface between the D1 and D2 domains.
from the 15 conformers deposited for the protein Future studies will investigate the functional role of
entry, clearly displays the residues simultaneously this bisꢀThr pair in p97 protein dynamics.
rotating in a turnstile manner to gear between polar
and hydrophobic subsite orientations.
Fig. 7. The bisꢀThr pair in NMR structure, PDB entry, 2FIN. (a) The PDB entry contains 15 conformers, with a bisꢀThr
pair formed by residues Thr 78 and Thr 95. Over the 15 conformers of the protein, the two Thr residues display a range
of χ torsion rotations. (b) An example of a polar bisꢀThr conformation as seen in conformer 12 of the protein ensemble.
In this example, the hydroxyl hydrogen atom of Thr 78 engages in a hydrogen bond (yellow dash) with the hydroxyl
oxygen atom of Thr 95. (c) An example of a hydrophobic (or apolar) bisꢀThr conformation as seen in conformer 1 of
the protein ensemble. In this example, the methyl groups of Thr 78 and Thr 95 engage in a favorable hydrophobic
contact (orange dash). Movie S1 shows the rotations observed for the 15 conformers in the PDB entry.
Experimental section
Materials and Methods
Conclusions
Synthetic summary schemes, computational
methods and any associated references and
additional discussion as well as a summary SAR
table are available in the online version in the
Supplementary Information of this paper.
Synthesis
We utilized a series of focused small molecule
probes and extensive structural information from the
PDB to develop a unified computational model that
is able to rationalize the divergent potency in a
series of phenyl indoleꢀbased p97 inhibitors. The
SAR of all 25 probe molecules can be
comprehensively explained by an allosteric binding
site model, with the dynamic turnstile nature of two
amphiphilic Thr side chains providing a compelling
rationalization for the observation that inhibitors
possessing either polar or hydrophobic substituents
at the same position are simultaneously
accommodated in the binding site. Furthermore, the
results from our PDB search identified a similar
amphiphilic bisꢀThr turnstile in ligand complexes of
protein kinase B, suggesting this previously
unrecognized feature may frequently occur in small
molecule protein binding sites. In support of this
hypothesis, an analysis of NMR/solution structures
provided further evidence for the concept that side
chains of Thr pairs are able to interact in a dynamic,
gearꢀlike manner in protein structures. We can
General Synthetic Methods. All nonꢀaqueous
reactions were carried out under
a nitrogen
atmosphere in ovenꢀ or flameꢀdried glassware unless
otherwise noted. Anhydrous tetrahydrofuran and
diethyl ether were distilled from sodium
benzophenone ketyl; anhydrous dichloromethane
and toluene were distilled from CaH2; alternatively,
the same solvents were obtained from a solvent
purification system using alumina columns. All
other solvents and reagents were used as obtained
from commercial sources without further
purification unless noted. Reactions were monitored
via TLC using 250 µm preꢀcoated silica gel 60 F254
plates, which were visualized with 254 nm and/or
365 nm UV light and by staining with KMnO4 (1.5 g
KMnO4, 10 g K2CO3, and 1.25 mL 10% NaOH in

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200 mL water), cerium molybdate (0.5 g 7.48ꢀ7.43 (m, 2 H), 7.16 (s, 1 H); 13C NMR (100
Ce(NH4)2(NO3)6, 12 g (NH4)6Mo7O24•4H2O, and 28 MHz, DMSOꢀd6) δ 138.9, 138.5, 133.5, 131.1, 130.8,
mL conc. H2SO4 in 235 mL water), or vanillin (6 g 128.1, 127.7, 125.8, 124.7, 124.4, 122.5, 120.5,
vanillin and 1.5 mL conc. H2SO4 in 100 mL EtOH). 112.6, 101.7, 100.5; HRMS (ESI+) m/z calcd for
Flash chromatography was performed with SiliCycle C15H10BrN2 297.0022 (M+H), found 297.0022.
silica gel 60 (230ꢀ400 mesh) or with ISCO MPLC.
Microwave reactions were performed using
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀIsopropylpiperazinꢀ1ꢀ
yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀ1Hꢀindoleꢀ
a
Biotage Initiator in glass microwave vials (cap 5ꢀcarbonitrile (1, UPCDC30283): A solution of 2ꢀ
sealed) with continuous magnetic stirring and an (3ꢀbromophenyl)ꢀ1Hꢀindoleꢀ5ꢀcarbonitrile (26, 0.15
external surface temperature sensor. 1H and 13C g, 0.50 mmol), LiHMDS (0.20 g, 1.2 mmol),
NMR spectra were recorded on Bruker Avance 300, Pd2(dba)3 (9.2 mg, 0.010 mmol), and CyJohnPhos
400, or 500 MHz spectrometers, using the residual (14.0 mg, 0.040 mmol) in anhydrous THF was
solvent as an internal standard. 19F NMR spectra treated with tertꢀbutyl (2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
were obtained using a protonꢀdecoupled pulse yl)ethyl)(piperidinꢀ4ꢀyl)carbamate3,11 (A, 0.213 g,
sequence without internal standard. IR spectra were 0.600 mmol). The reaction mixture was heated at
obtained on a Smiths IdentifyIR or PerkinElmer 75 °C overnight, cooled to room temperature,
Spectrum 100. HRMS data were obtained on a diluted with sat. NaHCO3, and extracted with
Thermo Scientific Exactive HRMS coupled to a CH2Cl2 (3x). The combined organic layers were
Thermo Scientific Accela HPLC system using a 2.1 washed with brine, dried (Na2SO4), filtered,
x 50 mm 3.5 µm Waters XTerra C18 column eluting concentrated, and purified by chromatography on
with MeCN/H2O containing 0.1% formic acid. SiO2 (2% MeOH/CH2Cl2 with 1% TEA) followed
Purity of compounds was assessed using the same by filtration through basic Al2O3 (CH2Cl2) to
HPLC system with either the PDA or an Agilent 385 provide
tertꢀbutyl
(1ꢀ(3ꢀ(5ꢀcyanoꢀ1Hꢀindolꢀ2ꢀ
ELSD. All final screening samples passed QC yl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
based on >95% purity by LC/MS/ELSD analysis.
Experimental Procedures.
2ꢀ(3ꢀBromophenyl)ꢀ1Hꢀindoleꢀ5ꢀcarbonitrile
yl)ethyl)carbamate (0.16 g, 0.28 mmol, 56%) as a
yellow foamy solid: IR (ATR) 2965, 2932, 2809,
2216, 1685, 1653, 1599, 1448, 1411, 1362, 1320,
(26): A solution of 3ꢀbromo acetophenone (4.6 g, 23 1245, 1172, 1146, 1010, 971, 898, 805, 775 cmꢀ1; 1H
mmol), 4ꢀaminobenzonitrile (2.5 g, 21 mmol), and NMR (400 MHz, CDCl3) δ 9.60 (br s, 1 H), 7.92 (s,
TsOH•H2O (36 mg, 0.20 mmol) in toluene (100 mL) 1 H), 7.44 (d, J = 8.4 Hz, 1 H), 7.34 (dd, J = 8.4, 1.6
was heated overnight under DeanꢀStark conditions. Hz, 1 H), 7.29 (t, J = 8.0 Hz, 1 H), 7.20 (br s, 1 H),
The reaction mixture was cooled to room 7.15 (d, J = 7.6 Hz, 1 H), 6.88 (br d, J = 7.2 Hz, 1 H),
temperature, concentrated, and purified by 6.81 (s, 1 H), 4.05 (br s, 1 H), 3.74 (br s, 2 H), 3.22
chromatography
EtOAc/hexanes)
on
to
SiO2
provide
(0
to
100% (br s, 2 H), 2.66ꢀ2.44 (m, 13 H), 1.85ꢀ1.64 (m, 4 H),
(E)ꢀ4ꢀ((1ꢀ(3ꢀ 1.48 (s, 9 H), 1.03 (d, J = 6.4 Hz, 6 H); 13C NMR
bromophenyl)ethylidene)amino)ꢀbenzonitrile (3.6 g, (100 MHz, CDCl3) δ 155.6, 151.9, 141.2, 138.7,
12 mmol, 57%) as a yellowish oil that was used 132.4, 129.9, 129.1, 125.9, 124.9, 121.0, 116.91,
without further purification.
116.85, 113.8, 111.9, 103.0, 100.0, 80.1, 54.6, 53.90,
A
solution
of
(E)ꢀ4ꢀ((1ꢀ(3ꢀ 53.88, 49.5, 48.7, 30.14, 30.14, 28.6, 18.6; HRMS
bromophenyl)ethylidene)amino)benzonitrile (3.0 g, (ESI+) m/z calcd for C34H47O2N6 571.3755 (M+H),
10 mmol), Pd(OAc)2 (225 mg, 1.0 mmol), and found 571.3759.
Cu(OAc)2 (5.58 g, 30 mmol) in DMSO (100 mL)
A solution of TFA (0.53 mL, 7.0 mmol) and
was heated at 40 °C for 24 h, cooled to room triethylsilane (0.11 mL, 0.70 mmol) in CH2Cl2 (1
temperature, diluted with EtOAc (150 mL), and mL) was added to a solution of tertꢀbutyl (1ꢀ(3ꢀ(5ꢀ
filtered through Celite. The filtrate was washed with cyanoꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
sat. NH4Cl and brine. The organic layer was dried isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (40 mg,
(MgSO4), filtered, concentrated, and purified by 0.07 mmol) in CH2Cl2 (0.5 mL). After 1 h, the
chromatography on SiO2 (20% EtOAc/hexanes) reaction mixture was concentrated, diluted with sat.
followed by trituration with EtOAc and hexanes to NaHCO3, and extracted with EtOAc (3x). The
give
2ꢀ(3ꢀbromophenyl)ꢀ1Hꢀindoleꢀ5ꢀcarbonitrile combined organic layer was washed with brine,
(26, 1.56 g, 5.25 mmol, 52%) as a yellow solid: IR dried (Na2SO4), filtered, concentrated, and purified
(ATR) 3301, 2216, 1599, 1584, 1560, 1512, 1457, by chromatography on SiO2 (8 to 10%
1441, 1437, 1340, 1322, 1247, 1170, 876, 825, 800, MeOH/CH2Cl2 with 1% TEA) followed by filtration
1
792, 774, 673 cmꢀ1; H NMR (400 MHz, DMSOꢀd6) through basic Al2O3 (0 to 10% MeOH/CH2Cl2) to
δ 12.22 (s, 1 H), 8.13 (br s, 1 H), 8.09 (br s, 1 H), provide
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
7.91 (d, J = 7.6 Hz, 1 H), 7.56 (d, J = 8.4 Hz, 2 H), yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀ

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DOI: 10.1039/C7OB00526A
carbonitrile 1 (UPCDC30283, 21 mg, 0.045 mmol,
65%) as a yellow oil: IR (ATR) 3334, 2936, 2930,
1ꢀ(3ꢀ(5ꢀFluoroꢀ1Hꢀindolꢀ2ꢀyl)ꢀ4ꢀmethylphenyl)ꢀ
Nꢀ(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ
2872, 2809, 2214, 1599, 1577, 1461, 1381, 1361, 4ꢀamine (3, UPCDC30346): A solution of 2ꢀ(5ꢀ
1241, 1176, 1144, 1124, 982, 859, 803, 775, 734 bromoꢀ2ꢀmethylphenyl)ꢀ5ꢀfluoroꢀ1Hꢀindole (27,
1
cmꢀ1; H NMR (500 MHz, acetoneꢀd6) δ 11.28 (br s, 0.100 g, 0.409 mmol), tertꢀbutyl (2ꢀ(4ꢀ
1 H), 8.00 (s, 1 H), 7.56 (d, J = 8.5 Hz, 1 H), 7.47 isopropylpiperazinꢀ1ꢀyl)ethyl)(piperidinꢀ4ꢀ
(br s, 1 H), 7.40 (dd, J = 8.3, 1.3 Hz, 1 H), 7.32ꢀ7.28 yl)carbamate (A, 0.160 g, 0.450 mmol), Pd2(dba)3 (8
(m, 2 H), 7.02 (s, 1 H), 6.97 (d, J = 7.5 Hz, 1 H), mg, 0.008 mmol), and CyJohnPhos (12 mg, 0.033
3.78ꢀ3.74 (m, 2 H), 2.90ꢀ2.84 (m, 2 H), 2.72 (t, J = mmol) in anhydrous THF (0.5 mL) in a microwave
6.3 Hz, 2 H), 2.66ꢀ2.55 (m, 4 H), 2.46ꢀ2.40 (m, 8 H), vial was degassed by bubbling argon for 20ꢀ30 min.
1.98ꢀ1.95 (m, 2 H), 1.48 (qd, J = 13.5, 3.5 Hz, 2 H), The reaction mixture was charged with LiHMDS
0.97 (d, J = 7.0 Hz, 6 H); 13C NMR (125 MHz, (0.180 g, 1.02 mmol) and the vial was sealed and
acetoneꢀd6) δ 153.2, 142.4, 139.8, 133.1, 130.5, heated at 80 °C overnight. The reaction mixture was
130.0, 126.2, 125.1, 121.2, 116.9, 116.8, 113.8, cooled to room temperature, diluted with water and
113.1, 103.5, 100.2, 59.0, 55.6, 54.9, 54.6, 49.4, extracted with EtOAc. The organic layer was
48.6, 44.4, 33.2, 18.8; HRMS (ESI+) m/z calcd for washed with brine, dried (Na2SO4), filtered,
C29H38N6 471.3231 (M+H), found 471.3231.
concentrated, and purified by chromatography on
SiO2 (5 to 15% MeOH/CH2Cl2) to provide tertꢀbutyl
2ꢀ(5ꢀBromoꢀ2ꢀmethylphenyl)ꢀ5ꢀfluoroꢀ1Hꢀ
indole (27):
A
solution of 1ꢀ(5ꢀbromoꢀ2ꢀ (1ꢀ(3ꢀ(5ꢀfluoroꢀ1Hꢀindolꢀ2ꢀyl)ꢀ4ꢀ
methylphenyl)ethanone 21 (5.00 g, 23.5 mmol), 4ꢀ methylphenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
fluoroaniline (2.77 g, 24.6 mmol), and TsOH•H2O isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (0.116 g,
1
(91 mg, 0.47 mmol) in toluene (150 mL) was heated 0.201 mmol, 49%): H NMR (300 MHz, CDCl3) δ
for 40 h under DeanꢀStark conditions. The reaction 8.23 (s, 1 H), 7.34ꢀ7.28 (m, 2 H), 7.18 (d, J = 8.4 Hz,
mixture was cooled to room temperature, 1 H), 7.01 (d, J = 2.1 Hz, 1 H), 6.97ꢀ6.86 (m, 2 H),
concentrated, and purified by chromatography on 6.54 (d, J = 0.9 Hz, 1 H), 4.11 (br s, 1 H), 3.72 (d, J
SiO2 (100% hexanes followed by 5% Et2O/hexanes = 12.0 Hz, 2 H), 3.24 (br s, 2 H), 2.81ꢀ2.48 (m, 13
with 1% TEA) to provide (E)ꢀ1ꢀ(5ꢀbromoꢀ2ꢀ H), 2.38 (s, 3 H), 1.78 (br s, 4 H), 1.47 (s, 9 H), 1.11
methylphenyl)ꢀNꢀ(4ꢀfluorophenyl)ethanꢀ1ꢀimine
(7.03 g, 23.0 mmol, 98%) as a yellow oil that was C34H49FN5O2 578.3845 (M+H), found 578.3864.
used without further purification. To a solution of tertꢀbutyl (1ꢀ(3ꢀ(5ꢀfluoroꢀ1Hꢀ
(br s, 6
H); HRMS (ESI+) m/z calcd for
A solution of (E)ꢀ1ꢀ(5ꢀbromoꢀ2ꢀmethylphenyl)ꢀNꢀ indolꢀ2ꢀyl)ꢀ4ꢀmethylphenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
(4ꢀfluorophenyl)ethanꢀ1ꢀimine (4.03 g, 13.2 mmol), isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (0.100 g,
Pd(OAc)2 (0.296 g, 1.32 mmol), and Cu(OAc)2 0.173 mmol) in CH2Cl2 (2 mL) at 0 °C was added
(7.32 g, 39.5 mmol) in DMSO (130 mL) was heated trifluoroacetic acid (0.64 mL, 8.6 mmol). The
at 40 °C for 45 h, cooled to room temperature, reaction mixture was allowed to warm to room
diluted with EtOAc (500 mL), and filtered through temperature and stirred for 3 h, treated with sat.
Celite. The filtrate was washed with water (3x) and NaHCO3, and extracted with EtOAc (3x). The
brine. The organic layer was dried (Na2SO4), filtered, combined organic layers were washed with brine,
concentrated, and purified by chromatography on dried (MgSO4), filtered, concentrated, and purified
SiO2 (10% Et2O/hexanes) followed by trituration by chromatography on SiO2 (2 to 5% MeOH/CH2Cl2
with Et2O and hexanes to give 2ꢀ(5ꢀbromoꢀ2ꢀ with 1% TEA) followed by filtration through basic
methylphenyl)ꢀ5ꢀfluoroꢀ1Hꢀindole (27, 2.01 g, 6.61 Al2O3 (0 to 5% MeOH/CH2Cl2) to provide 1ꢀ(3ꢀ(5ꢀ
mmol, 50%) as a white powder: IR (ATR) 3430, fluoroꢀ1Hꢀindolꢀ2ꢀyl)ꢀ4ꢀmethylphenyl)ꢀNꢀ(2ꢀ(4ꢀ
2993, 2980, 1769, 1758, 1586, 1480, 1372, 1245, isopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀamine
3
1
1241, 1055 cmꢀ1; H NMR (300 MHz, DMSOꢀd6) δ (UPCDC30346, 49 mg, 0.10 mmol, 60%) as an offꢀ
11.49 (s, 1 H), 7.73 (d, J = 2.1 Hz, 1 H), 7.47 (dd, J white solid: IR (ATR) 3157, 2930, 2807, 1607, 1491,
= 8.1, 2.1 Hz, 1 H), 7.39 (dd, J = 8.9, 4.7 Hz, 1 H), 1448, 1379, 1178, 1123, 1107, 982, 848, 785, 760
1
7.34ꢀ7.30 (m, 2 H), 6.97 (td, J = 9.2, 2.4 Hz, 1 H), cmꢀ1; H NMR (400 MHz, CD3OD) δ 7.33 (dd, J =
6.66 (d, J = 1.5 Hz, 1 H), 2.44 (s, 3 H); 13C NMR 8.8, 4.4 Hz, 1 H), 7.19 (dd, J = 10.0, 2.4 Hz, 1 H),
(100 MHz, DMSOꢀd6) δ 157.1 (d, JCF = 231.0 Hz), 7.16 (d, J = 8.4 Hz, 1 H), 7.11 (d, J = 2.4, 1 H), 6.88
137.4, 134.9, 134.3, 133.2, 133.1, 130.9, 130.3, (dd, J = 8.4, 2.8 Hz, 1 H), 6.86 (td, J = 9.2, 2.4 Hz, 1
128.4 (d, JCF = 10.0 Hz), 118.8, 112.3 (d, JCF = 10.0 H), 6.48 (br s, 1 H), 3.69 (app d, J = 12.6 Hz, 2 H),
Hz), 109.9 (d, JCF = 25.0 Hz), 104.6 (d, JCF = 23.0 2.78ꢀ2.74 (m, 4 H), 2.65ꢀ2.50 (m, 12 H), 2.38 (s, 3
Hz), 102.8 (d, JCF = 4.0 Hz), 20.6; 19F NMR (376 H), 2.00 (app d, J = 12.4 Hz, 2 H), 1.53 (qd, J = 11.9,
MHz, DMSOꢀd6) δ ꢀ124.6; HRMS (ESI+) m/z calcd 3.5 Hz, 2 H), 1.07 (d, J = 6.4 Hz, 6 H); 13C NMR
for C15H12BrFN 304.0132 (M+H), found 304.0131. (100 MHz, CD3OD) δ 159.2 (d, JCF = 230.3 Hz),

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151.0, 141.5, 134.6, 134.4, 132.6, 130.5 (d, JCF = 1 H), 4.08 (br s, 1 H), 3.71 (app d, J = 10.0 Hz, 2 H),
10.3 Hz), 128.6, 118.9, 118.0, 112.6 (d, JCF = 9.8 3.23 (br s, 2 H), 2.77ꢀ2.48 (m, 13 H), 2.37 (s, 3 H),
Hz), 110.3 (d, JCF = 26.3 Hz), 105.3 (d, JCF = 23.3 1.80ꢀ1.75 (m, 4 H), 1.47 (s, 9 H), 1.08 (d, J = 4.5 Hz,
Hz), 102.9 (d, JCF = 4.9 Hz), 58.4, 56.2, 55.9, 54.1, 6 H); 13C NMR (125 MHz, CDCl3) δ 155.5, 149.7,
50.5, 49.6, 43.8, 32.8, 20.4, 18.7; HRMS (ESI+) m/z 140.6, 137.9, 132.2, 132.0, 128.7, 127.3, 126.0,
calcd for C29H41FN5 478.3341 (M+H), found 124.9, 121.0, 117.5, 117.4, 111.8, 103.17, 103.13,
478.3340.
80.1, 58.4, 55.0, 53.6, 53.1, 50.0, 48.6, 30.2, 28.7,
20.0, 18.5; HRMS (ESI+) m/z calcd for C35H49O2N6
2ꢀ(5ꢀBromoꢀ2ꢀmethylphenyl)ꢀ1Hꢀindoleꢀ5ꢀ
carbonitrile (28): Prepared by the same 2 step 585.3912 (M+H), found 585.3911.
procedure as for the compound 27 using 1ꢀ(5ꢀ
To a solution of tertꢀbutyl (1ꢀ(3ꢀ(5ꢀcyanoꢀ1Hꢀ
bromoꢀ2ꢀmethylphenyl)ethanone18 (5.00 g, 23.5 indolꢀ2ꢀyl)ꢀ4ꢀmethylphenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
mmol, 1.0 equiv) and 4ꢀaminobenzonitrile (2.97 g, isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (0.070 g,
24.6 mmol, 1.05 equiv). The crude residue was 0.112 mmol) in CH2Cl2 (2 mL) at 0 °C was added
purified by chromatography on SiO2 (10 to 20% trifluoroacetic acid (0.36 mL, 4.8 mmol). The
EtOAc/hexanes) followed by trituration with EtOAc reaction mixture was allowed to warm to room
and hexanes to give 2ꢀ(5ꢀbromoꢀ2ꢀmethylphenyl)ꢀ temperature and stirred for 2 h, treated with sat.
1Hꢀindoleꢀ5ꢀcarbonitrile (28, 1.37 g, 4.39 mmol, NaHCO3, and extracted with CH2Cl2 (3x). The
step 1, 85% and step 2, 34%) as a yellow solid: IR combined organic layers were washed with brine,
(ATR) 3308, 2992, 2982, 2220, 1769, 1758, 1471, dried (MgSO4), filtered, concentrated, and purified
1
1372, 1241, 1094, 1049 cmꢀ1; H NMR (400 MHz, by chromatography on SiO2 (0 to 10%
DMSOꢀd6) δ 12.00 (s, 1 H), 8.10 (s, 1 H), 7.75 (d, J MeOH/CH2Cl2 with 1% TEA) followed by filtration
= 2.0 Hz, 1 H), 7.56 (d, J = 8.4 Hz, 1 H), 7.52ꢀ7.46 through basic Al2O3 (0 to 5% MeOH/CH2Cl2) to
(m, 2 H), 7.32 (d, J = 8.4 Hz, 1 H), 6.81 (d, J = 1.2 provide
2ꢀ(5ꢀ(4ꢀ((2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
Hz, 1 H), 2.43 (s, 3 H); 13C NMR (100 MHz, yl)ethyl)amino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethylphenyl)ꢀ1Hꢀ
DMSOꢀd6) δ 138.13, 138.07, 135.2, 133.5, 133.2, indoleꢀ5ꢀcarbonitrile 5 (UPCDC30361, 38 mg,
131.1, 130.7, 127.9, 125.8, 124.4, 120.7, 118.8, 0.078 mmol, 66%) as a light yellow solid: IR (ATR)
112.5, 103.3, 101.4, 20.5; HRMS (ESI+) m/z calcd 3293, 3120, 2956, 2924, 2811, 2705, 2214, 1603,
for C16H12BrN2 311.0178 (M+H), found 311.0176.
2ꢀ(5ꢀ(4ꢀ((2ꢀ(4ꢀIsopropylpiperazinꢀ1ꢀ
1560, 1500, 1459, 1379, 1333, 1320, 1273, 1232,
1
1176, 1113, 982, 803 cmꢀ1; H NMR (400 MHz,
CD3OD) δ 7.98 (d, J = 0.8 Hz, 1 H), 7.52 (d, J = 8.8
yl)ethyl)amino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethylphenyl)ꢀ
1Hꢀindoleꢀ5ꢀcarbonitrile (5, UPCDC30361): A Hz, 1 H), 7.38 (dd, J = 8.4, 1.6 Hz, 1 H), 7.18 (d, J =
solution of 2ꢀ(5ꢀbromoꢀ2ꢀmethylphenyl)ꢀ1Hꢀindoleꢀ 8.8 Hz, 1 H), 7.11 (d, J = 2.8 Hz, 1 H), 6.94 (dd, J =
5ꢀcarbonitrile (28, 0.100 g, 0.397 mmol), tertꢀbutyl 8.4, 2.8 Hz, 1 H), 6.64 (d, J = 0.8 Hz, 1 H), 3.71ꢀ
(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)(piperidinꢀ4ꢀ
yl)carbamate (A, 0.123 g, 0.437 mmol), Pd2(dba)3 (6 H), 2.37 (s, 3 H), 2.00 (d, J = 10.8 Hz, 2 H), 1.52 (qd,
mg, 0.006 mmol), and CyJohnPhos (9.0 mg, 0.025 J = 11.9, 3.6 Hz, 2 H), 1.07 (d, J = 6.4 Hz, 6 H); 13
3.68 (m, 2 H), 2.78ꢀ2.71 (m, 4 H), 2.66ꢀ2.50 (m, 12
C
mmol) in anhydrous THF (0.5 mL) in a microwave NMR (100 MHz, CD3OD) δ 151.1, 142.4, 139.8,
vial was degassed by bubbling argon for 20ꢀ30 min. 133.5, 132.7, 130.1, 128.6, 126.6, 125.1, 121.9,
The reaction mixture was charged with LiHMDS 118.7, 118.4, 113.0, 103.4, 102.9, 58.2, 56.3, 56.0,
(0.180 g, 1.02 mmol) and the vial was sealed and 54.1, 50.3, 49.6, 43.7, 32.8, 20.3, 18.7; HRMS
heated at 80 °C for 12 h. The reaction mixture was (ESI+) m/z calcd for C30H41N6 485.3387 (M+H),
cooled to room temperature, diluted with water and found 485.3388.
extracted with EtOAc (3x). The combined organic
layers were washed with brine, dried (Na2SO4),
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀIsopropylpiperazinꢀ1ꢀ
yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀ1Hꢀindoleꢀ
by 5ꢀcarboxamide (6, UPCDC30310): To a solution
filtered,
concentrated,
and
purified
chromatography on SiO2 (0 to 10% MeOH/CH2Cl2) of
to provide tertꢀbutyl (1ꢀ(3ꢀ(5ꢀcyanoꢀ1Hꢀindolꢀ2ꢀyl)ꢀ yl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
4ꢀmethylphenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ yl)ethyl)carbamate (62 mg, 0.11 mmol) in DMSO
tertꢀbutyl
(1ꢀ(3ꢀ(5ꢀcyanoꢀ1Hꢀindolꢀ2ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (0.093 g, was added K2CO3 (15 mg, 0.11 mmol) in water (0.2
0.16 mmol, 51%) as a brown solid: IR (ATR) 3301, mL) at 0 °C then a solution of 30% w/w hydrogen
2960, 2932, 2890, 2218, 1685, 1659, 1605, 1499, peroxide (1.2 mL) was added dropwise to the
1465, 1363, 1318, 1299, 1172, 1144, 1010, 898, 803, mixture. The resulting mixture was stirred at 5 °C
1
749, 728 cmꢀ1; H NMR (500 MHz, CDCl3) δ 8.83 for 5 min then the ice bath was removed. After
(br s, 1 H), 7.97 (s, 1 H), 7.47 (d, J = 8.0 Hz, 1 H), another 5 min, water (10 mL) was added to the
7.42 (d, J = 8.5 Hz, 1 H), 7.18 (d, J = 8.0 Hz, 1 H), reaction mixture and extracted with EtOAc
7.00 (br s, 1 H), 6.90 (d, J = 8.0 Hz, 1 H), 6.63 (br s, (3x10mL). The combined organic layers were

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washed with brine, dried (MgSO4), filtered, CH2Cl2 (15 mL) under nitrogen was added dropwise
concentrated, and purified by chromatography on boron tribromide (1.0 M solution in CH2Cl2, 0.49
SiO2 to provide tertꢀbutyl (1ꢀ(3ꢀ(5ꢀcarbamoylꢀ1Hꢀ mL, 0.49 mmol) at room temperature and stirred for
indolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
1 h. The reaction was quenched with MeOH,
isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (35 mg, concentrated and purified by chromatography on
0.059 mmol, 55%) as a yellow oil: IR (ATR) 3208, SiO2 (8 to 20% MeOH/CH2Cl2 with 1% TEA)
2973, 2962, 1668, 1653, 1586, 1474, 1448, 1431, followed by filtration through basic Al2O3 (0 to 10%
1363, 1333, 1245, 1146, 1103, 1049, 1020, 757, 710, MeOH/CH2Cl2)
to
provide
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀ
1
689 cmꢀ1; H NMR (500 MHz, CDCl3) δ 9.55 (br s, isopropylpiperazinꢀ1ꢀyl)ethyl)amino)piperidinꢀ1ꢀ
1 H), 8.15 (s, 1 H), 7.66 (d, J = 8.5 Hz, 1 H), 7.44 (d, yl)phenyl)ꢀ1Hꢀindolꢀ5ꢀol 7 (UPCDC30256, 55.0 mg,
J = 8.5 Hz, 1 H), 7.30 (t, J = 7.8 Hz, 2 H), 7.18 (d, J 0.119 mmol, 96%) as a colorless oil: IR (ATR) 3260,
= 7.5 Hz, 1 H), 6.88 (d, J = 8.0 Hz, 1 H), 6.83 (s, 1 2960, 2932, 2818, 1705, 1623, 1599, 1584, 1489,
H), 6.36 (br s, 1 H), 5.74 (br s, 1 H), 4.11 (br s, 1 H), 1452, 1420, 1381, 1363, 1312, 1295, 1273, 1253,
3.81 (app d, J = 8.1 Hz, 2 H), 3.18 (br s, 2 H), 2.80ꢀ 1221, 1200, 1178, 1146, 1118, 971 cmꢀ1; H NMR
1
2.64 (m, 11 H), 2.45ꢀ2.44 (m, 2 H), 1.78ꢀ1.73 (m, 4 (500 MHz, acetoneꢀd6) δ 10.49 (s, 1 H), 7.42 (s, 1 H),
H), 1.47 (s, 9 H), 1.10 (d, J = 6.0 Hz, 6 H); HRMS 7.26ꢀ7.21 (m, 3 H), 6.97 (d, J = 2.5 Hz, 1 H), 6.88ꢀ
(ESI+) m/z calcd for C34H48O3N6 589.3861 (M+H), 6.86 (m, 1 H), 6.71ꢀ6.69 (m, 2 H), 3.76ꢀ3.72 (m, 2
found 589.3859.
H), 2.86ꢀ2.81 (m, 2 H), 2.73 (t, J = 6.3 Hz, 2 H),
A solution of TFA (0.45 mL, 5.9 mmol) and 2.65ꢀ2.55 (m, 2 H), 2.48ꢀ2.41 (m, 10 H), 1.97ꢀ1.94
triethylsilane (0.10 mL, 0.59 mmol) in CH2Cl2 (1 (m, 2 H), 1.48 (qd, J = 14.0, 3.5 Hz, 2 H), 0.98 (d, J
mL) was added to a solution of tertꢀbutyl (1ꢀ(3ꢀ(5ꢀ = 6.5 Hz, 6 H); 13C NMR (125 MHz, acetoneꢀd6) δ
carbamoylꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ 153.1, 152.2, 140.0, 134.4, 133.0, 131.0, 130.2,
(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (35 mg, 116.6, 115.9, 113.5, 112.8, 112.3, 105.0, 99.1, 58.9,
0.059 mmol) in CH2Cl2 (0.5 mL). After 1 h, the 55.7, 54.9, 54.5, 49.4, 48.8, 44.2, 33.2, 30.6, 18.8;
reaction mixture was concentrated, diluted with sat. HRMS (ESI+) m/z calcd for C28H40ON5 462.3227
NaHCO3, and extracted with EtOAc (3x). The (M+H), found 462.3227.
combined organic layer was washed with brine,
dried (Na2SO4), filtered, concentrated, and purified carboxylate (29):
Methyl
2ꢀ(3ꢀbromophenyl)ꢀ1Hꢀindoleꢀ5ꢀ
solution of 3ꢀbromo
A
by chromatography on SiO2 (8 to 10% acetophenone (8.3 g, 41 mmol), 4ꢀaminobenzoate
MeOH/CH2Cl2 with 1% TEA) followed by filtration (5.7 g, 37 mmol), and 4 Å molecular sieves (24 g) in
through basic Al2O3 (0 to 10% MeOH/CH2Cl2) to toluene (180 mL) was refluxed for 28 h under Deanꢀ
provide
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ Stark conditions. A second portion of 4 Å molecular
yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀ sieves (16 g) was added and refluxed for an
carboxamide 6 (UPCDC30310, 19 mg, 0.039 mmol, additional 95 h. The reaction mixture was filtered
65%) as a yellow foam: IR (ATR) 3302, 2943, 2829, through a pad of Celite, concentrated and purified by
1637, 1603, 1540, 1474, 1435, 1384, 1338, 1250, chromatography on SiO2 (5 to 10% Et2O/hexanes
1176, 1146, 1020, 757, 738, 723, 710 cmꢀ1; 1H NMR with 2% TEA) to give methyl (E)ꢀ4ꢀ((1ꢀ(5ꢀbromoꢀ2ꢀ
(400 MHz, CDCl3) δ 9.71 (br s, 1 H), 8.10 (s, 1 H), methylphenyl)ethylidene)amino)ꢀbenzoate (2.40 g,
7.62 (d, J = 8.4 Hz, 1 H), 7.36 (d, J = 8.4 Hz, 1 H), ca 20%) as a yellowishꢀgreen solid.
7.24 (d, J = 8.4 Hz, 2 H), 7.12 (d, J = 7.2 Hz, 1 H),
A solution of methyl (E)ꢀ4ꢀ((1ꢀ(5ꢀbromoꢀ2ꢀ
6.87 (d, J = 8.0 Hz, 1 H), 6.78 (s, 1 H), 6.39 (br s, 1 methylphenyl)ethylidene)amino)ꢀbenzoate (2.34 g,
H), 5.98 (br s, 1 H), 3.69 (d, J = 12.0 Hz, 2 H), 2.80ꢀ 7.04 mmol), Pd(OAc)2 (0.158 g, 0.704 mmol), and
2.70 (m, 4 H), 2.62ꢀ2.45 (m, 12 H), 1.94 (app d, J = Cu(OAc)2 (3.84 g, 21.1 mmol) in DMSO (70 mL)
10.8 Hz, 2 H), 1.52ꢀ1.44 (m, 2 H), 1.02 (d, J = 6.4 was heated at 40 °C for 40 h, cooled to room
Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 171.1, temperature, diluted with water (280 mL), filtered
152.2, 140.6, 139.1, 132.9, 129.9, 129.0, 125.2, through Celite, and extracted with EtOAc (4x). The
121.6, 120.8, 116.8, 116.4, 113.8, 111.2, 100.5, 58.1, combined organic layer was washed with brine,
55.3, 54.6, 53.7, 48.9, 48.7, 43.5, 32.6, 18.9; HRMS dried (Na2SO4), filtered, concentrated, and purified
(ESI+) m/z calcd for C29H41ON6 489.3336 (M+H), by chromatography on SiO2 (6 to 10%
found 489.3335.
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀIsopropylpiperazinꢀ1ꢀ
yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀ1Hꢀindolꢀ
Et2O/hexanes) followed by trituration with EtOAc
and hexanes to give methyl 2ꢀ(3ꢀbromophenyl)ꢀ1Hꢀ
indoleꢀ5ꢀcarboxylate (29, 1.6 g, 4.8 mmol, 69%) as a
5ꢀol (7, UPCDC30256): To a solution of Nꢀ(2ꢀ(4ꢀ white solid: IR (ATR) 2993, 2911, 2227, 1433, 1404,
1
isopropylpiperazinꢀ1ꢀyl)ethyl)ꢀ1ꢀ(3ꢀ(5ꢀmethoxyꢀ1Hꢀ 1308, 1042, 951, 925, 725, 697, 667 cmꢀ1; H NMR
indolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀamine
(UPCDC30238, 60.0 mg, 0.124 mmol) in dry 1.2 Hz, 1 H), 8.11 (t, J = 1.8 Hz, 1 H), 7.91ꢀ7.88 (m,
151 (400 MHz, DMSOꢀd6) δ 12.02 (s, 1 H), 8.25 (d, J =

Organic & Biomolecular Chemistry
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1 H), 7.76 (dd, J = 8.6, 1.7 Hz, 1 H), 7.54 (ddd, J = MeOH/CH2Cl2 with 1% TEA) followed by filtration
8.0, 1.9, 0.9 Hz, 1 H), 7.46 (dt, J = 15.0, 7.8 Hz, 2 through basic Al2O3 (0 to 5% MeOH/CH2Cl2) to
H), 7.17 (s, 1 H), 3.85 (s, 3 H); 13C NMR (100 MHz, provide methyl 2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
DMSOꢀd6) δ 167.1, 139.8, 137.7, 133.9, 131.1, yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀ
130.5, 128.0, 127.5, 124.2, 123.0, 122.8, 122.5, carboxylate 8 (UPCDC30341, 30 mg, 0.060 mmol,
121.1, 111.4, 101.1, 51.7; HRMS (ESI+) m/z calcd 45%): IR (ATR) 3327, 2958, 2930, 2809, 1707,
for C16H13BrO2N 330.0124 (M+H), found 330.0123. 1691, 1599, 1577, 1545, 1433, 1379, 1344, 1310,
1
1249, 1169, 1122, 1088, 982, 768, 690 cmꢀ1; H
Methyl
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
NMR (300 MHz, CD3OD) δ 8.29 (s, 1 H), 7.79 (dd,
yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀ1Hꢀindoleꢀ
5ꢀcarboxylate (8, UPCDC30341): A solution of J = 8.4, 1.2 Hz, 1 H), 7.43 (app d, J = 9.0 Hz, 2 H),
methyl 2ꢀ(3ꢀbromophenyl)ꢀ1Hꢀindoleꢀ5ꢀcarboxylate 7.32ꢀ7.26 (m, 2 H), 6.96ꢀ6.92 (m, 1 H), 6.90 (s, 1 H),
(29, 0.150 g, 0.445 mmol), tertꢀbutyl (2ꢀ(4ꢀ 3.90 (s, 3 H), 3.79 (app d, J = 12.3 Hz, 2 H), 2.84ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)(piperidinꢀ4ꢀ
2.73 (m, 4 H), 2.66ꢀ2.49 (m, 12 H), 2.01 (app d, J =
yl)carbamate (A, 0.189 g, 0.534 mmol) and K3PO4 11.7 Hz, 2 H), 1.53 (qd, J = 11.8, 3.1 Hz, 2 H), 1.07
(0.146 g, 0.668 mmol) in deoxygenated dioxane (1 (d, J = 6.6 Hz, 6 H); 13C NMR (75 MHz, CD3OD) δ
mL) in a 2ꢀ5 mL microwave vial was degassed by 170.1, 153.4, 141.8, 141.5, 134.2, 130.7, 130.1,
bubbling with argon for 20ꢀ30 min. The reaction 124.1, 123.9, 122.4, 117.9, 117.5, 114.7, 111.8,
mixture was charged with Pd2(dba)3 (8 mg, 0.009 100.8, 58.4, 56.2, 55.9, 54.1, 52.3, 49.9, 43.8, 32.8,
mmol), and CyJohnPhos (13 mg, 0.036 mmol) and 18.7; HRMS (ESI+) m/z calcd for C30H41O2N5
the vial was sealed and heated at 110 °C for 18 h. 504.3333 (M+H), found 504.3330.
The reaction mixture was cooled to room
8ꢀ(3ꢀ(1HꢀIndolꢀ2ꢀyl)phenyl)ꢀ1,4ꢀdioxaꢀ8ꢀ
temperature, diluted with sat. NaHCO3 and extracted azaspiro[4.5]decane (30). A suspension of 2ꢀ(3ꢀ
with EtOAc (3x). The combined organic layer was bromophenyl)ꢀ1Hꢀindole11 (0.40 g, 1.5 mmol),
washed with brine, dried (Na2SO4), filtered, K3PO4 (0.48 g, 2.2 mmol), Pd2(dba)3 (32 mg, 0.034
concentrated, and purified by chromatography on mmol), CyJohnPhos (42 mg, 0.12 mmol) in dry,
SiO2 (0 to 15% MeOH/EtOAc) to provide methyl 2ꢀ degassed dioxane (10 mL) was treated with 1,4ꢀ
(3ꢀ(4ꢀ((tertꢀbutoxycarbonyl)(2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)amino)piperidinꢀ1ꢀ
dioxaꢀ8ꢀazaspiro[4.5]decane (0.30 mL, 2.4 mmol).
The flask was sealed and the reaction mixture was
yl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀcarboxylate (0.18 g, 0.30 heated at 110 °C for 6 h under microwave irradiation.
mmol, 68%) as a brown solid: IR (ATR) 2958, 2813, The reaction mixture was diluted with sat. NaHCO3
1707, 1685, 1601, 1577, 1446, 1435, 1308, 1247, and extracted with EtOAc (3x). The combined
1
1165, 1144, 1124, 1089, 1008, 917, 768 cmꢀ1; H organic layer was washed with brine, dried (Na2SO4),
NMR (300 MHz, CDCl3) δ 8.87 (br s, 1 H), 8.38 (s, filtered,
1 H), 7.89 (dd, J = 8.6, 1.4 Hz, 1 H), 7.40 (d, J = 8.7 chromatography
concentrated
on
and
SiO2
purified
(30 to
by
40%
Hz, 1 H), 7.32 (t, J = 8.0 Hz, 1 H), 7.21 (s, 1 H), EtOAc/hexanes) to provide 8ꢀ(3ꢀ(1Hꢀindolꢀ2ꢀ
7.14 (d, J = 7.5 Hz, 1 H), 6.91 (dd, J = 8.3, 1.4 Hz, 1 yl)phenyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (30,
H), 6.86 (s, 1 H), 4.13 (br s, 1 H), 3.94 (s, 3 H), 3.81 0.40 g, 1.2 mmol, 81%) as a pale yellow foam: Mp
(d, J = 12.3 Hz, 2 H), 3.25ꢀ3.24 (m, 2 H), 2.87ꢀ2.47 142ꢀ143 °C; IR (ATR) 3348, 2957, 2927, 2886,
1
(m, 13 H), 1.85ꢀ1.77 (m, 4 H), 1.48 (s, 9 H), 1.06 (d, 1600, 1484, 1354, 1219, 1096, 776, 746 cmꢀ1; H
J = 6.6 Hz, 6 H); 13C NMR (75 MHz, CDCl3) δ NMR (400 MHz, CDCl3) δ 8.38 (s, 1 H), 7.64 (d, J
168.3, 155.5, 151.9, 140.1, 139.5, 133.0, 130.0, = 7.6 Hz, 1 H), 7.40 (dd, J = 8.0, 0.4 Hz, 1 H), 7.32
129.0, 123.7, 123.6, 122.4, 116.8, 116.6, 113.7, (t, J = 8.0 Hz, 1 H), 7.27ꢀ7.26 (m, 1 H), 7.20 (td, J =
110.7, 101.0, 80.0, 54.7, 53.6, 52.0, 49.7, 48.5, 30.2, 7.6, 1.2 Hz, 1 H), 7.16ꢀ7.12 (m, 2 H), 6.93 (dd, J =
28.7, 18.5; HRMS (ESI+) m/z calcd for C35H50O4N5 8.4, 2.0 Hz, 1 H), 6.81 (app d, J = 1.2 Hz, 1 H), 4.02
604.3857 (M+H), found 604.3856.
To
solution of methyl 2ꢀ(3ꢀ(4ꢀ((tertꢀ 4 H); 13C NMR (100 MHz, CDCl3) δ 151.5, 138.6,
butoxycarbonyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ 136.8, 133.4, 129.9, 129.4, 122.3, 120.7, 120.3,
(s, 4 H), 3.42 (t, J = 5.8 Hz, 4 H), 1.90 (t, J = 5.8 Hz,
a
yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀ 116.7, 116.2, 113.8, 111.0, 107.2, 100.0, 64.5, 48.0,
carboxylate (80 mg, 0.13 mmol) in CH2Cl2 (2 mL) at 34.7; HRMS (ESI+) m/z calcd for C21H23O2N2
0 °C was added TFA (0.40 mL, 5.3 mmol). The 335.1754 (M+H), found 335.1758.
reaction mixture was allowed to warm to room
temperature and stirred for 2 h, treated with sat.
1ꢀ(3ꢀ(1HꢀIndolꢀ2ꢀyl)phenyl)ꢀNꢀ(2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀamine
NaHCO3, and extracted with EtOAc (2x). The (9, UPCDC30083): A solution of 8ꢀ(3ꢀ(1Hꢀindolꢀ2ꢀ
combined organic layer was washed with brine, yl)phenyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (30,
dried (MgSO4), filtered, concentrated, and purified 0.20 g, 0.60 mmol) in acetone (70 mL) and 3.5 M
by chromatography on SiO2 (0 to 10% HCl (60 mL) was heated at 80 °C for 3.5 h. The

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DOI: 10.1039/C7OB00526A
reaction mixture was cooled to 0 °C, quenched with yellowish oil (4.6 g, 15 mmol, 74%) that was used
Na2CO3 (14 g) and extracted with EtOAc (3x). The without further purification.
combined organic layers were washed with brine,
A
solution of (E)ꢀ1ꢀ(3ꢀbromophenyl)ꢀNꢀ(4ꢀ
dried (Na2SO4), filtered, concentrated, and purified chlorophenyl)ethanꢀ1ꢀimine (1.2 g, 3.9 mmol),
by chromatography on SiO2 (25 to 30% Pd(OAc)2 (87 mg, 0.39 mmol), and Cu(OAc)2 (2.2 g,
EtOAc/hexanes) to provide 1ꢀ(3ꢀ(1Hꢀindolꢀ2ꢀ 12 mmol) in DMSO (100 mL) was heated at 40 °C
yl)phenyl)piperidinꢀ4ꢀone (0.14 g, 0.48 mmol, 80%) for 24 h, cooled to room temperature, diluted with
as a pale yellow solid which is not stable under air EtOAc (150 mL), and filtered through Celite. The
and was used immediately: 1H NMR (300 MHz, filtrate was washed with sat. NH4Cl, and brine. The
CDCl3) δ 8.35 (s, 1 H), 7.63 (d, J = 7.8 Hz, 1 H), organic layer was dried (MgSO4), filtered,
7.42ꢀ7.35 (m, 2 H), 7.28ꢀ7.27 (m, 1 H), 7.23ꢀ7.10 (m, concentrated, and purified by chromatography on
3 H), 6.95 (app dd, J = 8.0, 2.3 Hz, 1 H), 6.82 (app d, SiO2 (20% EtOAc/hexanes) followed by trituration
J = 1.5 Hz, 1 H), 3.69 (t, J = 6.0 Hz, 4 H), 2.61 (t, J with EtOAc and hexanes to give 2ꢀ(3ꢀbromophenyl)ꢀ
= 6.2 Hz, 4 H); HRMS (ESI+) m/z calcd for 5ꢀchloroꢀ1Hꢀindole (31, 0.76 g, 2.5 mmol, 63%) as a
C19H19ON2 291.1492 (M+H), found 291.1489.
yellow solid: IR (ATR) 3431, 1683, 1564, 1452,
A
solution of crude 1ꢀ(3ꢀ(1Hꢀindolꢀ2ꢀ 1439, 1305, 1281, 1249, 1057, 922, 865, 803, 772,
yl)phenyl)piperidinꢀ4ꢀone (ca. 87 mg, 0.30 mmol) in 680 cmꢀ1; H NMR (300 MHz, CD3OD) δ 7.94 (t, J
1,2ꢀDCE (3 mL) was treated with 2ꢀ(4ꢀ = 1.7 Hz, 1 H), 7.72 (dt, J = 7.8, 1.4 Hz, 1 H), 7.50
isopropylpiperazinꢀ1ꢀyl)ethanamine 22 (B, 0.077 g, (d, J = 1.8 Hz, 1 H), 7.43 (ddd, J = 8.0, 1.8, 1.1 Hz,
0.45 mmol) followed by Ti(OiPr)4 (0.10 mL, 0.32 1 H), 7.35ꢀ7.29 (m, 2 H), 7.07 (dd, J = 8.7, 2.1 Hz, 1
mmol) and stirred at room temperature overnight. H), 6.78 (s, 1 H); 13C NMR (75 MHz, CD3OD) δ
The reaction mixture was treated with NaBH(OAc)3 139.2, 137.3, 135.9, 131.7, 131.40, 131.38, 129.0,
(130 mg, 0.60 mmol) in a single portion. After 2 h, 126.3, 125.0, 124.0, 123.3, 120.6, 113.4, 100.4.
1
the reaction mixture was diluted with sat. NaHCO3
and extracted with EtOAc (3x). The combined
1ꢀ(3ꢀ(5ꢀChloroꢀ1Hꢀindolꢀ2ꢀyl)phenyl)ꢀNꢀ(2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀamine
organic layers were washed with brine, dried (10, UPCDC30317):
A
solution of 2ꢀ(3ꢀ
(Na2SO4), filtered, concentrated and purified by bromophenyl)ꢀ5ꢀchloroꢀ1Hꢀindole (31, 77 mg, 0.25
chromatography on SiO2 (5 to 20% MeOH/CH2Cl2 mmol), LiHMDS (0.10 g, 0.60 mmol), Pd2(dba)3
with 1% TEA) followed by filtration through basic (4.6 mg, 0.005 mmol), and CyJohnPhos (7.0 mg,
Al2O3 (0 to 7% MeOH/CH2Cl2) to provide 1ꢀ(3ꢀ(1Hꢀ 0.020 mmol) in anhydrous THF was treated with
indolꢀ2ꢀyl)phenyl)ꢀNꢀ(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
tertꢀbutyl
(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
yl)ethyl)piperidinꢀ4ꢀamine 9 (UPCDC0083, 95 mg, yl)ethyl)(piperidinꢀ4ꢀyl)carbamate (A, 0.106 g,
0.21 mmol, 71% over 2ꢀsteps) as a pale yellow 0.300 mmol). The reaction mixture was heated at
foam: IR (ATR) 3422, 3222, 2957, 2931, 2808, 75 °C overnight, cooled to room temperature,
1
1600, 1450, 1294, 1144, 776, 746 cmꢀ1; H NMR diluted with sat. NaHCO3, and extracted with
(400 MHz, CD3OD) δ 7.52 (d, J = 8.0 Hz, 1 H), CH2Cl2 (3x). The combined organic layer was
7.40ꢀ7.37 (m, 2 H), 7.26ꢀ7.20 (m, 2 H), 7.11ꢀ7.07 (m, washed with brine, dried (Na2SO4), concentrated,
1 H), 7.00 (app t, J = 7.4 Hz, 1 H), 6.82 (dt, J = 7.5, and purified by chromatography on SiO2 (2 to 10%
1.9 Hz, 1 H), 6.77 (s, 1 H), 3.70ꢀ3.66 (m, 2 H), 2.66 MeOH/CH2Cl2) to give tertꢀbutyl (1ꢀ(3ꢀ(5ꢀchloroꢀ
(td, J = 12.2, 1.5 Hz, 2 H), 2.59ꢀ2.36 (m, 14 H), 1.87 1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
(br d, J = 11.2 Hz, 2 H), 1.41 (qd, J = 11.8, 3.5 Hz, 2 isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (68 mg,
H), 1.01 (d, J = 6.8 Hz, 6 H); 13C NMR (100 MHz, 0.12 mmol, 47%) as a foam: IR (ATR) 2962, 2956,
CD3OD) δ 153.3, 139.8, 138.7, 134.8, 130.6, 122.6, 2949, 2932, 2926, 2807, 1685, 1653, 1599, 1575,
121.2, 120.5, 117.8, 116.9, 114.5, 112.1, 99.7, 58.4, 1463, 1446, 1411, 1381, 1363, 1329, 1303, 1269,
56.0, 55.8, 54.0, 49.9, 49.5, 43.6, 32.9, 18.7; HRMS 1245, 1172, 1144, 1103, 1059, 1048, 1010, 993, 982,
(ESI+) m/z calcd for C28H40N5 446.3278 (M+H), 971, 915, 900, 861, 772, 755, 734, 718, 692 cmꢀ1; 1H
found 446.3277.
NMR (400 MHz, CDCl3) δ 9.13 (br s, 1 H), 7.55 (d,
2ꢀ(3ꢀBromophenyl)ꢀ5ꢀchloroꢀ1Hꢀindole (31): A J = 1.6 Hz, 1 H), 7.29ꢀ7.25 (m, 2 H), 7.17 (br s, 1 H),
solution of 3ꢀbromo acetophenone (4.0 g, 20 mmol), 7.13ꢀ7.08 (m, 2 H), 6.84 (br d, J = 7.2 Hz, 1 H), 6.69
4ꢀchloroaniline (2.6 g, 20 mmol), and TsOH•H2O (s, 1 H), 4.08 (br s, 1 H), 3.71ꢀ3.69 (m, 2 H), 3.21
(35 mg, 0.20 mmol) in toluene (50 mL) was heated (br s, 2 H), 2.69ꢀ2.44 (m, 13 H), 1.71 (s, 4 H), 1.49
overnight under DeanꢀStark conditions. The reaction (s, 9 H), 1.04 (d, J = 6.4 Hz, 6 H); 13C NMR (100
mixture was cooled to room temperature, MHz, CDCl3) δ 155.6, 151.8, 140.1, 135.3, 133.0,
concentrated, and purified by chromatography on 130.3, 129.8, 125.6, 122.3, 119.8, 118.2, 116.8,
SiO2 (0 to 5% EtOAc/hexanes) to provide (E)ꢀ1ꢀ(3ꢀ 116.5, 113.73, 113.72, 112.0, 99.3, 80.1, 58.4, 54.7,
bromophenyl)ꢀNꢀ(4ꢀchlorophenyl)ethanꢀ1ꢀimine as a 53.8, 49.5, 48.6, 30.1, 28.6, 18.6; HRMS (ESI+) m/z

Organic & Biomolecular Chemistry
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calcd for C33H47ClO2N5 580.3413 (M+H), found (ESI+) m/z calcd for C33H49O2N6 561.3912 (M+H),
580.3411.
found 561.3912.
A solution of TFA (0.84 mL, 11.2 mmol) and
triethylsilane (0.18 mL, 1.1 mmol) in CH2Cl2 (1 mL)
tertꢀButyl
yl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
(1ꢀ(3ꢀ(5ꢀazidoꢀ1Hꢀindolꢀ2ꢀ
was added to a solution of tertꢀbutyl (2ꢀ(4ꢀ isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate
(33).
isopropylpiperazinꢀ1ꢀyl)ethyl)(1ꢀ(3ꢀ(5ꢀ
Into a dried flask under nitrogen was added tertꢀ
(trifluoromethyl)ꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀ butyl
(1ꢀ(3ꢀ(5ꢀaminoꢀ1Hꢀindolꢀ2ꢀ
yl)carbamate (65 mg, 0.11 mmol) in CH2Cl2 (0.5 yl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
mL). After h, the reaction mixture was yl)ethyl)carbamate (32, 0.085 g, 0.15 mmol) in
1
concentrated, diluted with sat. NaHCO3, and AcOH (4 mL). The reaction mixture was cooled to 0
extracted with EtOAc (3x). The combined organic oC and protected from light (foil wrap) and treated
layer was washed with brine, dried (Na2SO4), with a solution of NaNO2 (0.012 g, 0.17 mmol) in
filtered,
concentrated,
and
purified
by H2O (0.4 mL). After 10 min, a solution of NaN3
chromatography on SiO2 (8 to 10% MeOH/CH2Cl2 (0.010 g, 0167 mmol) in H2O (0.4 mL) was added
with 1% TEA) followed by filtration through basic dropwise. After 45 min, the mixture was slowly
Al2O3 (0 to 10% MeOH/CH2Cl2) to provide 1ꢀ(3ꢀ(5ꢀ poured into H2O (10 mL) and sat. Na2CO3 was
chloroꢀ1Hꢀindolꢀ2ꢀyl)phenyl)ꢀNꢀ(2ꢀ(4ꢀ
added (8 mL) until neutral pH. The mixture was
isopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀamine 10 extracted with EtOAc (3x), washed with brine, dried
(UPCDC30317, 41 mg, 0.085 mmol, 76%) as a (Na2SO4), filtered, and concentrated. The crude
yellow foam: IR (ATR) 3181, 2960, 2932, 2926, product was purified by chromatography on SiO2 (0
2814, 1599, 1577, 1461, 1448, 1381, 1359, 1344, to 10 % MeOH/CH2Cl2) to provide tertꢀbutyl (1ꢀ(3ꢀ
1310, 1294, 1273, 1217, 1176, 1146, 1118, 1059, (5ꢀazidoꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
1
917, 861, 790, 775, 755, 738, 690 cmꢀ1; H NMR isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (33, 0.053
1
(400 MHz, CDCl3) δ 8.75 (s, 1 H), 7.56 (d, J = 2.0 g, 0.090 mmol, 60%) as an orange foam: H NMR
Hz, 1 H), 7.29 (t, J = 8.0 Hz, 2 H), 7.19 (t, J = 2.0 (400 MHz, acetoneꢀd6) δ 10.92 (br s, 1 H), 7.47ꢀ7.43
Hz, 1 H), 7.12ꢀ7.07 (m, 2 H), 6.90 (dd, J = 8.4, 2.0 (m, 2 H), 7.29ꢀ7.26 (m, 3 H), 6.95ꢀ6.92 (m, 1 H),
Hz, 1 H), 6.70 (d, J = 1.6 Hz, 1 H), 3.73ꢀ3.70 (m, 2 6.87 (br s, 1 H), 6.83 (dd, J = 8.6, 2.2 Hz, 1 H), 4.47
H), 2.85ꢀ2.75 (m, 4 H), 2.66ꢀ2.48 (m, 13 H), 2.01ꢀ (br s, 1 H), 3.91 (br d, J = 11.6 Hz 2 H), 3.24 (br s, 2
1.98 (m, 2 H), 1.52 (qd, J = 11.6, 3.0 Hz, 2 H), 1.04 H), 2.80 (t, J = 12.0 Hz, 2 H), 2.67 (dt, J = 13.1, 6.5
(d, J = 6.8 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ Hz, 1 H), 2.53 (br s, 8 H), 2.44 (t, J = 7.4 Hz, 3 H),
152.1, 140.2, 135.2, 132.9, 130.4, 129.8, 125.7, 1.93 (br s, 2 H), 1.75 (br s, 2 H), 1.45 (s, 9 H), 1.00
122.4, 119.9, 116.4, 116.3, 113.6, 111.9, 99.4, 58.1, (d, J = 6.4 Hz, 6 H); HRMS (ESI+) m/z calcd for
55.1, 54.6, 53.6, 48.8, 48.6, 43.5, 32.6, 18.8; HRMS C33H47O2N8 587.3816 (M+H), found 587.3815.
(ESI+) m/z calcd for C28H39ClN5 480.2889 (M+H),
1ꢀ(3ꢀ(5ꢀAzidoꢀ1Hꢀindolꢀ2ꢀyl)phenyl)ꢀNꢀ(2ꢀ(4ꢀ
found 480.2887.
tertꢀButyl
yl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀamine
(11, UPCDC30288). To a cooled solution of tertꢀ
butyl (1ꢀ(3ꢀ(5ꢀazidoꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ
(1ꢀ(3ꢀ(5ꢀaminoꢀ1Hꢀindolꢀ2ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (32). A 4ꢀyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)carbamate
solution of tertꢀbutyl (2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ (33, 0.050 g, 0.085 mmol) and lutidine (0.020 mL,
yl)ethyl)(1ꢀ(3ꢀ(5ꢀnitroꢀ1Hꢀindolꢀ2ꢀ
0.17 mmol) in dry CH2Cl2 (5 mL) was added
yl)phenyl)piperidinꢀ4ꢀyl)carbamate11 (0.102 g, 0.173 dropwise a solution of TMSOTf (0.023 mL, 0.127
°
mmol) in MeOH (5 mL) was evacuated, flushed mmol) in CH2Cl2 (5 mL) at 0 C. After 2 h, the
with argon (2x) and treated with 10% Pd/C (0.019 g, reaction mixture was quenched with sat. NaHCO3
0.17 mmol). The reaction mixture was evacuated and extracted with EtOAc (3x). The combined
and subjected to H2 (1 atm ꢀ balloon). After 2 h, the organic layer was washed with water followed by
solution was filtered through a plug of Celite, rinsed brine, dried (Na2SO4), filtered, concentrated, and
with MeOH and concentrated to provide tertꢀbutyl purified by chromatography on SiO2 (8 to 20%
(1ꢀ(3ꢀ(5ꢀaminoꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀ
yl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)carbamate
MeOH/CH2Cl2 with 1% TEA) followed by filtration
through basic Al2O3 (10% MeOH/CH2Cl2) to
(32, 0.070 g, 0.15 mmol, 72%) as an orange oil that provide 1ꢀ(3ꢀ(5ꢀazidoꢀ1Hꢀindolꢀ2ꢀyl)phenyl)ꢀNꢀ(2ꢀ
was used without further purification: 1H NMR (400 (4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀamine
MHz, CD3OD) δ 7.36 (br s, 1 H), 7.26ꢀ7.20 (m, 3 H), 11 (UPCDC30288, 0.025 g, 0.051 mmol, 60%) as
6.94 (br s, 1 H), 6.89 (br d, J = 5.2 Hz, 1 H), 6.69 (br an orange foam: IR (ATR) 2960, 2937, 2931, 2876,
d, J = 8.4 Hz, 1 H), 6.61 (br s, 1 H), 4.09ꢀ3.81 (m, 3 2811, 1599, 1582, 1577, 1476, 1458, 1452, 1381,
H), 3.35ꢀ3.25 (m, 2 H), 2.85ꢀ2.50 (m, 13 H), 2.01ꢀ 1359, 1342, 1331, 1305, 1266, 1217, 1176, 1146,
1
1.74 (m, 4 H), 1.47 (s, 9 H), 1.11 (br s, 6 H); HRMS 1117, 852, 777, 772, 751 cmꢀ1; H NMR (400 MHz,

Page 15 of 24
Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB00526A
acetoneꢀd6) δ 10.84 (br s, 1 H), 7.44ꢀ7.42 (m, 2 H),
A solution of trifluoroacetic acid (1.0 mL, 13.3
7.30ꢀ7.24 (m, 3 H), 6.94ꢀ6.91 (m, 1 H), 6.87ꢀ6.86 (m, mmol) and triethylsilane (0.046 mL, 0.29 mmol) in
1 H), 6.83 (dd, J = 8.6, 2.2 Hz, 1 H), 3.74 (dt, J = CH2Cl2 (1.0 mL) was added to a solution of tertꢀ
12.5, 3.4 Hz, 2 H), 2.86 (td, J = 11.9, 2.3 Hz, 2 H), butyl (1ꢀ(6,11ꢀ dihydroꢀ5Hꢀbenzo[a]carbazolꢀ2ꢀ
2.71 (t, J = 6.2 Hz, 2 H), 2.66ꢀ2.56 (m, 2 H), 2.47ꢀ yl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
2.38 (m, 10 H), 1.99ꢀ1.94 (m, 2 H), 1.50ꢀ1.40 (m, 2 yl)ethyl)carbamate (0.033 g, 0.058 mmol) in CH2Cl2
H), 0.98 (d, J = 6.4 Hz, 6 H); 13C NMR (100 MHz, (1.0 mL). After 2 h, the reaction mixture was
acetoneꢀd6) δ 153.2, 141.4, 141.3, 136.0, 135.9, concentrated, diluted with sat. NaHCO3, and
133.72, 133.68, 132.4, 131.1, 131.0, 130.4, 116.7, extracted with EtOAc (3x). The combined organic
116.4, 114.3, 113.6, 113.23, 113.18, 110.3, 99.5, layer was washed with brine, dried (Na2SO4) and
99.4, 59.0, 55.6, 54.9, 54.6, 49.4, 48.7, 44.3, 33.3, concentrated. The crude residue was purified by
18.8; HRMS (ESI+) m/z calcd for C28H39N8 chromatography on SiO2 (5 to 10% MeOH/CH2Cl2
487.3292 (M+H), found 487.3293.
with 1% TEA) followed by filtration through basic
Al2O3 (0 to 2% MeOH/CH2Cl2) to afford 13
2ꢀBromoꢀ6,11ꢀdihydroꢀ5Hꢀbenzo[a]carbazole
(34)23: A mixture of phenylhydrazine (0.506 g, 4.44 (UPCDC30206, 0.015 g, 0.032 mmol, 57%) as a
mmol) and 7ꢀbromoꢀ1ꢀtetralone (1.02 g, 4.44 mmol) pale yellow oil: IR (ATR) 3218, 2924, 2839, 2181,
1
in EtOH (10 mL) and conc. HCl (0.5 mL) was 1728, 1609, 1465, 1381, 1195, 740 cmꢀ1; H NMR
heated at reflux for 4 h. The reaction mixture was (500 MHz, acetoneꢀd6) δ 10.65 (br s, 1 H), 7.49 (d, J
concentrated and the resulting solid was suspended = 8.0 Hz, 1 H), 7.36 (d, J = 8.0 Hz, 1 H), 7.31 (d, J
in hexanes/CH2Cl2 (21 mL, 20:1) and stirred for 30 = 1.5 Hz, 1 H), 7.11ꢀ7.06 (m, 2 H), 7.03ꢀ6.99 (m, 1
min. The light red solid (34, 0.84 g, 2.8 mmol, 63%) H), 6.73 (dd, J = 1.5, 8.0 Hz, 1 H), 3.66 (d, J = 12.5
1
was collected by filtration and dried: H NMR (300 Hz, 2 H), 2.96ꢀ2.91 (m, 4 H), 2.89ꢀ2.76 (m, 2 H),
MHz, CDCl3) δ 8.11 (br s, 1 H), 7.56 (d, J = 7.5 Hz, 2.72ꢀ2.69 (m, 2 H), 2.61ꢀ2.56 (m, 3 H), 2.46ꢀ2.39 (m,
1 H), 7.44ꢀ7.43 (m, 1 H), 7.38 (d, J = 8.1 Hz, 1 H), 10 H), 1.96ꢀ1.93 (m, 2 H), 1.44 (qd, J = 13.5, 3.5 Hz,
7.29ꢀ7.28 (m, 1 H), 7.23ꢀ7.22 (m, 1 H), 7.20ꢀ7.12 (m, 2 H) 0.97 (d, J = 6.5 Hz, 6 H); 13C NMR (100 MHz,
1 H), 3.02ꢀ2.95 (m, 4 H); HRMS (ESI+) m/z calcd acetoneꢀd6) δ 151.8, 138.3, 134.8, 130.5, 129.5,
for C16H13NBr 298.0220 (M+H), found 298.0224.
1ꢀ(6,11ꢀDihydroꢀ5Hꢀbenzo[a]carbazolꢀ2ꢀyl)ꢀNꢀ
(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀ
128.3, 127.5, 122.4, 119.9, 119.1, 115.1, 112.3,
111.9, 110.2, 59.0, 55.7, 54.9, 54.6, 49.4, 49.2, 44.4,
33.4, 20.7, 18.8; HRMS (ESI+) m/z calcd for
amine (13, UPCDC30206): A suspension of 34 C30H42N5 472.3440 (M+H), found 472.3433.
(0.079 g, 0.26 mmol), K3PO4 (0.087 g, 0.40 mmol),
Pd2(dba)3 (0.005 g, 0.005 mmol) and CyJohnPhos
2ꢀ(3ꢀ(4ꢀ((tertꢀButoxycarbonyl)(2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)amino)piperidinꢀ1ꢀ
(0.0076 g, 0.021 mmol) in dry degassed dioxane (1 yl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀcarboxylic acid (35): To a
mL) was treated with A (0.11 g, 0.32 mmol) in solution of methyl 2ꢀ(3ꢀ(4ꢀ((tertꢀbutoxycarbonyl)(2ꢀ
dioxane (2.5 mL). The reaction mixture was (4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)amino)piperidinꢀ1ꢀ
degassed by bubbling argon for 15 min, sealed and yl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀcarboxylate (0.650 g, 1.08
heated at 120 °C for 48 h. The reaction mixture was mmol) in a mixture of THF (10 mL) and H2O (5
cooled to room temperature, diluted with sat. mL) was added LiOH (0.079 g, 3.23 mmol,
NaHCO3 and extracted with CH2Cl2 (3x). The portionwise), and the mixture was stirred at 60 °C
combined organic layer was washed with brine, for 24 h. The reaction mixture was cooled to room
dried (Na2SO4) and concentrated. The crude residue temperature and diluted with EtOAc and the phases
was purified by chromatography on SiO2 (2% were separated. The basic aqueous layer was
MeOH/CH2Cl2 with 1% TEA) followed by filtration acidified with 1N HCl and extracted with EtOAc
through basic Al2O3 (CH2Cl2) to afford tertꢀbutyl (1ꢀ (3x). The combined organic layer was washed with
(6,11ꢀdihydroꢀ5Hꢀbenzo[a]carbazolꢀ2ꢀyl)piperidinꢀ brine, dried (Na2SO4), filtered, concentrated to
4ꢀyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)carbamate
provide crude 2ꢀ(3ꢀ(4ꢀ((tertꢀbutoxycarbonyl)(2ꢀ(4ꢀ
(0.050 g, 0.087 mmol, 33%) as a pale yellow isopropylpiperazinꢀ1ꢀyl)ethyl)amino)piperidinꢀ1ꢀ
1
amorphous solid: H NMR (300 MHz, CDCl3) δ yl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀcarboxylic acid (35, 0.443 g,
8.28 (br s, 1 H), 7.54 (d, J = 7.5 Hz, 1 H), 7.37 (d, J 0.751 mmol, 70%) as a brown solid, which was used
= 8.1 Hz, 1 H), 7.20ꢀ7.09 (m, 3 H), 6.94 (d, J =1.5 without further purification: HRMS (ESI+) m/z calcd
Hz, 1 H), 6.75 (dd, J = 1.8, 8.1 Hz, 1 H), 3.75ꢀ3.72 for C34H48O4N5 590.3701 (M+H), found 590.3701.
(m, 2 H), 3.28ꢀ3.24 (m, 2 H), 2.96ꢀ2.80 (br s, 4 H),
2.83ꢀ2.76 (m, 2 H), 2.56ꢀ2.31 (m, 11 H), 1.81ꢀ1.72
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀIsopropylpiperazinꢀ1ꢀ
yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀNꢀmethylꢀ
(m, 6 H), 1.48 (s, 9 H), 1.04 (d, J = 6.3 Hz, 6 H); 1Hꢀindoleꢀ5ꢀcarboxamide (14, UPCDC30367): To
HRMS (ESI+) m/z calcd for C35H50O2N5 572.3959 a solution of 2ꢀ(3ꢀ(4ꢀ((tertꢀbutoxycarbonyl)(2ꢀ(4ꢀ
(M+H), found 572.3956.
isopropylpiperazinꢀ1ꢀyl)ethyl)amino)piperidinꢀ1ꢀ

Organic & Biomolecular Chemistry
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yl)phenyl)ꢀ1Hꢀindoleꢀ5ꢀcarboxylic acid (35, 0.100 g, 18.7; HRMS (ESI+) m/z calcd for C30H43ON6
0.169 mmol), methylamine hydrochloride (17.5 mg, 503.3493 (M+H), found 503.3492.
0.254 mmol) and TEA (0.191 mL, 1.36 mmol) in
((3ꢀBromophenyl)ethynyl)trimethylsilane
dry acetonitrile (0.5 mL) was slowly added T3P (36) 24 : To a solution of 1ꢀbromoꢀ3ꢀiodobenzene
(0.107 g, 0.144 mmol; 50% EtOAc solution) at 0 °C. (2.00 g, 6.72 mmol) in dry, degassed acetonitrile
After 1 h, another portion of T3P (0.107 g, 0.144 (20.0 mL) was added PdCl2(PPh3)2 (0.24 g, 0.34
mmol; 50% EtOAc solution) was added and the mmol) and CuI (0.065 g, 0.34 mmol). The mixture
reaction mixture was allowed to warm to room was degassed and backfilled with argon and charged
temperature overnight. The reaction mixture was with Et3N (0.95 mL, 6.7 mmol) and
quenched with slow addition of 0.5M NaOH and the trimethylsilylacetylene (1.94 mL, 13.4 mmol). The
mixture was stirred at room temperature for 1 h. The mixture was stirred at room temperature for 1 h. The
crude product was extracted with EtOAc (3x) and reaction mixture was concentrated and the crude
the combined organic layer was washed with water product was extracted with EtOAc, washed with
followed by brine, dried (Na2SO4), filtered and water, brine, dried (Na2SO4), filtered and
concentrated to provide crude tertꢀbutyl (2ꢀ(4ꢀ concentrated. The residue was purified by
isopropylpiperazinꢀ1ꢀyl)ethyl)(1ꢀ(3ꢀ(5ꢀ
chromatography on SiO2 (100% hexanes) to obtain
(methylcarbamoyl)ꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ 36 as an orange oil (1.7 g, 6.6 mmol, 99%): IR
4ꢀyl)carbamate (77.5 mg, 0.129 mmol, 76%), which (ATR) 2956, 2160, 1582, 1588, 1470, 1450, 1260,
1
was used without further purification: 1H NMR 1245, 1079 cmꢀ1; H NMR (500 MHz, CDCl3) 7.62
(500 MHz, CD3OD) δ 8.07 (s, 1 H), 7.60 (d, J = 8.5 (t, J = 2.0 Hz, 1 H), 7.45ꢀ7.43 (m, 1 H), 7.16 (app t,
Hz, 1 H), 7.43 (d, J = 9.0 Hz, 2 H), 7.32ꢀ7.27 (m, 2 J = 7.5 Hz, 1 H), 0.25 (s, 9 H); 13C NMR (CDCl3,
H), 6.96 (d, J = 7.5 Hz, 1 H), 6.89 (s, 1 H), 4.04 (br s, 125 MHz) 134.8, 131.7, 130.6, 129.7, 125.3 122.2,
1 H), 3.93ꢀ3.91 (m, 2 H), 3.27 (br s, 2 H), 2.95 (s, 3 103.4, 96.0, 0.00.
H), 2.86 (t, J = 12.3 Hz, 2 H), 2.69ꢀ2.50 (m, 11 H),
1.98ꢀ1.93 (m, 2 H), 1.77ꢀ1.76 (m, 2 H), 1.47 (s, 9 H), dioxaꢀ8ꢀazaspiro[4.5]decane (37): A solution of 36
1.10 (d, J = 6.0 Hz, 6 H); HRMS (ESI+) m/z calcd (200
mg, 0.790 mmol), 1,4ꢀdioxaꢀ8ꢀ
for C35H51O3N6 603.4017 (M+H), found 603.4013. azaspiro[4.5]decane (127 mg, 0.87 mmol), Pd2(dba)3
To solution of tertꢀbutyl
isopropylpiperazinꢀ1ꢀyl)ethyl)(1ꢀ(3ꢀ(5ꢀ
8ꢀ(3ꢀ((Trimethylsilyl)ethynyl)phenyl)ꢀ1,4ꢀ
a
(2ꢀ(4ꢀ (36 mg, 0.039 mmol), DavePhos (16 mg, 0.039
mmol) in dry THF (5.0 mL) was degassed by
(methylcarbamoyl)ꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ bubbling argon and backfilled with argon three
4ꢀyl)carbamate (72.5 mg, 0.120 mmol) in CH2Cl2 (2 times. To this solution was added LiHMDS (340 mg,
mL) at 0 °C was added trifluoroacetic acid (0.36 mL, 1.97 mmol). The reaction mixture was degassed and
4.8 mmol). The reaction mixture was allowed to the reaction vial was sealed and heated at 70 °C for
warm to room temperature and stirred for 2 h, 3 h. The reaction mixture was cooled to room
treated with sat. NaHCO3 and extracted with CH2Cl2 temperature, diluted with sat. NaHCO3 and extracted
(3x). The combined organic layers were washed with EtOAc (3x). The combined organic layer was
with brine, dried (Na2SO4), filtered, concentrated, washed with brine, dried (Na2SO4), filtered and
and purified by chromatography on SiO2 (0 to 5% concentrated. The residue was purified by
MeOH/CH2Cl2 to 10% MeOH/CH2Cl2 with 1% chromatography
on
SiO2
(10
to
30%
TEA) followed by filtration through basic Al2O3 (0 EtOAc/hexanes) to provide 37 (0.17 g, 0.54 mmol,
to 5% MeOH/CH2Cl2) to provide 2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀ 68%) as an offꢀwhite sticky solid: IR (ATR) 2957,
Isopropylpiperazinꢀ1ꢀyl)ethyl)amino)piperidinꢀ1ꢀ
2879, 2829, 2143, 1589, 1478, 1246, 1101, 833 cmꢀ
yl)phenyl)ꢀNꢀmethylꢀ1Hꢀindoleꢀ5ꢀcarboxamide 14 1; 1H NMR (300 MHz, CDCl3) δ 7.16 (t, J = 7.8 Hz,
(UPCDC30367, 30 mg, 0.059 mmol, 49%): IR 1 H), 7.03 (br s, 1 H), 6.94ꢀ6.88 (m, 2 H), 3.99 (s, 4
(ATR) 3275, 2923, 2813, 1627, 1599, 1547, 1458, H), 3.32 (t, J = 5.7 Hz, 4 H), 1.82 (t, J = 5.7 Hz, 4 H),
1407, 1176, 1148, 1117, 980, 805, 777, 764 cmꢀ1; 1H 0.24 (s, 9 H); HRMS (ESI+) m/z calcd for
NMR (500 MHz, CD3OD) δ 8.07 (s, 1 H), 7.60 (d, J C18H26NO2Si 316.1733 (M+H), found 316.1725.
= 8.5 Hz, 1 H), 7.43 (d, J = 8.5 Hz, 2 H), 7.32ꢀ7.27
8ꢀ(3ꢀEthynylphenyl)ꢀ1,4ꢀdioxaꢀ8ꢀ
(m, 2 H), 6.96 (app d, J = 7.0 Hz, 1 H), 6.88 (s, 1 H), azaspiro[4.5]decane (38): A solution of 37 (820 mg,
3.82 (app d, J = 12.5 Hz, 2 H), 2.95 (s, 3 H), 2.85ꢀ 2.47 mmol) in THF (10.0 mL) was treated with
2.79 (m, 4 H), 2.72ꢀ2.53 (m, 12 H), 2.05 (app d, J = TBAF (0.95 mL, 75 wt % H2O) at 0 °C. The
11.5 Hz, 2 H), 1.56 (qd, J = 11.8, 3.3 Hz, 2 H), 1.09 reaction mixture was warmed to room temperature.
(d, J = 6.5 Hz, 6 H); 13C NMR (125 MHz, CD3OD) After 20 min, the reaction mixture was extracted
δ 172.3, 153.4, 141.6, 140.6, 134.4, 130.6, 130.1, with EtOAc (2x), washed with H2O, brine, dried
126.8, 121.8, 121.0, 118.0, 117.4, 114.7, 111.8, (Na2SO4), filtered and concentrated. The residue was
100.6, 58.3, 56.3, 55.9, 54.1, 50.0, 43.8, 32.7, 27.0, purified by chromatography on SiO2 (10 to 30%

Page 17 of 24
Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB00526A
EtOAc/hexanes) to give 38 (0.508 g, 2.08 mmol, powder (89 mg, 0.27 mmol, 33%): IR (ATR) 3092,
85%) as a yellow oil: IR (ATR) 3282, 2956, 2926, 2956, 2829, 2726, 1595, 1573, 1541, 1494, 1494,
1
2881, 1664, 1591, 1569, 1235, 1097 cmꢀ1; H NMR
1
1224, 1095 cmꢀ1; H NMR (300 MHz, DMSOꢀd6)
δ
(400 MHz, CDCl3) δ 7.18 (t, J = 8.0 Hz, 1 H), 7.06
(dd, J = 1.6, 2.4 Hz, 1 H), 6.96ꢀ6.92 (m, 2 H), 3.99
(s, 4 H), 3.33 (t, J = 5.6 Hz, 4 H), 3.02 (s, 1 H), 1.82
(t, J = 5.6 Hz, 4 H); 13C NMR (100 MHz, CDCl3)
150.7, 129.2, 123.1, 122.8, 119.9, 117.3, 107.2, 84.4,
76.5, 64.5, 47.5, 34.5; HRMS (ESI+) m/z calcd for
C15H18O2N 244.1332 (M+H), found 244.1331.
11.88 (s, 1 H), 8.83 (br s, 1 H), 8.19 (br s, 1 H), 7.46
(s, 1 H), 7.38 (br s, 1 H), 7.29ꢀ7.28 (m, 2 H), 7.02 (s,
1 H), 6.94 (s, 1 H), 3.93 (s, 4 H), 3.39 (t, J = 5.4 Hz,
4 H), 1.75 (t, J = 5.1 Hz, 4 H); 13C NMR (75 MHz,
DMSOꢀd6)
δ 150.8, 140.3, 140.1, 139.5, 132.0,
129.6, 115.8, 115.3, 112.5, 106.4, 97.3, 63.7, 46.6,
33.9; HRMS (ESI+) m/z calcd for C20H22N3O2
336.1707 (M+H), found 336.1704.
3ꢀIodopyridinꢀ4ꢀamine (39) 25 : To a refluxing
solution of 4ꢀaminopyridine (2.00 g, 21.3 mmol) and
Na2CO3 (1.35 g, 12.8 mmol) in H2O (7.6 mL) was
slowly added a solution of KI (3.99 g, 23.8 mmol)
and I2 (4.09 g, 15.9 mmol) in H2O (16.8 mL) and the
mixture was heated at reflux for 22 h. The reaction
mixture was cooled to room temperature and
extracted with EtOAc (2x). The combined organic
layer was washed with satd Na2S2O3, followed by
brine, dried (MgSO4), filtered and concentrated. The
residue was purified by chromatography on SiO2 (50
to 75% EtOAc/hexanes) to give 39 as an offꢀwhite
solid (1.66 g, 7.54 mmol, 36%): 1H NMR (300 MHz,
CDCl3) δ 8.55 (s, 1 H), 8.08 (d, J = 5.4 Hz, 1 H),
1ꢀ(3ꢀ(1HꢀPyrrolo[3,2ꢀc]pyridinꢀ2ꢀyl)phenyl)ꢀNꢀ
(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀ
amine (17, UPCDC30345): To a solution of 41
(0.080 g, 0.24 mmol) in acetone (15 mL) was added
3M HCl (10 mL) and the reaction mixture was
heated at 80 °C for 6 h. The solution was cooled to
room temperature and kept at this temperature
overnight. The solution was cooled to 0 °C,
neutralized with solid Na2CO3 and extracted with
EtOAc (2x). The combined organic layer was
washed with brine, dried (Na2SO4), filtered and
concentrated to afford 1ꢀ(3ꢀ(1Hꢀpyrrolo[3,2ꢀ
c]pyridinꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀone as a yellow
solid that was used without further purification:
HRMS (ESI+) m/z calcd for C18H18ON3 292.1444
(M+H), found 292.1443.
6.57 (d, J = 5.4 Hz, 1 H), 4.72 (br s, 2 H).
Nꢀ(3ꢀIodopyridinꢀ4ꢀyl)acetamide (40)
26
: A
solution of 39 (1.65 g, 7.50 mmol) in CH2Cl2 (15
mL) was treated with acetic anhydride (0.71 mL, 7.5
mmol) and TEA (1.58 mL, 11.3 mmol) at room
temperature. After 17 h, the solution was
To
a
suspension of 1ꢀ(3ꢀ(1Hꢀpyrrolo[3,2ꢀ
c]pyridinꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀone (0.070 g, 0.24
mmol), B (0.045 g, 0.26 mmol) in a mixture of 1,2ꢀ
DCE and THF (2:1, 1.5 mL) was added Ti(OiPr)4
(0.080 mL, 0.26 mmol). After 1.5 h, the reaction
mixture was treated with NaBH(OAc)3 (0.031 g,
0.14 mmol). After 2 h, a second portion of
NaBH(OAc)3 (0.025 g, 0.012 mmol) was added.
After 1 h, the solution was treated with 0.5 M NaOH
(aq) and the resulting suspension was extracted with
EtOAc (2x). The combined organic layer was
washed with brine, dried (Na2SO4), filtered and
concentrated. The residue was purified by
chromatography on SiO2 (0 to 10% MeOH/CH2Cl2
with 1% TEA) followed by filtration through basic
Al2O3 (0 to 5% MeOH/CH2Cl2) to give 17 as an offꢀ
white solid (UPCDC30345, 47 mg, 0.11 mmol, 42%
ꢀ 2 steps): IR (ATR) 3081, 2943, 2924, 2808, 1772,
concentrated.
The residue was diluted with
THF/H2O (6.0 mL, 1/1) and treated with LiOH (0.20
g, 1.1 mmol) at room temperature. After 30 min, the
reaction mixture was extracted with EtOAc (3x).
The combined organic layer was washed with brine,
dried (MgSO4), filtered and concentrated to give 40
(1.68 g, 6.40 mmol, 85%, containing approx. 5%
1
residual solvent) as a light orange solid: H NMR
(400 MHz, CDCl3) δ 8.81 (s, 1 H), 8.39 (d, J = 5.6
Hz, 1 H), 8.32 (d, J = 5.6 Hz, 1 H), 7.61 (br s, 1 H),
2.28 (s, 3 H).
8ꢀ(3ꢀ(1HꢀPyrrolo[3,2ꢀc]pyridinꢀ2ꢀyl)phenyl)ꢀ
1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (41): To
a
microwave vial equipped with a magnetic stir bar
was added 38 (200 mg, 0.822 mmol), 40 (323 mg,
1.23 mmol), PdCl2(PPh3)2 (29 mg, 0.041 mmol), CuI
(8.0 mg, 0.04 mmol), 1,1,3,3ꢀtetraꢀmethylguanidine
(0.31 mL, 2.5 mmol) and DMF (1.0 mL). The
mixture was degassed by bubbling argon for 15 min,
sealed, and then heated at 80 °C for 1 h under
microwave irradiation. The reaction mixture was
diluted with H2O and extracted with EtOAc (2x).
The combined organic layer was washed with H2O,
brine, dried (Na2SO4), filtered and concentrated. The
1
1601, 1575, 1541, 1496 cmꢀ1; H NMR (400 MHz,
CD3OD)
δ 8.75 (s, 1 H), 8.11 (d, J = 5.6 Hz, 1 H),
7.43ꢀ7.41 (m, 2 H), 7.34ꢀ7.28 (m, 2 H), 7.00 (d, J =
7.2 Hz, 1 H), 6.96 (s, 1 H), 3.82 (d, J = 12.8 Hz, 2
H), 2.86ꢀ2.78 (m, 4 H), 2.70ꢀ2.52 (m, 12 H), 2.05ꢀ
2.01 (m, 2 H), 1.54 (qd, J = 12.0, 3.5 Hz, 2 H), 1.08
(d, J = 6.4 Hz, 6 H); 13C NMR (100 MHz, CD3OD)
δ
153.4, 143.3, 142.5, 142.2, 140.3, 133.6, 130.8,
127.7, 118.1, 117.8, 114.8, 107.9, 98.8, 58.4, 56.3,
55.9, 54.1, 49.9, 49.5, 43.8, 32.8, 18.7; HRMS
solid was precipitated from
a
solution of
MeOH/CH2Cl2/hexanes to afford 41 as a white

Organic & Biomolecular Chemistry
Page 18 of 24
View Article Online
DOI: 10.1039/C7OB00526A
(ESI+) m/z calcd for C27H39N6 447.3231 (M+H), 1 H), 7.52 (s, 1 H), 7.33ꢀ7.27 (m, 2 H), 6.95 (d, J =
found 447.3229. 8.0 Hz, 1 H), 6.91 (s, 1 H), 3.93 (s, 4 H), 3.39 (t, J =
5ꢀFluoroꢀ3ꢀiodopyridinꢀ2ꢀamine (42) 27 : To a 5.0 Hz, 4 H), 1.74 (t, J = 5.0 Hz, 4 H).
stirring solution of 5ꢀfluoropyridinꢀ2ꢀamine (1.5 g,
1ꢀ(3ꢀ(5ꢀFluoroꢀ1Hꢀpyrrolo[2,3ꢀb]pyridinꢀ2ꢀ
13.4 mmol) in H2SO4 (2M, 20.0 mL) was slowly yl)phenyl)piperidinꢀ4ꢀone (45): To a solution of 44
added KIO3 (1.4 g, 6.7 mmol). The reaction mixture (0.135 g, 0.382 mmol) in acetone (20 mL) was
was heated at 100 °C and a solution of KI (2.24 g, added 3M HCl (15 mL). The reaction mixture was
13.4 mmol) in H2O (10.0 mL) was added. After 30 heated at 80 °C for 6 h. The solution was cooled to
min, the reaction mixture was cooled to room 0 °C, neutralized with Na2CO3, and extracted with
temperature and the solution was treated with EtOAc (2x). The combined organic layer was
NaHCO3 until pH 8ꢀ9. The reaction mixture was washed with brine, dried (Na2SO4), filtered and
extracted with EtOAc (3x). The combined organic concentrated. The solid that formed upon
layers were washed with sat. NaHSO3, H2O, brine, concentration was collected by filtration, washed
dried (Na2SO4), filtered and concentrated to afford with hexanes and dried in vacuo to give 45 as a
42 (2.29 g, 9.62 mmol, 72%) that was used without brown solid (82 mg, 0.26 mmol, 69%, approx. 90%
further purification.
δ 11.51 (s, 1 H),
purity): 1H NMR (300 MHz, CDCl3)
Nꢀ(5ꢀFluoroꢀ3ꢀiodopyridinꢀ2ꢀyl)acetamide (43):
A solution of 42 (1.20 g, 5.04 mmol) in AcOH (10.0
mL) was treated with acetic anhydride (0.47 mL, 5.0
mmol) and the mixture was heated at 100 °C for 4 h.
The reaction mixture was cooled to room
temperature and carefully treated with sat. K2CO3.
The reaction mixture was extracted with EtOAc (3x).
The combined organic layer was washed with H2O,
brine, dried (Na2SO4), filtered and concentrated. The
product was purified by chromatography on SiO2 (0
to 70% EtOAc/hexanes) to obtain 43 as a white
powder (926 mg, 3.31 mmol, 66%): M.p. 148ꢀ
150 °C; IR (ATR) 3223, 3189, 3029, 3001, 1688,
8.19 (s, 1 H), 7.64 (dd, J = 2.7, 9.0 Hz, 1 H), 7.46
(app t, J = 7.8 Hz, 1 H), 7.40 (s, 1 H), 7.32 (d, J =
7.5 Hz, 1 H), 7.03 (dd, J = 2.1, 8.1 Hz, 1 H), 6.75 (d,
J = 2.1 Hz, 1 H), 3.69 (t, J = 6.3 Hz, 4 H), 2.62 (t, J
= 6.0 Hz, 4 H); HRMS (ESI+) m/z calcd for
C18H17FN3O 310.1356 (M+H), found 310.1348.
1ꢀ(3ꢀ(5ꢀFluoroꢀ1Hꢀpyrrolo[2,3ꢀb]pyridinꢀ2ꢀ
yl)phenyl)ꢀNꢀ(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
yl)ethyl)piperidinꢀ4ꢀamine (19, UPCDC30381):
To a suspension of 45 (0.070 g, 0.23 mmol) and B
(0.046 g, 0.27 mmol) in THF/1,2ꢀdichloroethane
(1/2, 1.5 mL) was added Ti(OiPr)4 (0.083 mL, 0.27
mmol) at room temperature. After 30 min,
NaBH(OAc)3 (0.030 g, 0.13 mmol) was added. After
1
1655, 1569, 1515, 1431, 1262 cmꢀ1; H NMR (400
MHz, CDCl3)
= 7.2, 2.8 Hz, 1 H), 7.66 (br s, 1 H), 2.33 (s, 3 H); mmol) was added. After
13C NMR (100 MHz, CDCl3)
170.1, 155.6 (d, JCF
δ
8.27 (d, J = 2.4 Hz, 1 H), 7.89 (dd, J 2 h, a second portion of NaBH(OAc)3 (0.031 g, 0.14
1
h, additional
NaBH(OAc)3 (0.030 g, 0.13 mmol) was added and
the reaction mixture was stirred overnight. The
reaction mixture was diluted with sat. NaHCO3 and
EtOAc/MeOH. The suspension was filtered through
Celite and the filtrate was concentrated. The residue
was extracted with EtOAc, washed with H2O, brine,
dried (Na2SO4), filtered and concentrated. The
residue was purified by chromatography on SiO2 (5
to 10% MeOH/CH2Cl2 with 1% TEA) followed by
filtration through basic Al2O3 (5% MeOH/CH2Cl2)
to give 19 as an offꢀwhite foamy solid
(UPCDC30381, 0.051 g, 0.11 mmol, 49%): IR
(ATR) 3170, 2930, 2807, 1610, 1586, 1489, 1446,
δ
= 259 Hz), 147.6, 135.6 (d, JCF = 24 Hz), 135.1 (d,
JCF = 21 Hz), 24.0; HRMS (ESI+) m/z calcd for
C7H7FIN2O 280.9587 (M+H), found 280.9579.
8ꢀ(3ꢀ(5ꢀFluoroꢀ1Hꢀpyrrolo[2,3ꢀb]pyridinꢀ2ꢀ
yl)phenyl)ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (44)28:
To a microwave vial equipped with a magnetic stir
bar was added 38 (300 mg, 1.23 mmol), 43 (432 mg,
1.47 mmol), PdCl2(PPh3)2 (44 mg, 0.062 mmol), CuI
(19.0
mg,
0.098
mmol),
1,1,3,3ꢀtetraꢀ
methylguanidine (0.47 mL, 3.7 mmol) and DMF
(1.0 mL). The reaction mixture was degassed for 10
min. by argon bubbling and the vial was sealed and
heated at 80 °C under microwave irradiation. The
1
1343, 1176, 980, 758 cmꢀ1; H NMR (500 MHz,
reaction mixture was diluted with H2O and extracted CD3OD)
δ 8.06 (s, 1 H), 7.68 (dd, J = 2.5, 9.5 Hz, 1
with EtOAc (2x). The combined organic layer was H), 7.45 (s, 1 H), 7.34ꢀ7.31 (m, 2 H), 7.01ꢀ6.99 (m,
washed with H2O, brine, dried (Na2SO4), filtered 1 H), 6.81 (s, 1 H), 3.85 (d, J = 12.5 Hz, 2 H), 2.90
and concentrated. The residue was purified by (t, J = 6.5 Hz, 2 H), 2.87ꢀ2.82 (m, 3 H), 2.73ꢀ2.57
chromatography
on
SiO2
(30
to
50% (m, 11 H), 2.09 (d, J= 12.0 Hz, 2 H), 1.62 (qd, J =
EtOAc/hexanes) to obtain 44 as a light yellow solid 11.5, 3.0 Hz, 2 H), 1.10 (d, J = 6.5 Hz, 6 H); 13C
(145 mg, 0.41 mmol, 33%): IR (ATR) 3211, 2872,
2821, 1614, 1576, 1489, 1446, 1287, 1196, 1131,
NMR (125 MHz, CD3OD)
153.2, 147.6, 143.4, 133.7, 131.3 (d, JCF = 30 Hz),
130.7, 123.5 (d, JCF = 8 Hz), 118.0 (d, JCF = 39 Hz),
δ 157.2 (d, JCF = 238 Hz),
1
1110, 876, 758 cmꢀ1; H NMR (500 MHz, DMSOꢀ
d6)
δ 12.22 (s, 1 H), 8.17 (s, 1 H), 7.78 (d, J = 9.5 Hz, 114.9, 114.8, 114.6, 98.3 (d, JCF = 4 Hz), 57.5, 56.4,

Page 19 of 24
Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB00526A
56.1, 53.8, 49.7, 43.5, 31.9, 18.6; 19F NMR (376
A solution of trifluoroacetic acid (0.83 mL, 11.0
mmol) and triethylsilane (0.09 mL, 0.52 mmol) in
δ ꢀ141.4; HRMS (ESI+) m/z calcd
MHz, CD3OD)
CH2Cl2 (1.5 mL) was added to a solution of tertꢀ
for C27H38FN6 465.3136 (M+H), found 465.3133.
2ꢀ(3ꢀBromophenyl)naphthalene (46) 29 : To a
flask containing 1ꢀbromoꢀ3ꢀiodobenzene (0.28 mL,
2.2 mmol), 2ꢀnaphthalene boronic acid (0.26 g, 1.5
mmol), Pd(PPh3)2Cl2 (0.023 g, 0.033 mmol) and
K2CO3 (0.405 g, 2.93 mmol) was added degassed
DMF (4.0 mL). The reaction mixture was heated at
80 °C overnight. The reaction mixture was cooled to
room temperature, diluted with H2O, and extracted
with EtOAc (3x). The combined organic layer was
washed with brine, dried (Na2SO4), filtered and
concentrated. The residue was purified by
chromatography on SiO2 (0 to 10% EtOAc/hexanes)
to give 46 (0.265 g, 0.94 mmol, 64%) as an offꢀ
butyl
(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)(1ꢀ(3ꢀ
(naphthalenꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)carbamate
(0.055 g, 0.099 mmol) in CH2Cl2 (1.5 mL). After 1 h,
the reaction mixture was concentrated, diluted with
sat. NaHCO3, and extracted with EtOAc (3x). The
combined organic layer was washed with brine,
dried (Na2SO4) and concentrated. The crude residue
was purified by chromatography on SiO2 (8 to 20%
MeOH/CH2Cl2 with 1% TEA) followed by filtration
through basic Al2O3 (0 to 10% MeOH/CH2Cl2) to
afford 20 (UPCDC30222, 0.030 g, 0.066 mmol,
67%, containing approx. 5% residual solvents) as a
light yellow oil: IR (ATR) 2957, 2932, 2805, 1702,
1593, 1461, 1379, 1232, 1176, 1129, 982, 854, 779
δ 8.01 (d, J
white solid: 1H NMR (400 MHz, CDCl3)
cmꢀ1; 1H NMR (500 MHz, acetoneꢀd6)
δ 8.15 (d, J =
= 1.2 Hz, 1 H), 7.94ꢀ7.90 (m, 2 H), 7.89ꢀ7.86 (m, 2
H), 7.70 (dd, J = 2.0, 8.4 Hz, 1 H), 7.66ꢀ7.63 (m, 1
H), 7.55ꢀ7.48 (m, 3 H), 7.35 (app t, J = 8.0 Hz, 1 H);
1.0 Hz, 1 H), 7.98ꢀ7.96 (m, 2 H), 7.91 (d, J = 8.0 Hz,
1 H), 7.82 (dd, J = 2.0, 8.5 Hz, 1 H), 7.54ꢀ7.48 (m, 2
H), 7.35ꢀ7.32 (m, 2 H), 7.19 (dd, J = 0.5, 7.5 Hz, 1
H), 6.99 (dd, J = 2.5, 8.5 Hz, 1 H), 3.77 (dt, J = 3.0,
12.5 Hz, 2 H), 2.90ꢀ2.85 (m, 2 H), 2.72 (t, J = 6.0
Hz, 2 H), 2.65ꢀ2.55 (m, 3 H), 2.47ꢀ2.40 (m, 10 H),
1.98ꢀ1.95 (m, 2 H), 1.47 (qd, J = 13.0, 4.0, 2 H),
0.98 (d, J = 6.5 Hz, 6 H); 13C NMR (125 MHz,
13C NMR (100 MHz, CDCl3)
δ 143.5, 130.6, 130.5,
130.4, 128.8, 128.4, 127.8, 126.6, 126.4, 126.2,
126.1, 125.4; HRMS (ESI+) m/z calcd for C16H11Br
282.0044 (M+), found 282.0062.
Nꢀ(2ꢀ(4ꢀIsopropylpiperazinꢀ1ꢀyl)ethyl)ꢀ1ꢀ(3ꢀ
(naphthalenꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀamine (20,
UPCDC30222): A suspension of 46 (0.100 g, 0.353
mmol), A (0.150 g, 0.424 mmol), K3PO4 (0.116 g,
0.529 mmol), Pd2(dba)3 (0.006 g, 0.007 mmol) and
CyJohnPhos (0.010 g, 0.028 mmol) in dry degassed
dioxane (5 mL) was heated at 110 °C overnight. The
reaction mixture was cooled to room temperature,
diluted with sat. NaHCO3 and extracted with EtOAc
(3x). The combined organic layer was washed with
brine, dried (Na2SO4) and concentrated. The crude
residue was purified by chromatography on SiO2 (10
to 30% EtOAc/CH2Cl2) to give tertꢀbutyl (2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)(1ꢀ(3ꢀ(naphthalenꢀ2ꢀ
yl)phenyl)piperidinꢀ4ꢀyl)carbamate (0.101 g, 0.18
mmol, 51%) as a light yellow oil: IR (ATR) 2959,
2928, 2807, 1679, 1592, 1448, 1410, 1383, 1362,
1178, 1146, 1010, 982, 907, 767 cmꢀ1; 1H NMR (400
acetoneꢀd6)
δ 153.3, 142.5, 140.1, 134.7, 133.6,
130.3, 129.1, 129.0, 128.4, 127.1, 126.7, 126.4,
126.3, 118.6, 116.2, 115.9, 59.0, 55.7, 54.9, 54.6,
49.4, 48.9, 44.4, 33.4, 18.8; HRMS (ESI+) m/z calcd
for C30H41N4 457.3331 (M+H), found 457.3322.
2ꢀ(3ꢀBromophenyl)benzofuran
Benzofuranꢀ2ꢀylboronic acid (0.408 g, 2.47 mmol),
1ꢀbromoꢀ3ꢀiodobenzene (0.482 mL, 3.70 mmol),
PdCl2(PPh3)2 (0.088 g, 0.12 mmol), K2CO3 (0.682 g,
4.94 mmol) was treated with DMF (4.0 mL) and
heated at 80 °C overnight. The reaction mixture was
diluted with H2O and extracted with EtOAc (3x).
The combined organic layer was washed with brine,
dried (Na2SO4), filtered and concentrated. The
residue was purified by chromatography on SiO2 (0
to 10% EtOAc/hexanes) to give 47 as an offꢀwhite
30
(47)
:
1
solid (0.55 g, 2.0 mmol, 82%): H NMR (400 MHz,
MHz, CDCl3)
δ
8.01 (d, J = 0.8 Hz, 1 H), 7.91ꢀ7.85
CDCl3) δ 8.02 (t, J = 1.2 Hz, 1 H), 7.80ꢀ7.77 (m, 1
H), 7.61ꢀ7.58 (m, 1 H), 7.54ꢀ7.51 (m, 1 H), 7.49ꢀ
7.46 (m, 1 H), 7.34ꢀ7.29 (m, 2 H), 7.27ꢀ7.23 (m, 1
H), 7.05 (d, J = 0.8 Hz, 1 H); 13C NMR (100 MHz,
(m, 3 H), 7.72 (dd, J = 2.0, 8.8 Hz, 1 H), 7.52ꢀ7.45
(m, 2 H), 7.36 (app t, J = 8.0 Hz, 1 H), 7.25ꢀ7.23 (m,
1 H), 7.19 (d, J = 7.6 Hz, 1 H), 6.95 (dd, J = 2.0, 8.0
Hz, 1 H), 4.17 (br s, 1 H), 3.85 (d, J = 12.4 Hz, 2 H),
3.25 (br s, 2 H), 2.88ꢀ2.84 (m, 2 H), 2.69ꢀ2.47 (m,
11 H), 1.86ꢀ1.79 (m, 4 H), 1.47 (s, 9 H), 1.05 (d, J =
CDCl3)
δ 155.1, 154.3, 132.6, 131.5, 130.5, 129.0,
128.0, 124.9, 123.6, 123.3, 123.1, 121.3, 111.4,
102.6; HRMS (ESI+) m/z calcd for C14H9OBr
271.9837 (M+), found 271.9858.
6.4 Hz, 6 H); 13C NMR (100 MHz, CDCl3)
δ 155.4,
151.8, 142.3, 139.3, 133.7, 132.7, 129.7, 128.4,
128.3, 127.8, 126.4, 126.0, 125.92, 125.91, 119.1,
116.1, 115.8, 79.9, 54.7, 53.9, 49.9, 48.7, 30.2, 28.7,
18.7; HRMS (ESI+) m/z calcd for C35H49N4O2
557.3850 (M+H), found 557.3849.
1ꢀ(3ꢀ(Benzofuranꢀ2ꢀyl)phenyl)ꢀNꢀ(2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀamine
(21, UPCDC30221): A suspension of A (187 mg,
0.527 mmol), 2ꢀ(3ꢀbromophenyl)benzofuran (47,
120 mg, 0.44 mmol) and K3PO4 (144 mg, 0.66

Organic & Biomolecular Chemistry
Page 20 of 24
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DOI: 10.1039/C7OB00526A
mmol) in dry degassed dioxane (1.5 mL) was
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀIsopropylpiperazinꢀ1ꢀ
degassed for 5 min with argon. To this mixture was
added Pd2(dba)3 (8 mg, 0.009 mmol) and
yl)ethyl)amino)piperidinꢀ1ꢀyl)phenyl)ꢀN,Nꢀ
dimethylꢀ1Hꢀindoleꢀ5ꢀcarboxamide
(23,
CyJohnPhos (12 mg, 0.035 mmol). The reaction vial UPCDC30368): Prepared according to the
was sealed and the mixture was heated at 110 °C for procedure for 14 using dimethylamine hydrochloride
12 h. The reaction mixture was diluted with sat. instead of methylamine hydrochloride to provide
NaHCO3 and extracted with EtOAc (3x). The crude tertꢀbutyl (1ꢀ(3ꢀ(5ꢀ(dimethylcarbamoyl)ꢀ1Hꢀ
combined organic layer was washed with brine, indolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
dried (Na2SO4), filtered and concentrated. The isopropylpiperazinꢀ1ꢀyl)ethyl)carbamate (77 mg,
residue was purified by chromatography on SiO2 (10 0.13 mmol, 74%), which was used for the next
1
to 30% EtOAc/CH2Cl2) to give tertꢀbutyl (1ꢀ(3ꢀ reaction without further purification: H NMR (500
(benzofuranꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
MHz, CD3OD) δ 7.65 (s, 1 H), 7.45 (d, J = 8.0 Hz, 1
isopropylpiperazinꢀ1ꢀyl)ethyl)ꢀcarbamate (0.19 g, H), 7.42 (br s, 1 H), 7.32ꢀ7.28 (m, 2 H), 7.19 (d, J =
0.36 mmol, 82%) as a light yellow oil: IR (ATR) 8.5 Hz, 1 H), 6.96 (app d, J = 7.0 Hz, 1 H), 6.87 (s, 1
2962, 2930, 2807, 1685, 1601, 1450, 162, 1143, H), 4.05 (br s, 1 H), 3.93ꢀ3.91 (m, 2 H), 3.28 (br s, 2
1
1008, 775, 749 cmꢀ1; H NMR (400 MHz, CDCl3)
δ
H), 3.11 (s, 6 H), 2.87 (t, J = 12.0 Hz, 2 H), 2.71ꢀ
2.51 (m, 11 H), 1.97ꢀ1.91 (m, 2 H), 1.78ꢀ1.76 (m, 2
H), 1.48 (s, 9 H), 1.08 (d, J = 6.5 Hz, 6 H); HRMS
(ESI+) m/z calcd for C36H53O3N6 617.4174 (M+H),
found 617.4172.
7.50 (dd, J = 0.8, 7.6 Hz, 1 H), 7.46 (d, J = 8.0 Hz,
1 H), 7.38 (s, 1 H), 7.28ꢀ7.14 (m, 4 H), 6.93 (d, J =
0.8 Hz, 1 H), 6.65 (dt, J = 7.2, 1.6 Hz, 1 H), 4.11 (br
s, 1 H), 3.76 (d, J = 12.0 Hz, 2 H), 3.19 (br s, 2 H),
2.86ꢀ2.75 (m, 2 H), 2.63ꢀ2.42 (m, 11 H), 1.80ꢀ1.76
(m, 4 H), 1.43 (s, 9 H), 0.99 (d, J = 6.8 Hz, 6 H); 13C
To
a
solution of
tertꢀbutyl (1ꢀ(3ꢀ(5ꢀ
(dimethylcarbamoyl)ꢀ1Hꢀindolꢀ2ꢀ
yl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
yl)ethyl)carbamate (82.3 mg, 0.133 mmol) in
CH2Cl2 (2 mL) at 0 °C was added trifluoroacetic
acid (0.39 mL, 5.3 mmol). The reaction mixture was
allowed to warm to room temperature and stirred for
2 h, treated with sat. NaHCO3, and extracted with
CH2Cl2 (3x). The combined organic layers were
washed with brine, dried (Na2SO4), filtered,
concentrated, and purified by chromatography on
SiO2 (0 to 10% MeOH/CH2Cl2 with 1% TEA)
followed by filtration through basic Al2O3 (0 to 5 %
NMR (100 MHz, CDCl3)
δ 156.3, 155.4, 154.8,
151.5, 131.2, 129.5, 129.3, 124.2, 122.9, 120.8,
116.8, 116.4, 112.9, 111.1, 101.3, 79.8, 58.4, 54.6,
53.8, 53.0, 49.6, 48.6, 39.9, 30.1, 28.5, 18.6; HRMS
(ESI+) m/z calcd for C33H47O3N4 547.3643 (M+H),
found 547.3643.
A
solution of tertꢀbutyl (1ꢀ(3ꢀ(benzofuranꢀ2ꢀ
yl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀ
yl)ethyl)ꢀcarbamate (0.100 g, 0.18 mmol) in CH2Cl2
(1.5 mL) was treated with trifluoroacetic acid (1.5
mL) and triethylsilane (0.15 mL, 0.95 mmol) in
CH2Cl2 (1.5 mL). After 1 h, the solution was
concentrated, diluted with sat. NaHCO3 and
extracted with EtOAc (3x). The combined organic
layer was washed with brine, dried (Na2SO4) and
concentrated. The crude residue was purified by
chromatography on SiO2 (0 to 10% MeOH/CH2Cl2)
to afford 21 as a yellow foam (UPCDC30221, 0.060
g, 0.13 mmol, 73%): IR (ATR) 2957, 2928, 2807,
1599, 1567, 1489, 1450, 1254, 1220, 1176, 1144,
MeOH/CH2Cl2)
to
provide
2ꢀ(3ꢀ(4ꢀ((2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)amino)piperidinꢀ1ꢀ
yl)phenyl)ꢀN,Nꢀdimethylꢀ1Hꢀindoleꢀ5ꢀcarboxamide
23 (UPCDC30368, 36 mg, 0.069 mmol, 52%): IR
(ATR) 3225, 2930, 2808, 1599, 1541, 1499, 1444,
1383, 1318, 1176, 1146, 1071, 982, 803, 779, 764
1
cmꢀ1; H NMR (400 MHz, CD3OD) δ 7.65 (s, 1 H),
7.45 (d, J = 8.4 Hz, 1 H), 7.42 (br s, 1 H), 7.32ꢀ7.27
(m, 2 H), 7.19 (dd, J = 8.2, 1.4 Hz, 1 H), 6.96ꢀ6.94
(m, 1 H), 6.86 (s, 1 H), 3.81 (app d, J = 12.4 Hz, 2
H), 3.11 (s, 6 H), 2.85ꢀ2.78 (m, 4 H), 2.70ꢀ2.52 (m,
12 H), 2.04 (app d, J = 12.0 Hz, 2 H), 1.55 (qd, J =
11.9, 3.2 Hz, 2 H), 1.08 (d, J = 6.4 Hz, 6 H); 13C
NMR (100 MHz, CD3OD) δ 175.6, 153.4, 141.5,
139.4, 134.4, 130.6, 129.9, 128.0, 121.8, 120.7,
118.2, 117.4, 114.7, 111.9, 100.2, 58.4, 56.3, 55.9,
54.1, 50.0, 43.8, 32.8, 18.7; HRMS (ESI+) m/z calcd
for C31H45ON6 517.3649 (M+H), found 517.3649.
1ꢀBenzhydrylꢀ2ꢀ(3ꢀbromophenyl)ꢀ1Hꢀ
1
1122, 982, 747 cmꢀ1; H NMR (500 MHz, acetoneꢀ
d6) δ 7.62 (d, J = 7.5 Hz, 1 H), 7.55 (d, J = 8.0 Hz, 1
H), 7.52ꢀ7.51 (m, 1 H), 7.35ꢀ7.28 (m, 3 H), 7.26ꢀ
7.22 (m, 1 H), 7.00 (dt, J = 7.5, 2.0 Hz, 1 H), 3.75
(dt, J = 12.5, 3.0 Hz, 2 H), 2.92ꢀ2.87 (m, 3 H), 2.73
(app t, J = 6.0 Hz, 3 H), 2.67ꢀ2.56 (m, 3 H), 2.48ꢀ
2.41 (m, 9 H), 2.00ꢀ1.96 (m, 2 H); 1.46 (qd, J = 13.5,
4.0 Hz, 2 H), 0.98 (d, J = 6.5 Hz, 6 H); 13C NMR
(125 MHz, acetoneꢀd6) δ 157.4, 155.6, 153.0, 131.8,
130.3, 130.2, 125.1, 123.9, 121.8, 117.4, 116.1,
112.9, 111.8, 102.2, 59.0, 55.6, 54.9, 54.6, 49.4,
48.6, 44.4, 32.3, 18.8; HRMS (ESI+) m/z calcd for
C28H39N4O 447.3118 (M+H), found 447.3118 .
benzo[d]imidazole (48):
A
mixture of 3ꢀ
bromobenzaldehyde (0.216 g, 2.00 mmol), oꢀ
phenylenediamine (0.388, 2.10 mmol), boric acid
(0.006 g, 0.09 mmol) and glycerin (1 drop) in H2O

Page 21 of 24
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DOI: 10.1039/C7OB00526A
(3 mL) was heated at 80 °C until disappearance of H), 7.21 (t, J = 7.5 Hz, 2 H), 7.16 (app t, J = 3.5 Hz,
starting material. The water was decanted off and 4 H), 7.10 (d, J = 7.5 Hz, 1 H), 7.04ꢀ6.97 (m, 4 H),
MeOH (ca 10 mL) was added. The mixture was 6.82 (d, J = 8.5 Hz, 1 H), 4.10 (br s, 1 H), 3.57 (d, J
stirred for 2 h at room temperature. The formed = 11.5 Hz, 2 H), 3.28ꢀ3.20 (m, 2 H), 2.67ꢀ2.47 (m,
precipitate was collected by filtration and washed 13 H), 1.69 (br s, 5 H), 1.49 (s, 9 H), 1.10 (d, J = 6.5
with cold MeOH to afford 2ꢀ(3ꢀbromophenyl)ꢀ1Hꢀ Hz, 6 H); HRMS (ESI+) m/z calcd for C45H57N6O2
benzo[d]imidazole31 as a white solid (0.32 g, 1.2 713.4538 (M+H), found 713.4531.
mmol, 59%): Mp. 264ꢀ265 °C; IR (ATR) 3038,
A solution of tertꢀbutyl (1ꢀ(3ꢀ(1ꢀbenzhydrylꢀ1Hꢀ
2957, 2799, 1562, 1436, 1398, 1357, 742, 727 cmꢀ1; benzo[d]imidazolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀ
1H NMR (500 MHz, DMSOꢀd6) δ 13.04 (s, 1 H), isopropylꢀpiperazinꢀ1ꢀyl)ethyl)carbamate (0.089 g,
8.37 (t, J = 2.0 Hz, 1 H), 8.20ꢀ8.18 (m, 1 H), 7.69 0.13 mmol) in CH2Cl2 (2 mL) was treated with
(ddd, J = 8.0, 1.0, 1.0 Hz, 2 H), 7.56ꢀ7.50 (m, 2 H), trifluoroacetic acid (2.0 mL) followed by
7.27ꢀ7.19 (m, 2 H); 13C NMR (125 MHz, DMSOꢀd6) triethylsilane (0.40 mL, 2.5 mmol). The reaction was
δ 150.1, 144.1, 135.5, 132.9, 132.8, 131.6, 129.3, stirred at room temperature overnight. The solution
125.8, 123.4, 122.7, 122.4, 119.5, 112.0; HRMS was then heated at 70 °C overnight, concentrated,
(ESI+) m/z calcd for C13H10N2Br 273.0027 (M+H), diluted with sat. NaHCO3 and extracted with EtOAc
found 273.0023. To
a
solution of 2ꢀ(3ꢀ (3x). The combined organic layer was washed with
bromophenyl)ꢀ1Hꢀbenzo[d]imidazole (0.273 g, 0.99 brine, dried (Na2SO4) and concentrated. The crude
mmol) in anhydrous THF (8 mL) was added NaH residue was purified by chromatography on SiO2 (10
(0.048 g, 1.2 mmol, 60% dispersion). The reaction to 25% MeOH/CH2Cl2) to afford a pale yellow foam
mixture was stirred at room temperature for 20 min which was filtered through basic Al2O3 (0 to 10%
then diphenylmethyl chloride (0.41 g, 1.9 mmol) MeOH/CH2Cl2) to afford 24 as a pale yellow foam
was added. The mixture was stirred at room (UPCDC30250, 0.023 g, 0.051 mmol, 41%): IR
temperature for 30 min and then heated to 70 °C. (ATR) 3069, 2938, 2912, 2811, 1600, 1450, 1357,
1
After 24 h, the reaction mixture was cooled to room 1176, 1115, 776, 736 cmꢀ1; H NMR (500 MHz,
temperature, diluted with sat. NaHCO3, extracted CD3OD) δ 7.74 (t, J = 2.0 Hz, 1 H), 7.61 (dd, J = 6.0,
with EtOAc, dried (Na2SO4), and concentrated. The 3.5 Hz, 2 H), 7.53 (d, J = 7.5 Hz, 1 H), 7.38 (t, J =
crude residue was purified by chromatography on 8.0 Hz, 1 H), 7.27 (dd, J = 6.0, 3.5 Hz, 2 H), 7.11
SiO2 (10 to 20% EtOAc/hexanes) to afford 48 as a (dd, J = 8.5, 2.5 Hz, 1 H), 3.85 (d, J = 12.5 Hz, 2 H),
1
white foam (0.50 g, 1.1 mmol, 89%): H NMR (500 2.84 (t, J = 12.0 Hz, 2 H), 2.77 (t, J = 7.0 Hz, 2 H),
MHz, CDCl3) δ 7.85ꢀ7.82 (m, 2 H), 7.66ꢀ7.64 (m, 1 2.68ꢀ2.51 (m, 11 H), 2.02 (d, J = 11.5 Hz, 2 H), 1.53
H), 7.51 (d, J = 7.5 Hz, 1 H), 7.36 (app q, J = 3.5 Hz, (qd, J = 12.0, 3.5 Hz, 2 H), 1.09 (d, J = 6.5 Hz, 6 H);
7 H), 7.33 (t, J = 8.0 Hz, 2 H), 7.27ꢀ7.24 (m, 2 H), 13C NMR (125 MHz, CD3OD, several signals
7.18ꢀ7.15 (m, 5 H), 7.06ꢀ7.03 (m, 1 H), 6.96 (s, 1 H), missing) δ 152.5, 151.9, 130.2, 129.4, 122.5, 118.1,
6.82 (d, J = 8.5 Hz, 1 H).
117.1, 114.2, 57.0, 54.8, 54.5, 52.7, 48.2, 48.1, 42.4,
31.3, 17.3; HRMS (ESI+) m/z calcd for C27H39N6
447.3231 (M+H), found 447.3230.
1ꢀ(3ꢀ(1HꢀBenzo[d]imidazolꢀ2ꢀyl)phenyl)ꢀNꢀ(2ꢀ
(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)piperidinꢀ4ꢀ
amine (24, UPCDC30250): A suspension of A (87
2ꢀ(3ꢀBromophenyl)ꢀ1ꢀmethylꢀ1Hꢀindole
(49):
mg, 0.24 mmol), 1ꢀbenzhydrylꢀ2ꢀ(3ꢀbromophenyl)ꢀ To a solution of 2ꢀ(3ꢀbromophenyl)ꢀ1Hꢀindole11
1Hꢀbenzo[d]imidazole (48, 90 mg, 0.21 mmol) and (0.499 g, 1.83 mmol) in DMF (9 mL) was added
K3PO4 (67 mg, 0.31 mmol) in dry degassed dioxane NaH (0.110 g, 2.75 mmol, 60% dispersion) at 0 °C.
(1.5 mL) was degassed for 20 min with argon. To The reaction mixture was warmed to room
this mixture was added Pd2(dba)3 (4 mg, 0.004 temperature. After 45 min, the reaction mixture was
mmol) and CyJohnPhos (6 mg, 0.02 mmol). The cooled to 0 °C and treated with iodomethane (0.12
reaction vial was sealed and the mixture was heated mL, 1.9 mmol). The reaction mixture was warmed
at 110 °C for 12 h. The reaction mixture was diluted to room temperature and stirred overnight. The
with sat. NaHCO3 and extracted with EtOAc (3x). reaction mixture was diluted with H2O and extracted
The combined organic layer was washed with brine, with EtOAc (3x). The combined organic layer was
dried (Na2SO4), concentrated and purified by washed with brine, dried (Na2SO4), filtered and
chromatography
on
SiO2
(10
to
20% concentrated. The residue was purified by
MeOH/CH2Cl2) to afford tertꢀbutyl (1ꢀ(3ꢀ(1ꢀ chromatography on SiO2 (40% hexanes/CH2Cl2) to
benzhydrylꢀ1Hꢀbenzo[d]imidazolꢀ2ꢀ
give 49 (0.52 g, 1.8 mmol, 99%) as a white solid: 1H
yl)phenyl)piperidinꢀ4ꢀyl)(2ꢀ(4ꢀisopropylꢀpiperazinꢀ
1ꢀyl)ethyl)carbamate as a pale yellow solid (89 mg,
0.12 mmol, 61%): H NMR (500 MHz, CDCl3) δ
NMR (400 MHz, CDCl3)
δ 7.68 (s, 1 H), 7.65 (d, J
= 8.0 Hz, 1 H), 7.54 (d, J = 8.0 Hz, 1 H), 7.44 (d, J
= 8.0 Hz, 1 H), 7.38 (d, J = 8.8 Hz, 1 H), 7.33 (d, J
= 8.0 Hz, 1 H), 7.28 (d, J = 7.6 Hz, 1 H), 7.16 (t, J
1
7.82 (d, J = 8.0 Hz, 1 H), 7.32 (app t, J = 3.5 Hz, 6

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DOI: 10.1039/C7OB00526A
= 7.6 Hz, 1 H), 6.59 (s, 1 H), 3.75 (s, 3 H); HRMS 2.86 (td, J = 12.4, 2.8 Hz, 2 H), 2.71 (t, J = 6.0 Hz, 2
(ESI+) m/z calcd for C15H13NBr 286.0226 (M+H), H), 2.69ꢀ2.62 (m, 2 H), 2.61ꢀ2.39 (m, 10 H), 1.96ꢀ
found 286.0225.
Nꢀ(2ꢀ(4ꢀIsopropylpiperazinꢀ1ꢀyl)ethyl)ꢀ1ꢀ(3ꢀ(1ꢀ
methylꢀ1Hꢀindolꢀ2ꢀyl)phenyl)piperidinꢀ4ꢀamine
1.92 (m, 2 H), 1.45 (qd, J = 13.5, 4.0 Hz, 2 H), 0.98
(d, J = 6.5 Hz, 6 H); 13C NMR (100 MHz, acetoneꢀ
d6) δ 152.6, 143.0, 139.3, 134.3, 129.9, 129.0, 122.1,
(25, UPCDC30201): A suspension of A (105 mg, 120.9, 120.3, 120.2, 117.6, 116.4, 110.5, 101.8, 59.0,
0.297 mmol), 2ꢀ(3ꢀbromophenyl)ꢀ1ꢀmethylꢀ1Hꢀ 55.6, 54.9, 54.6, 49.4, 48.6, 44.4, 33.2, 31.5, 18.8;
indole (49, 71 mg, 0.25 mmol) and K3PO4 (81 mg, HRMS (ESI+) m/z calcd for C29H42N5 460.3440
0.37 mmol) in dry degassed dioxane (2.0 mL) was (M+H), found 460.3443.
treated with Pd2(dba)3 (5 mg, 0.005 mmol) and
Acknowledgements. This project was funded in whole or
in part with federal funds from the National Cancer
Institute, National Institutes of Health, under Chemical
Biology Consortium Contract No. HHSN261200800001E
Agreement No. 29XS127TO15. The authors thank Mr.
Yongzhao Yan and Dr. Zhizhou Yue for synthetic
contributions and Ms. Mary Liang and Taber Lewis for
analytical and administrative support. The authors also
greatly appreciate the stimulating discussions and valuable
suggestions from all members of the CBC p97 team,
particularly, Drs. Eric Baldwin (Leidos Biomedical
Research), TsuiꢀFen Chou (UCLA), Raymond Deshaies
(Caltech), Andrew Flint (Leidos Biomedical Research),
Gunda Georg (U. Minnesota), Neal Green (Leidos
Biomedical Research), William J. Moore (Leidos
Biomedical Research), Barbara Mroczkowski (NIH/NCI),
R. Jeffrey Neitz (UCSF), Shizuko Sei (Leidos Biomedical
Research), Gordon Stott (Leidos Biomedical Research),
Sriram Subramaniam (NIH/NCI), and Michael Walters (U.
Minnesota).
CyJohnPhos (7 mg, 0.02 mmol). The reaction vial
was sealed and the mixture was heated at 110 °C for
11 h under microwave irradiation conditions. The
reaction mixture was diluted with sat. NaHCO3 and
extracted with EtOAc (3x). The combined organic
layers were washed with brine, dried (Na2SO4),
concentrated and purified by chromatography on
SiO2 (10 to 20%, MeOH/CH2Cl2) to afford tertꢀ
butyl(2ꢀ(4ꢀisopropylpiperazinꢀ1ꢀyl)ethyl)(1ꢀ(3ꢀ(1ꢀ
methylꢀ1Hꢀindolꢀ2ꢀyl)phenyl)ꢀpiperidinꢀ4ꢀ
yl)carbamate as a pale yellow oil (100 mg, 0.17
mmol, 72%): 1H NMR (500 MHz, CDCl3) δ 7.63 (d,
J = 7.5 Hz, 1 H), 7.36ꢀ7.32 (m, 2 H), 7.26ꢀ7.22 (m, 1
H), 7.15ꢀ7.12 (m, 1 H), 7.05 (d, J = 1.5 Hz, 1 H),
6.96 (d, J = 8.5 Hz, 2 H), 6.55 (s, 1 H), 4.15 (br s, 1
H), 3.81 (d, J = 12.5 Hz, 2 H), 3.75 (s, 3 H), 3.25 (br
s, 2 H), 2.84ꢀ2.82 (m, 2 H), 2.71ꢀ2.48 (m, 11 H),
1.85 (br s, 4 H), 1.48 (s, 9 H), 1.06 (d, J = 6.5 Hz, 6
H); 13C NMR (125 MHz, CDCl3) δ 155.4, 151.2,
142.1, 138.3, 133.7, 129.2, 128.0, 121.6, 120.5,
120.4, 119.8, 117.6, 116.0, 109.6, 101.4, 79.8, 58.5,
54.6, 53.8, 53.1, 49.5, 48.6, 40.0, 31.2, 30.1, 28.6,
18.6; HRMS (ESI+) m/z calcd for C34H50O2N5
560.3965 (M+H), found 560.3959.
Author contributions. R.G., D.M.H. and P.W. devised
the experiments, J.C.B., L.P.S., M.K., R.C., C.F.M.,
A.R.H, B.D.P., S.L.B., C.L., M.G.L. and M.R.A.
conducted the theoretical and experimental investigations,
and everybody interpreted results and contributed to the
preparation of parts of this manuscript.
A solution of trifluoroacetic acid (1.5 mL) and
triethylsilane (0.15 mL, 0.93 mmol) in CH2Cl2 (1.5
mL) was added to a solution of tertꢀbutyl(2ꢀ(4ꢀ
isopropylpiperazinꢀ1ꢀyl)ethyl)(1ꢀ(3ꢀ(1ꢀmethylꢀ1Hꢀ
indolꢀ2ꢀyl)phenyl)ꢀpiperidinꢀ4ꢀyl)carbamate (0.10 g,
0.18 mmol) in CH2Cl2 (1.5 mL). The reaction
mixture was stirred at room temperature for 1 h,
concentrated, diluted with sat. NaHCO3 and
extracted with EtOAc (3x). The combined organic
layers were washed with brine, dried (Na2SO4) and
concentrated. The crude residue was purified by
chromatography on SiO2 (8 to 20% CH2Cl2/MeOH
with 1% TEA) followed by filtration through basic
Al2O3 (0 to 10% MeOH/CH2Cl2) to afford 25 as a
pale yellow solid (UPCDC30201, 0.061 g, 0.13
mmol, 74%): IR (ATR) 2981, 2808, 1596, 1462,
Competing financial interests. The authors declare no
competing financial interests.
Additional information. Supporting Information,
including copies of 1H and 13C NMR spectra, reprints and
permissions information is available at www.xxx.xxx. The
authors declare no competing financial interests.
Correspondence and requests for materials should be
addressed to P.W. (pwipf@pitt.edu).
Notes and References
1. Xia, D.; Tang, W. K.; Ye, Y. Gene 2016, 583, 64ꢀ77.
2. Li, G.; Huang, C.; Zhao, G.; Lennarz, W. J. Proc. Nat.
Acad. Sci. USA, 2012, 109, 3737ꢀ3741.
3. Banerjee, S.; Bartesaghi, A.; Merk, A.; Rao, P.; Bulfer,
S. L.; Yan, Y.; Green, N.; Mroczkowski, B.; Neitz, R.
J.; Wipf, P.; Falconieri, V.; Deshaies, R. J.; Milne, J. L.
S.; Huryn, D.; Arkin, M.; Subramaniam, S. Science
2016, 351, 871ꢀ875.
1
1339, 1178, 1145, 984, 776 cmꢀ1; H NMR (400
MHz, acetoneꢀd6) δ 7.56 (d, J = 7.6 Hz, 1 H), 7.40
(dd, J = 8.4, 0.8 Hz, 1 H), 7.34 (t, J = 8.0 Hz, 1 H),
7.19 (td, J = 8.0, 0.8 Hz, 1 H), 7.11ꢀ7.07 (m, 2 H),
7.03 (dd, J = 8.0, 2.0 Hz, 1 H), 6.94 (d, J = 7.6 Hz, 1
H), 6.52 (d, J = 0.8 Hz, 1 H), 3.75ꢀ3.71 (m, 5 H),
4. Zhou, H.ꢀJ.; Wang, J.; Yao, B.; Wong, S.; Djakovic,
S.; Kumar, B.; Rice, J.; Valle, E.; Soriano, F.; Menon,

Page 23 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB00526A
M.ꢀK.; Madriaga, A.; Kiss von Soly, S.; Kumar, A.;
Parlati, F.; Yakes, F. M.; Shawver, L.; Le Moigne, R.;
Anderson, D. J.; Rolfe, M.; Wustrow, D. J. Med. 21. Good, J. A. D.; Wang, F.; Rath, O.; Kaan, H. Y. K.;
Workman, P.; Garrett, M. D.; Collins, I. J. Med. Chem.
2008, 51, 2147ꢀ2157.
Chem. 2015, 58, 9480ꢀ9497.
Talapatra, S. K.; Podgórski, D.; MacKay, S. P.;
5. Halawani, D.; Tessier, S.; Anzellotti, D.; Bennett, D.
Kozielski, F. J. Med. Chem. 2013, 56, 1878ꢀ1893.
A.; Latterich, M.; Leblanc, A. C. J. Neurosci. 2010, 30, 22. Tapia, I.; AlonsoꢀCires, L.; LópezꢀTudanca, P. L.;
6132ꢀ6142.
Mosquera, R.; Labeaga, L.; Innerárity, A.; Orjales, A.
J. Med. Chem. 1999, 42, 2870ꢀ2880.
23. BuuꢀHoï, N. P.; Hoán, N.; Khôi, N. H.; Xuong, N. D.
J. Org. Chem. 1951, 16, 309ꢀ314.
6. Chapman, E.; Maksim, N.; De La Cruz, F.; La Clair, J.
J. Molecules 2015, 20, 3027ꢀ3049.
7. Alverez, C.; Bulfer, S. L.; Chakrasali, R.; Chimenti, M.
S.; Deshaies, R. J.; Green, N.; Kelly, M.; LaPorte, M. 24. Malamas, M. S.; Barnes, K.; Johnson, M.; Hui, Y.;
G.; Lewis, T. S.; Liang, M.; Moore, W. J.; Neitz, R. J.;
Peshkov, V. A.; Walters, M. A.; Zhang, F.; Arkin, M.
R.; Wipf, P.; Huryn, D. M. ACS Med. Chem. Lett.
2016, 7, 182ꢀ187.
Zhou, P.; Turner, J.; Hu, Y.; Wagner, E.; Fan, K.;
Chopra, R.; Olland, A.; Bard, J.; Pangalos, M.;
Reinhart, P.; Robichaud, A. J. Bioorg. Med. Chem.
2010, 18, 630ꢀ639.
8. Gui, L.; Zhang, X.; Li, K.; Frankowski, K. J.; Li, S.; 25. Livecchi, M.; Calvet, G.; Schmidt, F. J. Org. Chem.
Wong, D. E.; Moen, D. R.; Porubsky, P. R.; Lin, H. J.; 2012, 77, 5006ꢀ5016.
Schoenen, F. J.; Chou, T.ꢀF. ChemMedChem 2016, 11, 26. Calvet, G.; Livecchi, M.; Schmidt, F. J. Org. Chem.
953ꢀ957. 2011, 76, 4734ꢀ4740.
9. Tillotson, J.; Bashyal, B. P.; Kang, M.; Shi, T.; De La 27. Dyke, H. J.; Gazzard, L. J.; Williams, K. 1,7ꢀ
Cruz, F.; Gunatilaka, A. A. L.; Chapman, E. Org.
Biomol. Chem. 2016, 14, 5918ꢀ5921.
10. Her, N. ꢀG.; Toth, J. I.; Ma, C. ꢀT.; Wei, Y.;
Motamedchaboki, K.; Sergienko, E.; Petroski, M. D.
Cell Chem. Biol. 2016, 23, 517ꢀ528.
Diazacarbazoles as chk1 inhibitors, their preparation,
pharmaceutical compositions, and use as anticancer
agents and their use in the treatment of cancer. 2011,
WO2011/073263.
28. Hopkins, C. R.; Collar, N. Tetrahedron Lett. 2004, 45,
8631ꢀ8633.
11. Alverez, C.; Arkin, M. R.; Bulfer, S. L.; Colombo, R.;
Kovaliov, M.; LaPorte, M. G.; Lim, C.; Liang, M.; 29. Wagner, A. M.; Hickman, A. J.; Sanford, M. S. J. Am.
Moore, W. J.; Neitz, R. J.; Yan, Y.; Yue, Z.; Huryn, D. Chem. Soc. 2013, 135, 15710ꢀ15713.
M.; Wipf, P. ACS Med. Chem. Lett. 2015, 6, 1225ꢀ 30. Ruan, L.; Shi, M.; Mao, S.; Yu, L.; Yang, F.; Tang, J.
1230. Tetrahedron 2014, 70, 1065ꢀ1070.
12. Chou, T. ꢀF.; Bulfer, S. L.; Weihl, C. C.; Li, K.; Lis, L. 31. Mukhopadhyay, C.; Tapaswi, P. K.; Butcher, R. J.
G.; Walters, M. A.; Schoenen, F. J.; Lin, H. J.;
Deshaies, R. J.; Arkin, M. R. J. Mol. Biol. 2014, 426,
2886ꢀ2899.
Aust. J. Chem. 2009, 62, 140ꢀ144.
13. See SI for graphs.
14. Davies, J. M.; Brunger, A. T.; Weis, W. I. Structure
2008, 16, 715ꢀ726.
15
.
Hänzelmann, P.; Schindelin, H. Structure 2016, 24,
127ꢀ139.
16. Burnett, J. C.; Ruthel, G.; Stegmann, C. M.; Panchal,
R. G.; Nguyen, T. L.; Hermone, A. R.; Stafford, R. G.;
Lane, D. J.; Kenny, T. A.; McGrath, C. F.; Wipf, P.;
Stahl, A. M.; Schmidt, J. J.; Gussio, R.; Brunger, A.
T.; Bavari, S. J. Biol. Chem. 2007, 282, 5004ꢀ5014.
17. Nguyen, T. L.; McGrath, C.; Hermone, A. R.; Burnett,
J. C.; Zaharevitz, D. W.; Day, B. W.; Wipf, P.; Hamel,
E.; Gussio, R. J. Med. Chem. 2005, 48, 6107ꢀ6116.
18. Donald, A.; McHardy, T.; Rowlands, M. G.; Hunter,
L.ꢀJ. K.; Davies, T. G.; Berdini, V.; Boyle, R. G.;
Aherne, G. W.; Garrett, M. D.; Collins, I. J. Med.
Chem. 2007, 50, 2289ꢀ2292.
19. McHardy, T.; Caldwell, J. J.; Cheung, K. ꢀM.; Hunter,
L. J.; Taylor, K.; Rowlands, M.; Ruddle, R.; Henley,
A.; de Haven Brandon, A.; Valenti, M.; Davies, T. G.;
Fazal, L.; Seavers, L.; Raynaud, F. I.; Eccles, S. A.;
Aherne, G. W.; Garrett, M. D.; Collins, I. J. Med.
Chem. 2010, 53, 2239ꢀ2249.
20. Caldwell, J. J.; Davies, T. G.; Donald, A.; McHardy,
T.; Rowlands, M. G.; Aherne, G. W.; Hunter, L. K.;
Taylor, K.; Ruddle, R.; Raynaud, F. I.; Verdonk, M.;

Organic & Biomolecular Chemistry
Page 24 of 24
View Article Online
DOI: 10.1039/C7OB00526A
A Threonine Turnstile Defines a Dynamic Amphiphilic
Binding Motif in the AAA ATPase p97 Allosteric Binding Site
James C. Burnett,§ Chaemin Lim,‡ Brian D. Peyser,& Lalith P. Samankumara,‡ Marina Kovaliov,‡
Raffaele Colombo,‡ Stacie L. Bulfer,∥ Matthew G. LaPorte,‡ Ann R. Hermone,§ Connor F.
McGrath,§ Michelle R. Arkin,∥ Rick Gussio,*,& Donna M. Huryn,*,‡,† and Peter Wipf*,‡,†
§Leidos Biomedical Research, Inc., P.O. Box B, Frederick, MD 21702, United States
‡University of Pittsburgh Chemical Diversity Center, University of Pittsburgh, Pittsburgh, PA 15260,
United States
&Frederick National Laboratory for Cancer Research, Developmental Therapeutics Program, P.O. Box B,
Frederick, MD 21702, United States
∥Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, University of California,
San Francisco, CA 94158, United States
†Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, United States
TOC. The turnstile motion of two neighboring threonines accommodates both polar and apolar ligands in
an allosteric binding site.