Optimization of Phenyl Indole Inhibitors of the AAA+ ATPase p97

Article abstract of DOI:10.1021/acsmedchemlett.8b00372

Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.

Full text of DOI:10.1021/acsmedchemlett.8b00372

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Letter
Optimization of Phenyl Indole Inhibitors of the AAA ATPase p97
Matthew G. LaPorte, James C Burnett, Raffaele Colombo, Stacie L. Bulfer,
Celeste Alverez, Tsui-Fen Chou, R. Jeffrey Neitz, Neal Green, William J. Moore,
Zhizhou Yue, Shan Li, Michelle R. Arkin, Peter Wipf, and Donna M. Huryn
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00372 • Publication Date (Web): 18 Sep 2018
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Optimization of Phenyl Indole Inhibitors of the AAA+
ATPase p97
Matthew G. LaPorte,‡ James C. Burnett,‡ Ω Raffaele Colombo,‡ Stacie Bulfer,§ Celeste Alverez,‡† Tsui-
Fen Chou,¶ R. Jeffrey Neitz,§ Neal Green,& William J. Moore,& Zhizhou Yue,‡ Shan Li,¶ Michelle R.
Arkin,§* Peter Wipf, ‡† * Donna M. Huryn‡†*
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‡
University of Pittsburgh Chemical Diversity Center, University of Pittsburgh, Pittsburgh, PA 15260; Ω Computational Drug
Development Group, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
§
Institute, Bethesda, MD 20892; Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, University of
†
California, San Francisco, CA 94158; Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA
15260; ¶ Division of Medical Genetics, Department of Pediatrics, Harbor–UCLA Medical Center and Los Angeles Biomedi-
&
cal Research Institute, Torrance, CA, USA; Leidos Biomedical Research, Inc., Frederick, MD 21702 United States.
KEYWORDS: AAA+ ATPase, p97, allosteric inhibitor, protein homeostasis modulator, anti-cancer, phenyl indole
ABSTRACT: Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for
inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a bio-
chemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently
obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.
The AAA+ (ATPase associated with various cellular activities)
p97 is a hexameric, multidomain protein that plays a key role in
protein homeostasis. Energy from the hydrolysis of ATP is me-
chanically relayed by p97 for the extraction of polypeptides from
membranes, the ribosome or DNA, facilitating polypeptide deg-
radation by the proteasome. In addition, p97 is hypothesized to
play a role in other protein degradation pathways, including au-
tophagy.1-2 Its essential role in protein homeostasis and the clinical
success of proteasome inhibitors suggests the potential for target-
ing certain cancers with p97 inhibitors. Towards this end, a
number of small molecule inhibitors of p97 have been reported to
show promising anti-proliferative effects.3-10 One ATP-
competitive active site inhibitor, CB-5083, advanced to Phase I
clinical trials for multiple myeloma and advanced solid tumors,
but was recently discontinued due to off-target retinal toxicity
related to inhibition of PDE6.10-12
ing mechanism.15 Properties (MW, CLogP, tPSA; ChemDraw) of
1 were all in the drug-like range.
For further optimization, we used an approach that kept the 2-
(3-(piperidin-1-yl)phenyl)-1H-indole scaffold constant and varied
the amine side-chain (Scheme 1). Our synthetic strategy, which
focused on late-stage diversification, relied on previously opti-
mized Buchwald-Hartwig couplings of 3-bromo-2-phenyl indoles
(2).13-14,16-18 The intermediate, 3, was used for rapid further diver-
sification via reductive aminations19 to afford, in some cases after
additional modifications, final products of general structure 4. An
alternative strategy relied on direct coupling of an intact side-
chain (or a related analog) with 2 to generate 4.
The first rounds of SAR were generated from the unsubstituted
2-phenyl indole core and explored a wide array of side-chain
amines that varied the terminal group as well as the linker to the
piperidine. Replacement of the terminal triazole in 1 with a phe-
nyl group, resulting in analog 5, led to a loss of potency, indicating
the requirement for a polar group at this position. Replacement
with a pyridine (6) restored activity to near original levels.
As part of our efforts to identify small molecule allosteric inhibi-
tors of p979,13-14 we embarked on a medicinal chemistry program
to optimize a 2-phenyl indole scaffold discovered via a high-
throughput screening (HTS) effort.15 At the time our work was
conducted, no structural information on the compound’s binding
site was available, although it was determined to bind to an allo-
steric site.15 A structure activity relationship (SAR) study illustrat-
ed the importance of the indole moiety for potency.13-14 In this
report, we disclose additional SAR data and the optimization of
the flexible side-chain, as well as biological characterization of key
analogs.
N
H
N
Previous study
HN
N
N
1
N
MW = 400.53
CLogP = 2.2
tPSA = 55 Å2
Compound 1 (Figure 1 and Table 1) is representative of early
active compounds in the phenyl indole series. Compound 1 inhib-
ited the ATPase activity of p97 with an IC50=2.4 µM at low ATP
concentration (20 µM). At higher ATP concentrations (100 µM),
the IC50 improved to 0.99 µM, indicating an uncompetitive bind-
This study
Figure 1. First-generation phenyl indole inhibitor 1 and areas of focused
SAR explorations.
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O
O
spiro analog 20; piperidine-piperazine 21; and fused bicy-
clo[3.3.0] ring system 22) was not productive. These compounds
inhibited p97 in the micromolar range.
HN
H , F
1
2
3
4
5
6
7
8
1) Pd2(dba)3 (cat.),
CyJohnPhos (cat.)
K3PO4, dioxane,
110 oC, 6-9 h
N
H
N
3
(47-64%, 2 steps)
As an alternate approach to conformational constraint, we in-
corporated small cyclic moieties as “kinks” into the flexible linker.
This strategy led to the some of the most potent analogs in this
series. Cyclobutane 23 inhibited p97 with an IC50=20 nM, i.e.
over ten times more potent than the parent analog 9. Based on
SAR trends, this difference could not be attributed solely to the
presence of the 5-F indole in 23. Unlike the flexible linker series
(e.g. 9-11), within this subseries, the N-Me piperazine terminus
(23) was 5-fold more potent than the N-iPr analog (24). The ox-
etane analog 25 (IC50=90 nM) was similar in potency to the cy-
clobutane 24.
2) Acetone, HCl,
reflux, 2.5-3.5 h
H , F
O
reductive
amination
N
H
H , F
Buchwald-Hartwig
coupling
Br
2
N
H
N
4
N
R1
R2
9
Scheme 1. General synthesis of phenyl indoles using late-stage diversifi-
cation strategies.
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The length of the linker appeared to be insignificant, as the 1-
carbon linker (7) possessed equivalent potency to the 2-carbon
analog in this series. Replacement of the aromatic heterocycle
with a piperazine group provided a first significant (~3-fold) im-
provement in potency (8; IC50=330 nM) compared to compound
1.20 Further functionalization by capping the terminal nitrogen
with an aliphatic substituent (e.g. Me, Et, iPr, tBu; 9-12), provided
another boost in potency. In these examples, the general trend
was that greater lipophilicity seemed to correlate with improved
potency, with iPr exhibiting 160 nM potency. In contrast to com-
pounds 6 and 7, the length of the linker between the piperidine
and the terminal moiety modestly affected potency: a 3-carbon
linker (13) was ~2-fold less potent than its 2-carbon homolog 11.
Combining the preferred 5-F indole14,16 with the N-iPr pipera-
zine afforded 14, the first analog with potency below 100 nM
(IC50=70 nM). Previous SAR data13 indicated that the indole
moiety was highly sensitive to modification, and a cryo-EM co-
structure16 and extensive molecular modeling14 showed the indole
deeply embedded in an allosteric binding site in p97’s D2 domain,
located very near the D1 - D2 interface, with the fluorine atom
engaging in key interactions with surrounding residues.14,16 Given
the importance of the 5-fluoroindole to binding and potency, our
next iterations maintained this feature and explored additional
amine groups that contained a N- iPr moiety. (see SI)
To develop structure-based hypotheses to rationalize the bio-
chemical activity of this series, we utilized a recently reported
cryo-EM co-structure of 14 bound to p97; however, while strong
electron-density for the phenyl-indole component of the inhibitor
was observed in this structure, there was no density detected for
the conformationally flexible side-chain.16 As shown in Figure 2a,
the side-chain in the cryo-EM model was positioned extending out
of the allosteric binding cleft and directly into solvent, making no
contacts with the protein.16 This binding mode was inconsistent
with the side-chain SAR, which indicated a preference for specific,
basic groups at this position (e.g., piperazine preferred over pyri-
dine and phenyl, as in 8 vs. 6, 5), as well as a preference for lipo-
philic groups at the piperazine terminus (e.g., ethyl, iPr, tBu: 10-12
vs. H, Me: 8, 9). We therefore developed a binding model that is
consistent with the available SAR (Figure 2b). Placing the piperi-
dine ring in a chair conformation (vs the twist chair conformation
in the cryo-EM co-structure) allows the basic side-chain of 14 to
be oriented toward the protein and to engage in multiple hydro-
gen bonds, instead of extending out into solvent. Favorable inter-
actions develop between: 1) the terminal piperazine nitrogen atom
and the Glu534 carboxylate, 2) the internal piperazine nitrogen
atom and the side-chain amide carbonyl of Gln494, 3) the sec-
ondary amine and Glu498, and 4) the piperidine ring nitrogen
atom (an aniline) and the thiol of Cys535 (an interaction also ob-
served in the twist chair conformation). Using this refined model,
we were able to rationalize much of the SAR, particularly that of
the side-chain.
The terminal amine in the piperazine moiety contributed to po-
tency, as the truncated dimethyl amino analog 15, while main-
taining some activity (IC50=800 nM), was >10-fold less potent
than 14. Modulation of the piperazine nitrogen’s basicity by in-
troducing a carbonyl group into a 3-carbon linker as in 16 was
tolerated, but decreased potency compared to 14. While 16
(IC50=190 nM) was slightly more potent than 13 (IC50=380 nM),
we attributed this difference to the presence of the potency-
enhancing 5-F-indole moiety rather than a preference for the
modified linker. We concluded that the length of the linker was
not critical to potency, and that the pKa of the internal piperazine
nitrogen could be manipulated without detriment to biological
activity. Taking this information into account, we attempted to
simultaneously constrain the conformation of the flexible side-
chain and reduce the number of basic nitrogen atoms. This led to
the design of analogs such as 17, containing an amine-linked bis-
piperidine side-chain. This compound exhibited strong inhibition
of p97 (IC50=50 nM) and was somewhat more potent than com-
pounds containing a flexible linker, such as 14. While we had no
data to suggest specific liabilities, the elimination of a basic nitro-
gen was anticipated to address potential liabilities such as hERG
receptor binding and efflux. To further explore this truncated
scaffold, we replaced the internal piperidine with cis- and trans-
cyclohexane groups (see SI). The trans-analog 18 retained activity
towards p97, albeit ~3-4 fold less than the piperidine 17. In con-
trast, the cis-isomer 19 was >20-fold less potent compared to the
trans-isomer and >100-fold less potent than 17, indicating a signif-
icant conformational preference. Further shortening and con-
straining the side-chain by eliminating the nitrogen linkage (e.g.,
Figure 3 shows the proposed binding modes of trans- and cis-
cyclohexanes 18 and 19. The trans-orientation in 18 results in a
favorable binding mode for the side-chain component; hydrogen
bonds with acidic residues Glu498 and Glu534 are maintained,
and the cyclohexyl component engages in hydrophobic contacts
with side-chains of Val493 and Ile531. Additionally, the iPr group
orients away from the solvent toward the aromatic indole side-
chain of residue Trp476. In contrast, binding of the cis-isomer 19
results in a significantly more solvent-exposed side-chain orienta-
tion that forces the cyclohexyl and iPr groups toward the solvent,
an energetically unfavorable pose. Moreover, the secondary nitro-
gen atom engages in unfavorable hydrophobic-polar clashes with
the side-chains of residues Val493 and Ile531. Finally, the con-
strained orientation of the cis-isomer results in the loss of a hydro-
gen bond with Glu498. The increased potency of 23 compared to
compounds resembling 14 can be rationalized by the binding pose
in Figure 4, and the observation that the kinked side-chain is em-
bedded more deeply into the p97 helix 16-17 interface than the
straight-chain analogs, and more fully occupies a hydrophobic
pocket formed by Val493, Ile531 (Figure 4), and, to a lesser de-
gree, Leu 527. We hypothesize that desolvation of the cyclobutyl
group drives this deeper binding mode. Interestingly, the more
buried binding mode of the side-chain of 23 results in the loss of
the H-bond with Glu498 predicted for 14 and a weakening of the
H-bond with Glu534 that is observed for 14 (Figure 2b).
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R2
1
2
3
4
R1
N
H
Table 1. p97 Inhibitory activity of phenyl indole
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p97 IC50
(µM) (n)
p97 IC50
(µM) (n)
Compound
1
R1
H
R2
Compound
15
R1
F
R2
H
N
H
N
0.80±0.34
(5)
0.99± 0.56
(44)
N
N
N
N
N
N
N
9
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
39
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H
N
0.19±0.15
(4)
N
2.6± 0.66
(2)
H
N
16
17
18
19
20
21
22
23
24
25
F
F
F
F
F
F
F
F
F
F
N
5
6
7
H
H
H
O
N
H
N
H
N
0.05±0.04
(12)
1.05±1.2
(4)
N
N
N
HN
N
N
0.18±0.06
(5)
H
N
1.06±0.25
(2)
N
HN
N
H
N
4.26±1.15
(5)
0.33±0.14
(4)
N
8
9
H
H
N
NH
H
N
0.28±0.12
(5)
1.13±0.17
(2)
N
N
N
N
N
H
N
0.20±0.09
(4)
4.76±2.13
(5)
N
N
10
11
12
13
14
H
H
H
H
F
N
N
N
N
H
N
0.16±0.10
(17)
19.0
(1)
N
N
N
N
N
H
N
H
N
0.21±0.12
(13)
0.02±0.02
(15)
N
N
N
N
N
N
H
N
0.38±0.05
(2)
0.10±0.09
(5)
N
H
N
N
N
N
N
N
H
N
O
0.07±0.11
(65)
H
N
0.09±0.08
(6)
N
N
N
N
N
N
Assay conditions: ADP-Glo with 20 nM WT p97 in the presence of 100 µM ATP. n denotes number of independent replicates, each as-
sayed in duplicate or triplicate. In the same assay, the allosteric p97 inhibitor, NMS-8739 exhibited an IC50=11 nM ± 4.9 (n= 13).
Rather, the side-chain of 23 engages in H-bonds with the amide
side-chains of Gln490 and 494, and forms a favorable cation-
π interaction with the side-chain of Trp476. As noted above,
within this gem-disubstituted cyclobutane subseries, N-Me pipera-
zine is favored over the N-iPr analog (23 vs. 24), in contrast to
other members of the class (e.g. 9 vs. 11), where the larger group
is preferred. We hypothesize that the presence of the smaller N-
Me substituent is more sterically compatible with the formation of
the cation-π interaction than larger terminal moieties.
aqueous (pH 7.4) solubility (>300 µM) and half-life in in vitro met-
abolic stability assays (t1/2 >270 min) in human (HLM) and mouse
liver microsomes (MLM) (Table 2) of 17 and 23 are significantly
better; the corresponding value for NMS-873’s solubility is 86 µM
and its metabolic half-life is <10 min in both HLM and MLM.
The improvement in properties for the phenyl indoles has facili-
tated biophysical studies (e.g. cryo-EM analyses) with these com-
pounds that were not feasible with NMS-873. Another key differ-
ence between this series of inhibitors and NMS-873 appears to be
the nature of the allosteric inhibition. While NMS-873 exhibits
mostly non-competitive inhibition,9 the phenyl indoles, as a class,
The biochemical potencies of compounds 17 and 23 compare
favorably to another allosteric inhibitor of p97, NMS-873 (IC50=
11 nM)8 in side-by-side comparisons. However, the physical
properties of the phenyl indoles are superior. Specifically, the
are
uncompetitive
inhibitors.15,16
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Figure 2. (a) Representation of cryo-EM structure of 14 (carbon
Figure 3. Molecular modeling comparison of the binding modes
of 18 (a, carbon = cyan) and 19 (b, carbon = orange); yellow
dashes = H-bond; red dashes and spheres = unfavorable interac-
tions. (a) The side-chain of 18 retains two H-bonds to Glu498
and Glu534, the cyclohexyl group engages in hydrophobic inter-
actions with Val493 and Ile531, and the terminal iPr group is
located near, and solvent shielded by, the side-chain indole moiety
of Trp476. (b) In contrast, in the model of the cis-isomer (19) the
H-bond to Glu498 is lost; unfavorable interactions (red dashes and
spheres) between the secondary nitrogen and hydrophobic
sidechains Val493 and Ile531 are observed, and the cyclohexyl
and iPr groups are significantly more solvent exposed.
= magenta; nitrogen = blue; fluorine = light green; key hydrogens
= white; yellow dashes = H-bonds) bound to p97 at an allosteric
site in the D2 domain.16 The phenyl indole binds in a deep pock-
et, but the side-chain points directly into solvent and engages in
no contacts with protein residues. (b) A molecular modeling de-
rived binding mode that accounts for side-chain SAR. Key inhib-
itor (14) side-chain:protein interactions include H-bonds (yellow
dashes) with Gln494, Glu498, Glu534, and Cys535, as well as
solvent shielding of the terminal iPr group by the side-chain indole
of Trp476.
Figure 4. Predicted binding mode of 23 (carbon = pink; yellow dashes = H-bonds). Like other inhibitors of this chemotype, the side-chain
binds down the helix 16/17 interface and engages in multiple hydrogen bonds with side-chain residues. However, unlike the more flexible
parent 14, the cyclobutyl moiety in 23 binds more deeply in a hydrophobic pocket at the helical interface. As a result: 1) new H-bond
patterns are formed with the amide side-chains of Gln490 and 494; 2) the cyclobutyl moiety engages in closer, favorable hydrophobic con-
tacts with the side-chains of Val493 and Ile531 (purple dash with terminal sphere) and with the side-chain indole of Trp476.
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We then evaluated whether compounds inhibited p97-
tial to be developed into novel cancer therapeutics and to be used
as chemical biology tools to interrogate the function of p97 in
cancer and other p97-dependent diseases. Further studies on this
series of p97 inhibitors will be reported in due course.
dependent activities in cells. In an assay that measured accumula-
tion of a ubiquitinated substrate,21-23 17 and 23 showed modest
effects at 1 h (EC50=19 and 15 µM, respectively), but the effect
was not observed at 6 h even at 40 µM. In contrast, NMS-873
showed robust effects at both 1 and 6 h (EC50 = 1.42 and 2.06
µM, respectively) (Table 2). We considered several hypotheses to
explain this lack of correlation between biochemical activity
(ATPase) and cellular activity (ubiquitin-mediated degradation).
MDCK permeability measurements were uninterpretable, but
poor cellular penetration was considered unlikely based on the
high intracellular concentrations measured for representative
analogs (including 14; data not shown).24 A second possibility was
that the uncompetitive mechanism of the phenyl indole inhibitors
was not as efficacious as ATP-competitive (such as CB-5083) or
non-competitive (such as NMS-873) mechanisms at blocking p97’s
unfoldase activity.15,25-27 Finally, an intriguing possibility is that
the UbG76V-GFP cellular assay does not recapitulate all of the
diverse functions of p97, and that this phenyl indole class selective-
ly inhibits only certain p97-dependent functions, such as autopha-
gy. We have started to evaluate whether other p97-dependent
cellular activities are affected by these phenyl indoles. Preliminary
data suggests this may be the case; these results will be reported in
due course.15
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2
3
4
5
6
7
8
Supporting Information
Synthetic procedures and spectral data for key compounds, com-
putational methods, biological assay protocols, NCI-60 data, and
NMR spectra of final products. This material is available free of
charge via the Internet at http://pubs.acs.org.
9
AUTHOR INFORMATION
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Corresponding Authors
* Huryn@pitt.edu; pwipf@pitt.edu; michelle.arkin@ucsf.edu
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script.
Funding Sources
The 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.
Consistent with blockade of p97, 17 and 23 inhibited cell
growth in the NCI-60 cell line panel.28 Both demonstrated broad
activity in leukemia (pGI50 =-5.7 to -6.7 for 17 & 23); NSCL can-
cer (pGI50 = -5.7 to -6.0 for 17; -5.7 to -5.8 for 23); colon cancer
(pGI50 = 5.8 to -6.4 for 17; 5.8 to -6.3 for 23) CNS tumor (pGI50
= -5.7 to -5.8 for 17 & 23); melanoma (pGI50 = -5.7 to -5.9 for 17;
-5.7 to -6.5 for 23; ovarian cancer (pGI50 = -5.7 to -5.8 for 17 &
23; renal cancer (pGI50 = -5.7 to -5.8 for 17 & 23:, prostate can-
cer (pGI50 = -5.7 for 17; -5.7 to -5.8 for 23) and breast cancer
(pGI50 = -5.8 for 17; -5.7 to -5.9 for 23) cell lines (see SI).
HHSN261200800001E Agreement No. 29XS127TO15.
Notes
The authors declare no competing interest.
ACKNOWLEDGMENT
The authors gratefully acknowledge Marina Kovaliov (University
of Pittsburgh) for technical support and advice; Chaemin Lim
(University of Pittsburgh) for data retrieval and collation; Mary
Liang (University of Pittsburgh) for compound management and
extensive assistance with manuscript preparation; Taber Maskrey
(University of Pittsburgh) for analytical chemistry support; Clifford
Bryant (UCSF) for preliminary chemistry; Gregory Lee (UCSF)
for help with data compilation; Sean Marcsisin, CTP US Army;
Jason Sousa and Brittney Potter (WRAIR) for solubility data;
AMRI for microsomal stability data; Richard Gussio, CAPT
USPHS (NCI) for contributions to computational and molecular
modeling studies; and the NCI for access to the NCI-60 cell line.
The authors also appreciate the helpful discussion and suggestions
of all the CBC p97project team members particularly Raymond
DeShaies (CalTech), Eric Baldwin (Leidos), Andrew Flint (Leidos),
Barbara Mroczowski (NCI), Shizuko Sei (Leidos), Gordon Stott
(Leidos), Gunda Georg (University of Minnesota) and Michael
Walters (University of Minnesota).
Table 2. Characterization of Compounds 17 and 23,
and comparison to NMS-873
Properties
17
50
23
20
NMS-873
11
p97 IC50 (nM)
380
330
86
Solubility (µM)
HLM (t , min)
569
386
475
277
<10
<10
½
MLM (t , min)
½
UbG76V-GFP EC50 (µM)
19
15
1.4
2.1
1 h
6 h
>40
>40
ABBREVIATIONS
NCI-60
AAA+, ATPase associated with diverse activities; CLogP, calcu-
lated logP; CNS, central nervous system; cryo-EM, cryo electron
microscopy, dba, dibenzylideneacetone; CyJohnPhos, (2-
biphenyl)dicyclohexylphosphine; GI50, growth inhibition 50%;
HLM, human liver microsomes; IC50, inhibitory concentration
50%; MLM, mouse liver microsomes; NSCL, non-small cell lung;
PDE6, phosphodiesterase 6; tPSA, total polar surface area;
UbG76V-GFP, ubiquitin Gly76 to Val-green fluorescent protein.
Mean logGI50
-5.8
-5.7
-6.1
In conclusion, by applying systematic medicinal chemistry
strategies, we optimized the side-chain region of a series of 2-
phenylindole p97 inhibitors. SAR studies combined with struc-
tural information and molecular modeling were used to develop a
refined binding model suitable for future analog design iterations.
The most potent compounds in this series, such as 17 and 23,
exhibit low nanomolar biochemical inhibition of the ATPase,
excellent properties, but showed variable effects in cellular assays
that are commonly used to evaluate certain p97-dependent activi-
ties. However, these compounds exhibited anti-proliferative ef-
fects in the NCI-60 panel in the high nanomolar to low micromo-
lar range. Allosteric inhibitors such as 17 and 23 have the poten-
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