Discovery of a First-in-Class, Potent, Selective, and Orally Bioavailable Inhibitor of the p97 AAA ATPase (CB-5083)

Article abstract of DOI:10.1021/acs.jmedchem.5b01346

The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.

Full text of DOI:10.1021/acs.jmedchem.5b01346

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Drug Annotation
Discovery of a First-in-Class, Potent, Selective and Orally
Bioavailable Inhibitor of the p97 AAA ATPase (CB-5083)
Han-Jie Zhou, Jinhai Wang, Bing Yao, Steve Wong, Stevan Djakovic, Brajesh Kumar,
Julie Rice, Eduardo Valle, Ferdie Soriano, Mary-Kamala Menon, Antonett Madriaga,
Szerenke Kissvonsoly, Abhinav Kumar, Francesco Parlati , Michael F. Yakes, Laura
Shawver, Ronan Le Moigne, Daniel J. Anderson, Mark Rolfe, and David Wustrow
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01346 • Publication Date (Web): 13 Nov 2015
Downloaded from http://pubs.acs.org on November 15, 2015
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Discovery of a First-in-Class, Potent, Selective and Orally Bioavailable Inhibitor of the
p97 AAAATPase (CB-5083)
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Han-Jie Zhou*, Jinhai Wang, Bing Yao, Steve Wong, Stevan Djakovic, Brajesh Kumar, Julie
Rice, Eduardo Valle, Ferdie Soriano, Mary-Kamala Menon, Antonett Madriaga, Szerenke
Kissvonsoly, Abhinav Kumar, Francesco Parlati, Michael F. Yakes, Laura Shawver, Ronan Le
Moigne, Daniel J. Anderson, Mark Rolfe and David Wustrow
Cleave Biosciences Inc., 866 Malcom Road, Burlingame CA 94010 USA.
Abstract:
The p97 AAA-ATPase plays vital roles in mechanisms of protein homeostasis, including
ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-
associated degradation (ERAD) and autophagy. Herein we describe our lead optimization
efforts focused on in vitro potency, ADME and pharmaceutical properties that led to the
discovery of a potent, ATP-competitive, D2-selective and orally bioavailable p97 inhibitor 71,
CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers
associated with inhibition of UPS and ERAD functions which induces irresolvable proteotoxic
stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid
accumulation of markers of the unfolded protein response (UPR) and subsequently induces
apoptosis leading to sustained anti-tumor activity in in vivo xenograft models of both solid and
hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple
myeloma and solid tumors.
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Introduction:
The protein p97 (also called valosin-containing protein (VCP), or CDC48 in yeast) is an
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abundant AAA+ ATPase associated with a variety of cellular activities. Working together with
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various cofactors, p97 is involved in multiple biological processes including protein
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homeostasis, ERAD, autophagy, chromatin remodeling, and Golgi reassembly, where it
supplies the mechanical force required for extracting proteins by hydrolyzing ATP. Under
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physiological conditions, p97 forms a ring-shaped homo hexamer.
The ATPase activity of
p97 is crucial for conversion of the potential energy in ATP into mechanical energy via
conformational changes in the p97 hexamer. Each p97 protomer consists of three domains:
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two ATPase domains (D1 and D2) and one N-terminal domain. The N-terminal domain binds
various cofactors that interact with a variety of substrate proteins. The D1 domain has low
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basal ATPase activity owing in part to a very low off rate of ADP. The D2 domain is thought
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to be responsible for most of the ATPase activity of p97 under physiological conditions. The
D2 ATPase region has been shown to have both a higher Km for ATP and a faster hydrolysis of
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ATP to ADP. Numerous studies have implicated p97’s role in promoting ERAD in
collaboration with the UPS. For instance, p97 in combination with substrate recruiting
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cofactors Ufd1 and Np14 extracts misfolded poly-ubiquitinated proteins from the endoplasmic
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reticulum (ER) into the cytosol and then delivers them to the proteasome for degradation.
Expression of p97 is essential to maintain protein homeostasis, especially under stressed
conditions. Indeed, siRNA knockdown of p97 causes irresolvable ER stress and activates the
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UPR, leading to apoptosis via UPS inhibition and activation of caspases. This observation
has led to the hypothesis that p97 inhibition (p97i) could preferentially kill those cancer cells
which have a high protein synthesis burden. Small molecules which inhibit the ATPase
function of p97 could prevent the mechanical action of various p97 containing complexes and
therefore inhibit the UPS, activate the UPR and induce apoptosis. A number of small molecule
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inhibitors of p97 activity have been previously described. A high-throughput screening
(HTS) campaign for inhibition of p97 ATPase activity followed by hit-to-lead optimization led
to the discovery of a series of 2-anilino-thiazole analogs such as 3-(2-((4-
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hydroxyphenyl)amino)thiazol-4-yl)phenol 1 with submicromolar p97 inhibitory potency.
However, this series of compounds has also been reported to be active against other enzymes,
such as neuropeptide Y5 receptor and sphingosine kinase with a similar potency; therefore,
selectivity with this series of compounds could be problematic. 2-Chloro-N-(3-((1,1-
dioxidobenzo[d]isothiazol-3-yl)amino)phenyl)acetamide 2 (NMS-859) was identified from a
different HTS campaign as a covalent p97 inhibitor with moderate biochemical and cellular
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potency (p97i IC , 0.37M). A more potent allosteric p97 inhibitor came from a series of
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substituted triazoles, which was discovered by the same group. 3-(Isopropylthio)-5-
(phenoxymethyl)-4-phenyl-4H-1,2,4-triazole 3 was the initial hit and structure and activity
relationship (SAR) optimization led to its analogue, 3-(3-(cyclopentylthio)-5-(((2-methyl-4'-
(
methylsulfonyl)-[1,1'-biphenyl]-4-yl)oxy)methyl)-4H-1,2,4-triazol-4-yl)pyridine 4 (NMS-
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73) with the reported IC of 24 nM against p97 in the biochemical assay and 380 nM of cell
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killing against HCT 116 cells. However this compound suffered from extremely poor
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metabolic stability. A series of cyclohexylamides has also been described.
Cyclopentylthio)-5-(((2-methyl-4'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl)oxy)methyl)-4H-
1,2,4-triazol-4-yl)pyridine 5 has an IC of 74 nM in the p97 biochemical assay and an IC of
3-(3-
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5 M in an HCT 116 cytotoxicity assay. However, no in vivo anti-tumor activity has been
reported for these molecules.
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N ,N -dibenzylquinazoline-2,4-diamine 6 (DBeQ) was identified from a HTS for inhibitors of
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p97 ATPase activity using the NIH compound library. It reversibly inhibits the ATPase
function of p97 in an ATP competitive manner. Hit-to-lead optimization efforts resulted in the
identification
of
two
analogs,
2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-benzyl-8-
methoxyquinazolin-4-amine 7 (ML240) and 2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-
5,6,7,8-tetrahydroquinazolin-4-amine 8 (ML241) with almost 10-fold improvement of p97i
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potency. Compound 7 had good selectivity for inhibition of p97 over a panel of other
ATPases and kinases. Recently it has been reported that compounds 7 and 8 preferentially
2
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inhibit the D2 ATPase domain of p97. While these compounds were valuable research tools,
their potency and pharmaceutical properties were insufficient to determine the impact of p97
inhibition in vivo. Herein, we report our lead optimization efforts and SAR analysis leading to
the identification of compound 71 (CB-5083), which, to our knowledge, is the first selective
p97 inhibitor with the requisite pharmacological properties to allow for testing in clinical
trials.
Results:
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Chemistry: The compound reported to have structure 7 was prepared by coupling of 2-
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chloroquinazoline 74a with 2-aminobenzoimidazole (75a) as shown in Scheme 1. However, it
was never unambiguously determined whether the coupling took place on the nitrogen of the
imidazole or at the 2-amino group. To verify this, intermediate 74a was coupled with 1,2-
diaminobenzene 76 to yield the intermediate 77a, which was treated with cyanic bromide to
unambiguously form molecule 7 thus confirming this structural assignment. Analogs (9-11) were
prepared using a similar approach from the corresponding 4-aminoquinazolines 74b-d. 2-Chloro-
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4-amino-substituted derivative 78 was acylated with benzoyl chloride to give 79. Using a
similar two step procedure as outlined above, analog 12 was obtained in a modest yield. The 4-
chloro of 73, in the presence of a strong base, can be selectively replaced with alcohols such as
BnOH to yield intermediate 80, which was converted into compound 13 through the previously
described coupling and cyclization reactions.
Intermediate 73 was reacted with styrylboronic acid 81 in the presence of Pd(PPh ) as a catalyst
3 4
to regioselectively give 4-styrylquinazoline 82, which was readily converted into the desired
22
compound 14. Nitrile 84 was reacted with benzylmagnesium bromide followed by cyclization
with methyl chloroformate resulting in pyrimidinone 85 which was chlorinated and converted to
the corresponding 2-aminobenzimidazole derivative 15 as described previously.
The synthesis of target molecules (16-21, 26, 27) is summarized in Scheme 2. 75a was
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acylated and coupled with intermediate 74a to yield compound 16. Intermediate 77a was
reacted with isothiocyanatomethane followed by methyl iodide to yield monomethyl compound
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7. Treatment of the intermediate 77a with 2-chloro-1,1,3,3-tetramethylformamidinium
2
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chloride produced the dimethyl analog 18. 77a was reacted with carbonyldiimidazole to form
the 2-hydroxybenzoimidazole derivative 19. N-(4,5,6,7-Tetrahydro-1H-benzo[d]imidazol-2-
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yl)acetamide 87 was coupled with 74a and the acetyl group was removed with hydrazine to
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form compound 20. 3,4-Dimethylimidazole 88 was reacted with 74a to give compound 21. A
Boc-protecting group was regioselectively installed onto a nitrogen in the imidazole ring of 75a
to yield intermediate 89; the latter was then coupled with 74a and deprotected to yield the
regioisomer 26 which had distinct spectroscopic properties from 7. Treatment of 2-amino-3-
methoxybenzoate 90 with 2-bromoacetonitrile under an acidic condition provided 2-
bromomethyl-4-hydroxyquinazoline 91 in modest yield. The latter was reacted with 1,2-
diaminobenzene followed by cyclization with trimethoxyethane to yield intermediate 92 and the
hydroxyl group was converted to a benzylamino group to provide compound 27.
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The synthesis of target molecules (22-25, 28-38 and 40-72) is illustrated in Scheme 3 and was
achieved through a variety of palladium-catalyzed coupling reactions between 2-chloro-4-
benzylamino (substituted) quinazolines, fused pyrimidines or their derivatives (74a-l, herein
referred to as the cores) with the 5,6-bicycloaromatic rings (75, 93-96, herein referred to as P2-
moieties) to introduce this functionality at the P-2 position.
Representative synthetic routes of the (substituted) quinazolines or fused pyrimidines 74e-l (also
referred to as the cores) which possess a chlorine at the 2-position and benzylamino group at the
4
-position are illustrated in Scheme 4. Demethylation of 8-methoxyquinazoline 74a was effected
by treatment with boron tribromide. The resulting hydroxyl group on 97 was reacted with 1-
bromo-2-methoxyethane to afford the quinazoline 74e. Dichlorination of the thieno[2,3-
28
d]pyrimidin-4-one 98a followed by condensation with benzylamine gave key intermediate 74f.
2
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In an analogous way, thiazolo[5,4-d]pyrimidine diol 98b was used to prepare 74g. 2-
oxocyclohexanecarboxylate 99 was easily converted into 2,4-dihydroxypyrimidine 100 which
was transformed into the intermediate 74h. Pyrimidine diols (102a, 102b) were prepared from
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ketoesters (101a, 101b) and then converted to their corresponding dichlorides using standard
methodology. Because of concerns about removal of the benzyl protecting group on the nitrogen
of the saturated ring later in the sequence, it was removed at this point using 1-chloroethyl
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chlorofomate giving 103a and 103b containing free amine functionality. The amine
functionalities were protected as their t-butyl carbonate derivative and an N-benzyl group was
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introduced as before to give 74i and 74j. Unlike other keto esters, 104a could not withstand
the strongly acidic or basic conditions required to catalyze condensation with urea to form a
fused pyrimidine. However it was found that 104a could be converted to enamine 105a followed
by reaction with 2,2,2-trichloroacetyl isocyanate and cyclization with ammonia to give
pyranopyrimidine diol 106a. Similarly 104b was converted to pyrimidine diol 106b. Pyrimidine
diols 106a and 106b were both converted to their 4-N-benzyl-2-chloro derivatives (74k and 74l
respectively) using the previously described conditions.
The 5,6-bicyclicheteroaromatic functionalities (also referred to as P2-moieties) that were coupled
to the 2-position of the (substituted) quinazolines or fused pyrimidine, were either commercially
available or were prepared via the approaches summarized in Scheme 5. 2-Methoxy-1H-
benzo[d]imidazole 75b was prepared by reaction of benzene-1,2-diamine 76 with tetramethyl
orthocarbonate under acidic conditions. Methylation of indolin-2-one 107 afforded 2-
methoxyindole 93a. Reduction of ethyl 1H-indole-2-carboxylate 93b using lithium aluminum
hydride yielded 93c, which was methylated to give intermediate 93d. Reduction of the amide
bond of intermediate 93e, which was prepared from 93b, followed by Boc-protection gave
intermediate 93f. Bromination followed by N-Boc protection of 2-methyl indole 93g yielded
o
intermediate 108. Halogen metal exchange with n-butyl lithium at -78 C followed by reaction
33
with isopropoxypinacolborate yielded 3-indolyl boronate 94. Compound 109 was hydrolyzed
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and the resulting acid was converted into the bromide by NBS and de-bromination using n-butyl
lithium at a low temperature yielded the desired 95. 2-Methylimidazo[1,2-a]pyridine 96 was
prepared in a one-step process wherein 2-aminopyridine 110 was reacted with 1-bromopropan-2-
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A variety of 4, 5 and 6-substituted 2-methyl indole derivatives 93h-t were prepared by a three-
step process. Indoles 111h-t were converted to their corresponding 1-benzenesulfonyl derivatives
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12h-t which were reacted at a low temperature with n-butyl lithium to effect directed metalation
at the 2-position of the indole. Quenching of the resulting anions with methyl iodide produced
the 2-methyl derivatives 113h-t. The benzenesulfonyl group was then removed to give the target
indoles. The 2-methyl-1H-indole-4-carboxylate derivative 93u was prepared from 4-bromo-2-
methyl indole 93r via a palladium catalyzed carboxylation reaction.
Palladium-catalyzed coupling of the fused pyrimidines 74a-l with the aforementioned P2
moieties (75, 93-96) yielded the target molecules (22-25, 28-38 and 40-72) as exemplified in
Scheme 6. The substituted quinazolines or fused pyrimidine (74a, 74e-i) were reacted with either
the aforementioned benzoimidazoles (75a,75b) or commercially available benzoimidazole 75c,
2-methylbenzoimidazole 75d, 2-ethylbenzoimidazole 75e in the presence of Pd (dba) , X-Phos
2
3
and cesium carbonate to give target molecules (22-25 and 28-35). In addition to these steps
removal of the Boc protecting group was required for compounds 36-38. Similar coupling
reactions between 74h and commercially available indoles such as 2-methylindole 93g, 2-
ethylindole 93v or 2-trifluoromethylindole 93w or the indoles (93a, 93c, 93d, 93f) yielded the
target molecules 40-46. The 3-indolyl regioisomer 47 was prepared by Suzuki coupling of the
fused pyrimidine 74h with boronate 94 followed by deprotection of the indole nitrogen. 2-
methylpyrazolopyridine-containing analogs (48, 49) were made via palladium catalyzed Heck-
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type coupling between 2-chloropyrimidine 74h and pyrazolopyridines (95, 96), respectively.
Similar conditions (Pd (dba) , X-Phos and cesium carbonate) were used to carry out Buchwald-
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type coupling reactions of 74i with the indoles 93g-u to give targets 50-62 and the intermediate
14, respectively. The latter was hydrolyzed into the acid 63 which was subsequently converted
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into amides 66-68. Using similar coupling conditions, the 4-carbamoyl-1-indole containing
molecules (65, 69-72) were prepared through introduction of the 4-cyano-2methyl-1-indole
(93p) to the 2-position of the fused pyrimidines 74h-l. These intermediates were reacted with
palladium acetate and acetaldehyde oxime in the presence of triphenylphosphine to convert the
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nitrile into the primary carboxamide. This methodology was required because coupling with 4-
carbamoyl-2-methyl-indoles either completely failed or resulted in extremely poor yields of the
desired products. The intermediate nitrile 115 was reacted with sodium azide followed by acid
mediated removal of the t-butyl-carbonyl protecting group to provide tetrazine 64.
X-ray crystal structure of compound 71: a crystal hydrate was obtained from a 1:1 ethanol-
water solution; the unit cell contains two molecules of water per molecule of 71 as shown in
Figure 2. Anisotropic atomic displacement ellipsoids for the non-hydrogen atoms are shown at
the 50% probability level. The inter-molecular hydrogen bonds are shown as dashed lines and
hydrogen atoms are displayed with an arbitrarily small radius.
TM
Biological assays: The primary biochemical assay used was the ADP-Glo assay (Promega)
TM
with purified human p97 enzyme. Cell-based assays included a 72-hour Cell Titre-Glo
(CTG) viability assay and 6-hour pharmacodynamic (PD) marker immunofluorescence assays
which measured K48 poly-ubiquitinated protein accumulation for target engagement,
CCAAT/enhancer-binding protein homologous protein (CHOP) accumulation and
sequestosome 1 (p62) reduction for pathway inhibition, p53 accumulation and cleaved caspase
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37
3
/7 activation for death induction (data not shown).
In vitro ADME and in vivo pharmacokinetics and pharmacodynamics (PK/PD): Chemical
stability was evaluated in simulated gastric and intestinal fluids (SGF and SIF), and the
percentage of parent remaining was determined after 15-minute incubation. Metabolic stability
was assessed in liver microsomes from mouse, rat, dog, monkey and human; clearance and T1/2
were calculated. Permeability was assessed by Caco-2 permeability assays with cultured Caco-
0
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2
monolayers; PA-B, and PB-A were determined and efflux ratio was calculated. Solubility was
assessed in a variety of pH buffer solutions. In vivo biological activity was determined in
tumor and tissues by PD measurements of the following markers: poly-ubiquitin, CHOP and
cleaved poly ADP ribose polymerase (cPARP) after oral (p.o.) administration in mice.
Absolute bioavailability (F%) was determined by the PK assessment of areas under the plasma
concentration versus time curves following i.v. and p.o. administration. Anti-tumor efficacy
was assessed in immunocompromised mice bearing established human tumor xenografts.
Discussion:
Initial investigations into the SAR of the 8-methoxy quinazoline series focused on improving
potency, selectivity and pharmaceutical properties so that the postulate of p97 inhibition
resulting in anti-tumor activity in vivo could be tested. During this lead optimization process, we
were mindful of the effect that molecular changes could have on physical parameters such as
cLogP, lipE and PSA. The goal was to identify an optimal range of these parameters and
consequently to increase potency significantly. The initial strategy involved a systematic
evaluation of the functionality of compound 7. We started by investigating the possibility of
changing the benzylamino group at the 4-position of the quinazoline. This resulted in compounds
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9
which were essentially inactive in the p97 biochemical assay (9-15) as shown in Table 1. Neither
simple methylation on methenyl 9 nor nitrogen 10 was tolerated. The benzylamino group could
not be replaced by either phenylethylamine 11 or amide 12. Replacement of nitrogen with either
oxygen 13 or carbon 14 as well as deletion of the nitrogen 15 failed to retain the p97 activity.
Therefore, the 4-benzylamino group was retained for further optimization. Attention was next
focused on the modification of the 2-amino-benzoimidazole moiety found in compound 7 as
illustrated in Table 2. Acetylation of the amino functionality at the 2-position of the
benzoimidazolyl group resulted in an inactive compound 16. In contrast, either mono or double
methylation of the amino group (17 and 18 respectively) increased biochemical potency
approximately 4-fold. Replacement of the imidazole ring of benzoimidazole with imidazolone 19
or its phenyl ring by either cyclohexyl 20 or dimethyl 21 failed to increase the p97 potency. The
absence of a substituent on the 2-position of benzoimidazolyl 22 lost p97 activity completely.
Among the best replacements of the amino group was either methyl 23 or methoxy 25 which led
to an approximately 6-fold increase in biochemical potency. Notably, these had only a modest
increase in cLogP, but did have a significant drop in PSA compared to compound 7. However,
larger alkyl groups such as ethyl found in compound 24 were not tolerated. Linking the
benzoimidazole and quinazoline functionalities through an amino group on the 2-position of the
imidazole or insertion of methenyl as a bridge between them resulted in inactive compounds (26
and 27, respectively).
10
11
12
13
14
15
16
17
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19
20
21
22
23
24
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59
60
A variety of substituents at the 8-position of the quinazoline and alternatives to the quinazoline
core were investigated as exemplified in Table 3. Extensive investigation demonstrated that
ether groups were preferred at the 8-position of the quinazoline (data not shown). Solubility was
improved by either introducing additional heterofunctionality such as substituted ether groups at
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the 8-position (28, 29, 30) or replacing the phenyl ring of the quinazoline with 5 or 6-membered
heteroaromatic rings such as thiophene or thiazole (31 and 32 respectively); however, this only
resulted in compounds with either similar or only moderately better potency. Efforts to replace
the phenyl ring of the quinazoline with saturated 5 or 6-membered rings led us to identify some
more attractive leads that had both enhanced potency and better pharmaceutical properties. The
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4
,5,6,7-tetrahydroquinazoline core produced compounds that were the most potent p97 inhibitors
that we had synthesized to that point (33-35). Compounds 34 and 35 were the first compounds
we had seen with an IC under 100 nM against p97. Replacement of the quinazoline ring with
5
0
the 4,5,6,7-tetrahydropyridyl [4,3-d]pyrimidine functionality, which inserted a basic nitrogen
into the saturated ring, resulted in compounds with unimpressive p97 biochemical potency (36-
38). However, these compounds had markedly lower cLogP and better aqueous solubility,
especially in low pH buffer solutions (data not shown).
Compound 35 was notable in that it possessed a 10-fold increase in p97 inhibitory potency
compared to the starting point 7. To investigate how 35 interacted with the p97 D1 and D2
ATPase sites, ATP probe 39 (ActivX, San Diego, CA) was utilized to irreversibly label ATP
binding sites found in kinases and ATPases. The labeling of ATP sites was measured by mass
spectral detection (Figure 3A). Labeling of the proteins existing in the cellular lysates of A549
tumor cells in the presence or absence of 35 was assessed by measuring the abundance of labeled
peptide signals after tryptic digest and purification. Compound 35 selectively inhibited labeling
at the D2 site of p97 (Figure 3B). This specific interaction with the D2 domain of p97 is
consistent with its ability to inhibit the ATPase activity of p97 since the D2 region has been
shown to be primarily responsible for the ATPase activity of p97. In addition, 35 also exhibited
in vitro PD effects in A549 tumor cells that are a downstream consequence of specific inhibition
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of p97 activity (Table 7). These PD effects included dose-dependent accumulation of K48 poly-
ubiquitinated proteins as an indication of target engagement and CHOP accumulation and p62
reduction as an indication of pathway inhibition. CHOP is a key transcriptional regulator that is
10
11
12
13
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38, 39
activated through protein accumulation as a result of activation of the UPR.
p62 is an
4
0
adaptor protein that binds to aggregated proteins to target them to the autophagosome. When
autophagy is activated, p62 protein is degraded as it is processed through the autophagosome,
and therefore measuring p62 protein levels allows for the monitoring of autophagic activity after
3
7
compound treatment. The potency at which these changes occurred was in the same range
required to cause A549 cell death. More importantly, 35 demonstrated measurable anti-tumor
activity in mouse xenograft studies when administrated orally at the dose of 300 mg/kg on a
daily basis (See supporting information S1). Taken together, these data indicated that potent and
specific binding to the D2 ATPase domain could lead to p97 ATPase inhibition which in turn
could induce tumor cell death both in vitro and in vivo by interfering with this vital protein
homeostasis pathway.
While compound 35 provided important proof of concept, further improvement of potency and
metabolic stability was still required to achieve a viable drug candidate. Therefore, further
optimization of the potency and pharmaceutical properties of this series of p97 inhibitors was
undertaken. Since analogs with either methyl or methoxy groups at the 2-position of
benzoimidazole consistently showed more potent p97 activity, we decided to investigate whether
benzoimidazole itself could be further optimized by replacement with other 5,6-bicycloaromatic
rings with either methyl or methoxy on the 2-position using 4,5,6,7-tetrahydroquinazoline as the
core (Table 4). Indeed, the benzoimidazole was found to be replaceable. Both compounds (40,
4
1) containing1-indolyl functionality on their 2-position were approximately 3-fold more potent,
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with IC s under 50 nM, compared to their benzoimidazole analogs (13, 15 respectively). As in
5
0
the benzoimidazole subseries, at the 2-position of the indole, the bigger alkyl groups such as
ethyl 42 or electron withdrawing moieties such as trifluoromethyl 43 caused a loss of potency.
However, the hydroxymethyl analog 44 retained a similar potency in terms of p97 inhibition and
cell killing, though its methyl ether derivative 45 had dramatically reduced potency most likely
due to the limited tolerability of the size of the substituent at the 2-postion of indole. We next
turned our efforts to introducing basic moieties with the aim of increasing solubility. An
aminomethyl in compound 46 is tolerated with slightly weaker potency but retained its capacity
for killing cells. It was also found that 3-indole 47 or regional isomers of 1-benzoimidazole, 3-
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(2-methylpyrazolo[1,5-a]pyridinyl) 48 and 3-(2-methylimidazo[1,2-a]pyridinyl) 49 were all
tolerated with slightly less activity. Compound 50 had lower biochemical potency but had
excellent solubility and liver microsomal stability (Table 7). The introduction of the basic amino
group caused a significant decrease in cLogP and despite its decreased potency it had one of the
largest LipE values observed to that point. In addition, it was noticed that most of these
compounds still possessed relatively low PSA.
In an effort to improve the potency of compound 50, the substitution pattern on the phenyl ring
of the indole moiety was systematically investigated (Table 5). Initially, we intended to
introduce neutral, electron donating or electron withdrawing substituents (methyl, methoxy and
cyano, respectively) into all positions of its phenyl ring (4, 5, 6 and 7-position). However, it
turned out to be a challenge to couple 7-substituted 1-indoles with the 2-chloro-substituted core,
most likely due to steric factors, and therefore only nine derivatives (51-59) were prepared
successfully. It was found that substitution on either 5 or 6 position of the indole nucleus resulted
in complete or substantial losses in p97 potency. While substitution was generally better
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tolerated at the 4-position of the indole, only the 4-cyano substituted indole 59 showed somewhat
greater potency. Therefore, a variety of other electron withdrawing moieties (60-68) were
introduced into this position. The primary amide 65 turned out to be the best in terms of
biochemical and cell killing potency. The acid 63 and tetrazole derivatives 64 were
biochemically potent but inactive in cell killing, most likely owing to their zwitter-ionic nature.
Consistent with the observation, the PSA of these more potent compounds increased to a range
of 70~100.
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With this finding, we decided to keep the moiety of 1-(2-methyl-4-carbamoyl-indolyl) as the
substituent on the 2-position and the N-benzylamino moiety at the 4-position of the pyrimidine
and then screened a number of fused pyrimidine cores; representatives of which are summarized
in Table 6. All of these compounds demonstrated good p97 potency. Among them, 5,6,7,8-
tetrahydroquinazoline 69 and 7,8-dihydro-5H-pyrano[4,3-d]pyrimidine 71 were the most potent
in terms of both p97 enzyme inhibition (IC50 <15 nM) and tumor cell killing (sub-micromolar
IC ). They also caused significant K48 poly-ubiquitinated protein and CHOP accumulation as
5
0
well as p62 reduction at submicromolar concentrations after 6-hour treatment as a consequence
of p97 inhibition in cells (Table 7). They also possessed reasonable cLogP and PSA.
LipE is an easily calculated metric that assesses the contribution of nonspecific hydrophobic
4
1
interactions to potency and representative molecules’ LipEs are shown in Table 7. Use of this
metric assumes that compounds whose biochemical potency is driven by a specific interaction
with the biological target to a degree greater than would be expected by a simple increase in
4
2
lipophilicity will tend to be more “drug-like”. An analysis of LipE of key compounds in this
SAR study showed a continual increase in this parameter from the initial starting point of
compound 7 (LipE = 1.50) to compound 35 (LipE = 1.95) and compound 50 (LipE=2.87) which
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had improved potency with similar or somewhat lower cLogP values. The introduction of the
amide on to the 4-position of the indole produced a series of compounds with consistently higher
LipE values. Compound 65 had a significantly improved LipE and the best overall cellular
potency within its sub-series (see Table 7), while compound 69 possessed a slightly lower
overall LipE but higher cellular potency. Compound 71 had the highest LipE value (LipE = 5.20)
and excellent cellular potency.
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The two most in vitro potent molecules (69, 71) were then profiled in vivo using tumor-bearing
mice to evaluate their PK and PD effect and anti-tumor activity. Both molecules were
administered orally as a suspension in 0.5% methylcellulose aqueous suspensions at the fixed
dose strength of 150 mg/kg; plasma and tumor samples at multiple time points (2, 6, 16 and 24
hours) were harvested for PK/PD analysis (Figure 4). 71 had an approximately 2-fold higher
exposure in both plasma and tumor compared to 69. 71 also achieved a more sustained PD effect
of poly-ubiquitinated protein accumulation in tumor, especially at later time points. Anti-tumor
activity of the two compounds was assessed in an HCT 116 tumor xenograft model. 71 was
administered orally using every day (qd) dosing, whereas 69 was administered every other day
(q2d). 71 showed more profound anti-tumor activity in this study (Figure 5). In addition, 71 has
a better aqueous solubility than 69 (Table 7) and has good metabolic stability with a 102 minute
T1/2 in a mouse liver microsomal stability study and a 172 minute T1/2 in a hepatocyte stability
study. It also has excellent permeability as assessed in a Caco-2 assay (Table 8). PK studies
revealed that 71 has moderate absolute oral bioavailability (41%) in mouse (Table 9), making it
suitable for preclinical development. Therefore, 71 was selected for further in vitro and in vivo
evaluation.
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The specificity of the interaction of 71 with p97 was assessed in multiple ways. In A549 cellular
lysates, 71 selectively blocks the interaction of the irreversible ATP probe 39 with the D2 region
of p97 at 10 M concentration to a greater extent than was observed with 35 with no interaction
with the p97 D1 site (Figure 3B). It showed little or no interaction with a panel of over 300 other
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50
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59
60
3
7
ATPases, helicases and kinases that were also assessed in this assay (Table 8). A modest
interaction was observed with the kinase DNAPK. The biochemical IC of 71 was determined to
5
0
be > 60-fold weaker for DNAPK compared to p97 and no evidence of cellular effects of
3
7
inhibition of this kinase have been observed.
Cell lines that were resistant to 71 were generated and were found to contain specific mutations
in the p97 D2 ATPase region, N660 and T688 (data not shown). 71 had a ~50-fold reduction in
3
7
potency when tested on recombinant p97 carrying these mutations. Combining this information
with the aforementioned SAR analysis allowed us to investigate possible binding modes of 71
with p97. 71 was docked into the D2 ATP binding site. AutoDock Vina is the software used to
execute docking of the compound into the active site of p97. The search was performed within a
3
box of 30 x 30 x 30 Å centered at the ATP binding site. The side chains of a set of protein
residues lining the ATP binding pocket were allowed to adjust during the docking procedure.
The top 15 poses ranked by binding energy were examined visually and the best pose consistent
with the SAR and mutation data was selected for further analysis. The obtained docking pose
(
Figure 6) shows that not only multiple hydrogen bonds are potentially formed between the 2-
nitrogen of the pyrimidine core and the NH of the benzylamino group with the aforementioned
two amino acids, but also that the benzyl group fits into a tight hydrophobic pocket, which is
1
7
consistent with our SAR and the reported 5 binding model. The profound potency improvement
of the primary amide on the 4-position of the indole, compared to its cyano-substituted or non-
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substituted analogs, may suggest that the capacity of this amide as both a hydrogen bond donor
and acceptor is critical. Indeed, according to this model, this amide may interact with both
amino acids S664 and K663. Therefore, 71 may compete with ATP for the same binding site, but
perhaps through a slightly different orientation.
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The oral anti-tumor activity of 71 was compared to the proteasome inhibitor, bortezomib, in both
a multiple myeloma model (AMO-1) and a solid tumor model (A549 lung carcinoma) as
summarized in Figure 7. Bortezomib was administered at its reported efficacious dose strength,
administration route (i.v.) and schedule. 71 was administered orally at a dose of 100 mg/kg as a
suspension in 0.5% methylcellulose aqueous solution on a qd4on/3off weekly schedule. Both
compounds were active in the AMO-1 multiple myeloma model. However, only 71 was active
in the A549 lung carcinoma model and bortezomib was inactive. This provides preclinical
evidence that p97 inhibitors can potentially be effective against both hematologic and solid
tumors.
Conclusions:
Through a systematic SAR optimization, we have discovered 71, a potent and selective inhibitor
of the p97 D2 site with nanomolar biochemical and submicromolar cellular potency and
moderate oral bioavailability. In vivo 71 caused rapid and sustained accumulation of poly-
ubiquitinated proteins and markers of the UPR and apoptosis as well as demonstrating significant
tumor growth inhibition in solid tumor and hematological xenograft models. 71 (CB-5083) was
nominated as a drug candidate for the treatment of cancer and is currently being tested in
ongoing phase 1 clinical trials for patients with relapsed/refractory multiple myeloma and
advanced solid tumors.
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Experimental Section:
General Methods: chemicals, reagents and solvents were obtained from commercial sources and
they were used as received. NMR spectra were obtained in CDCl , DMSO-d , CD OD, or
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59
60
3
6
3
o
acetone-d at 25 C at 300 MHz on an OXFORD (Varian) with chemical shift (δ, ppm) reported
6
relative to TMS as an internal standard. HPLC-MS chromatograms and spectra were obtained
with Shimadzu LC-MS-2020 system and UV absorption was recorded at wavelengths of 214 and
254 nm using acetonitrile and water under either acidic conditions (i.e. 0.1% HCO H, HCl or
2
TFA) or neutral conditions (i.e. 0.1% NH OAc) as the mobile phases. Preparative reverse phase
4
HPLC instruments were Gilson GX-281(Gilson) and P230 Preparative Gradient System (Elite)
using the aforementioned mobile phases. Microwave instrument was CEM Discover SP. Normal
phase flash chromatography was performed on silica gel 60. All final compounds were purified
1
to >95% purity as determined by HPLC and HNMR spectra.
Chemistry:
N-Benzyl-2-chloro-5,6,7,8-tetrahydroquinazolin-4-amine
(74h):
Step
1:
5,6,7,8-
Tetrahydroquinazoline-2,4-diol (100). To a room temperature HCl solution in ethanol (3 N, 250
mL) were added urea (26.5 g, 441 mmol) and 2-oxocyclohexanecarboxylate (99) (50.0 g, 294
mmol), and the resulting solution was refluxed overnight. It was then cooled to room temperature
and the precipitated white solids were collected to yield the diol (100) (14.0 g, 28.6%) which was
+
used in the next step without further purification. LRMS (M+H ) m/z: calcd. 167.1; found 167.1.
Step 2: 2,4-Dichloro-5,6,7,8-tetrahydroquinazoline. A mixture of the crude diol (100) (14 g,
8
4.3 mmol) in POCl (100 mL) was refluxed for 2 hours. After being cooled to room
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temperature, the mixture was concentrated in vacuo. DCM (200 mL) and ice water (100 mL)
were added, the separated organic layer was dried over sodium sulfate and concentrated in
vacuo, and the residue was purified by flash chromatography (silica gel, petroleum ether,
0
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ethyl acetate) to afford the dichloride-5,6,7,8-tetrahydroquinazoline (16.3 g, 95 %). HNMR
(300 MHz, CDCl ): δ 2.85-2.82 (m, 2H, CH ), 2.56-2.53 (m, 2H, CH CN), 1.84-1.78 (m,
3
2
2
4
H, (CH ) ).
2 2
Step 3: N-Benzyl-2-chloro-5,6,7,8-tetrahydroquinazolin-4-amine (74h). To a room
temperature solution of the aforementioned crude dichloride (16 g, 79 mmol) in acetonitrile
(
200 ml) was added phenylmethanamine (25 g, 240 mmol) and the reaction mixture was
stirred at the same temperature overnight. The solvents were then removed in vacuo and the
residue was dissolved with dichloromethane (200 ml) and washed with saturated ammonium
chloride solution. The separated organic layer was concentrated and the residue was purified
by column chromatography (silica gel, petroleum ether, ethyl acetate) to give the key
1
intermediate (74h) (20 g, 93%). HNMR (300 MHz, CDCl ): δ 7.33-7.20 (m, 5H, Ph), 4.64
3
(
s, 2H, CH Ph), 2.58 (t, J = 5.1 Hz, 3H, CH ), 2.35 (t, J = 5.1 Hz, 2H, CH ), 1.84-1.78 (m,
2 2 2
4H, (CH ) ).
2
2
N-tert-Butyl
4-(benzylamino)-2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-
carboxylate (74i): Step 1: 6-Benzyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine-2,4-diol (102a).
To a room temperature solution of ethyl 1-benzyl-4-oxopiperidine-3-carboxylate (101a) (50 g,
0
0
.19 mol) in methanol (100 mL) were added urea (23 g, 0.38 mol) and sodium methoxide (21 g,
.38 mol), then the reaction mixture was refluxed for 48 hours. it was then cooled to room
temperature, the precipitated solids were collected, washed with water (50 mL x 3) and dried to
+
1
yield the diol (102a) (32 g, 65%). LRMS (M+H ) m/z: calcd. 258.1; found 258.1. HNMR (300
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MHz, DMSO-d ): δ 7.32-7.23 (m, 5H, Ph), 3.57 (s, 2H, CH Ph), 2.97 (s, 2H, NCH ), 2.56 (t, J =
6
2
2
6
Hz, 2H, NCH CH ), 2.29 (t, J = 6 Hz, 2H, NCH CH ).
2 2 2 2
Step 2: 6-Benzyl-2,4-dichloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine. A solution of the
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aforementioned diol (102a) (15 g, 0.058 mol) in POCl (200 mL) was refluxed and stirred for 3
3
hours. After being cooled to room temperature, the mixture was concentrated in vacuo. The
residue was diluted with dichloromethane (200 mL) and water (100 mL), and neutralized with
sodium hydroxide. The aqueous phase was separated and extracted with dichloromethane (50
mL x 2). The combined organic layers were washed with brine, dried over anhydrous sodium
sulfate, and concentrated in vacuo. The residue was dried to give 6-benzyl-2,4-dichloro-5,6,7,8-
tetrahydropyrido[4,3-d]pyrimidine (13.5 g, yield: 79%, purity: >95%), which was used in the
+
next step without further purification. LRMS (M+H ) m/z: calcd. 294.05; found 294.1.
Step 3: 2,4-Dichloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (103a). To a room temperature
solution of the aforementioned crude dichloride (13.5 g, 46 mmol) in 1,2-dichloroethane (120
mL) was added 1-chloroethyl carbonochloridate (19.7 g, 138 mmol). Then the solution was
refluxed for 3 hours. The solution was cooled and concentrated in vacuo. The residue was
dissolved in methanol (120 mL) and refluxed for another 30 minutes. It was cooled and
concentrated in vacuo to give the crude 2,4-dichloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine
+
(
103a) (9.0 g, 97%), which was used in the next step without further purification. LRMS (M+H )
m/z: calcd. 204.0; found 204.0.
Step 4: N-tert-Butyl 2,4-dichloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine. To a room
temperature solution of the aforementioned crude intermediate (103a) (9.0 g, 44.3 mmol) in
dichloromethane (90 mL) were added (Boc) O (11.5 g, 53 mmol) and Et N (18.5 mL, 133
2
3
mmol). Then the mixture was stirred at the same temperature for 2 hours. The reaction solution
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was washed with water (100 mL x 2) and brine (50 mL); the separated organic layer was
concentrated in vacuo to give the crude N-tert-butyl 2,4-dichloro-5,6,7,8-tetrahydropyrido[4,3-
d]pyrimidine (13.0 g, yield: 97%, purity: 95%), which was used in the next step without further
0
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8
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4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
+
purification. LRMS (M+H ) m/z: calcd. 304.1; found 303.9.
Step 5: N-tert-Butyl 4-(benzylamino)-2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-
carboxylate (74i). To a room temperature solution of the aforementioned crude Boc-protected
2
,4-dichloride (13.0 g, 43 mmol) in acetonitrile (90 mL) were added phenylmethanamine (7.0 g,
65 mmol) and triethylamine (18 mL, 129 mmol). The resulting solution was then stirred at the
same temperature overnight and concentrated in vacuo; the residue was purified by flash
chromatography (silica gel, petroleum ether, ethyl acetate) to afford N-tert-butyl 4-
(benzylamino)-2-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (74i) (13.0 g,
+
1
yield: 81%). LRMS (M+H ) m/z: calcd. 375.2; found 375.1. HNMR (300 MHz, CDCl ): δ 7.36-
3
7
.34 (m, 5H, Ph), 4.84 (br, 1H, NH), 4.70 (s, 2H, CH Ph), 4.19 (s, 2H, NCH ), 3.68 (t, J = 5.7
2 2
Hz, 2H, NCH CH ), 2.79 (t, J = 5.7 Hz, 2H, NCH CH ), 1.49 (s, 9H, C(CH ) ).
2
2
2
2
3 3
N-Benzyl-2-chloro-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (74k): Step 1: 7, 8-
Dihydro-5H-pyrano[4, 3-d]pyrimidine-2,4-diol (106a). Methyl 4-oxotetrahydro-2H-pyran-3-
carboxylate (104a) (1.58 g, 10 mmol) and ammonium acetate (2.3 g, 30 mmol) in methanol (20
mL) was stirred at room temperature overnight. The mixture was concentrated in vacuo,
dichloromethane (100 mL) and water (20 mL) were added, and the separated organic layer was
dried over sodium sulfate and concentrated in vacuo. The resulted crude methyl 4-amino-5,6-
dihydro-2H-pyran-3-carboxylate (105a) was then dissolved in acetontrile (20 mL) and 2,2,2-
trichloro-acetyl isocyanate (3.76 g, 20 mmol) was added. The resulting mixture was stirred for
3
0 minutes and the precipitated solids were collected and dissolved in a solution of ammonia in
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methanol (8 mL, 7 N), then the resulting mixture was heated at 70°C for 2 hours. The reaction
was cooled down and the precipitated solids were collected and dried to afford the diol (106a)
+
1
(
1.2 g, 71%, purity: ~ 99%). LRMS (M+H ) m/z: calcd. 169.1; found 169.0. HNMR (300 MHz,
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DMSO-d ): δ 10.98 (br, 2H, 2OH), 4.19 (s, 2H, OCH ), 3.76 (t, J = 5.4 Hz, 2H, OCH CH ), 2.38
6
2
2
2
(t, J = 5.4 Hz, 2H, OCH CH ).
2
2
7
4k was then prepared in a good yield following the aforementioned three-step procedure.
1
HNMR (300 MHz, CDCl ): δ 7.36-7.34 (m, 5H, Ph), 4.70 (d, J = 5.1 Hz, 2H, CH Ph), 4.61 (br,
3
2
1H, NH), 4.42 (s, 2H, OCH ), 3.96 (t, J = 5.4 Hz, 2H, OCH CH ), 2.79 (t, J = 5.4 Hz, 2H,
2
2
2
OCH CH ).
2
2
2
-Methyl-1H-indole-4-carbonitrile (93p): Step 1: 1-(Phenylsulfonyl)-1H-indole-4-carbonitrile
o
(112p). To a 0 C solution of 1H-indole-4-carbonitrile (111p) (1.00 g, 7.0 mmol) in THF (20
mL) was added NaH (0.42 g, 10.5 mmol, 60%). The mixture was stirred for 5 minutes, and
benzenesulfonyl chloride (1.49 g, 8.4 mmol) was then added at the same temperature. The
reaction mixture was stirred at room temperature for an additional 30 minutes and then poured
o
into a 0 C saturated aqueous NH Cl solution (50 mL). The aqueous phase was separated and
4
extracted with ethyl acetate (100 mL x 2); the combined organic layers were washed with water
(
50 mL) and brine (50 mL), dried over Na SO , and evaporated in vacuo. The residue was
2 4
recrystallized (heptane, EtOAc) to give the intermediate (112p) (1.6 g, yield: 81%, purity: 99%)
+
1
as a yellow solid. LRMS (M+H ) m/z: calcd. 283.1; found 283.0. HNMR (300 MHz, DMSO-
d6): δ 8.31 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 3.9 Hz, 1H, 2-H of indole), 8.08-8.05 (m, 2H), 7.82-
7
1
.80 (m, 1H), 7.76-7.71 (m, 1H), 7.65-7.60 (m, 2H), 7.53 (t, J = 8.1 Hz, 1H), 7.00 (d, J = 3.9 Hz,
H, 3-H of indole).
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o
Step 2: 2-Methyl-1-(phenylsulfonyl)-1H-indole-4-carbonitrile (113p). To a -40 C solution of the
aforementioned indole intermediate (112p) (1.00 g, 3.5 mmol) in THF (30 mL) was slowly
added n-BuLi (1.6 mL, 3.8 mmol, 2.4 M). The mixture was stirred for an additional one hour and
then MeI (0.27 mL, 4.25 mmol) was added at the same temperature. The resulting mixture was
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then allowed to warm to room temperature and stirred for an extra three hours. The mixture was
o
poured into a 0 C saturated aqueous NH Cl solution (100 mL). The aqueous phase was
4
separated and extracted with EtOAc (100 mL x 2). The combined organic layers were washed
with water (50 mL) and brine (50 mL x 2), dried over Na SO , and evaporated in vacuo. The
2
4
residue was recrystallized (EtOAc, hexane) to give 2-methyl-1-(phenylsulfonyl)-1H-indole-4-
+
carbonitrile (113p) (500 mg, yield: 47.6%, purity: 96%). LRMS (M+H ) m/z: calcd. 297.1; found
1
297.1. HNMR (300 MHz, DMSO-d ): δ 8.37 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.74
6
(
t, J = 6.9 Hz, 2H), 7.64-7.59 (m, 2H), 7.46 (t, J = 8.4 Hz, 1H), 6.82 (s, 1H, 3-H of indole), 2.68
s, 3H, 2-Me).
(
Step 3: 2-Methyl-1H-indole-4-carbonitrile (93p). Method A: To a room temperature solution of
the intermediate (113p) (18.5 g, 62.5 mmol) in ethanol (125 mL) was added aqueous sodium
o
hydroxide solution (4 M, 47 mL, 188 mmol). Then the mixture was stirred at 40 C for 3 hours.
The resulting solution was concentrated in vacuo and diluted with water (50 mL) and ethyl
acetate (100 mL); the organic phase was separated, dried over sodium sulfate and concentrated in
vacuo. The residue was purified by column chromatography (silica gel, petroleum ether, ethyl
+
acetate) to give the compound (93p) (6.7 g, yield: 69%). LRMS (M+H ) m/z: calcd. 157.1; found
1
1
8
57.1. HNMR (300 MHz, DMSO-d ): δ 11.59 (s, 1H, NH), 7.61 (d, J = 8.1 Hz, 1H), 7.43 (d, J =
6
.1 Hz, 1H), 7.13 (t, J = 8.1 Hz, 1H), 6.31 (s, 1H, 3-H of indole), 2.45 (s, 3H, 2-Me).
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Step 3: 2-Methyl-1H-indole-4-carbonitrile (93p). Method B: To a room temperature solution of
the intermediate (113r) (1.14 g, 5.4 mmol) in NMP (30 mL) were added Zn(CN) (0.7 g, 6.0
2
mmol), Zn (75 mg, 1.1 mmol), dppf (1.2 g, 2.2 mmol), and Pd (dba) (1.0 g, 1.1 mmol). Then the
2
3
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resulting mixture was heated under an argon atmosphere at 120 °C for 18 hours. It was cooled to
room temperature and diluted with water (50 mL), then extracted with ethyl acetate (100 mL x
3
). The combined organic layers were washed with water and brine, dried over anhydrous
MgSO and concentrated in vacuo. The residue was purified by flash chromatography (silica gel,
4
petroleum ether, ethyl acetate) to give the desired compound (93p).
1
-(4-(Benzylamino)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-
carboxamide (65): Step 1: N-tert-Butyl 4-(benzylamino)-2-(4-cyano-2-methyl-1H-indol-1-yl)-
7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate. To a room temperature solution of 2-
methyl-1H-indole-4-carbonitrile (93p) (4.2 g, 26.7 mmol) and N-tert-butyl 4-(benzylamino)-2-
chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (74i) (10 g, 26.7 mmol) in 1,4-
dioxane (250 mL) was added cesium carbonate (13 g, 40 mmol). The mixture was degassed and
filled with nitrogen three times. Pd (dba) (3.66 g, 4 mmol), X-Phos (1.9 g, 4 mmol) then were
2
3
o
added. The resulting mixture was stirred at 100 C for 12 hours and cooled to room temperature.
The volatiles was evaporated in vacuo and the resulting residue was dissolved in methylene
dichloride (500 mL), washed with water (50 mL) and brine (30 mL x 2), dried over Na SO ,
2
4
filtered and evaporated in vacuo. The residue was purified by column chromatography (silica
gel, petroleum ether, ethyl acetate) to give the give N-tert-butyl 4-(benzylamino)-2-(4-cyano-2-
methyl-1H-indol-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (12.0 g, yield:
+
9
1%). LRMS (M+H ) m/z: calcd. 495.2; found 495.2.
Step
2:
tert-Butyl
4-(benzylamino)-2-(4-carbamoyl-2-methyl-1H-indol-1-yl)-7,8-
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dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (115) . To a room temperature mixture of the
aforementioned crude nitrile intermediate (150 mg, 0.30 mmol), PPh (9.4 mg, 0.036 mmol) and
3
Pd(OAc) (6.7 mg, 0.03 mmol) in ethanol (4 mL) and water (0.5 mL) was added acetaldehyde
2
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oxime (35.4 mg, 0.60 mmol). The resulting mixture was refluxed for 2 hours, cooled down to
room temperature and concentrated in vacuo. The residue was purified by flash chromatography
(silica gel, petroleum ether, ethyl acetate) to afford crude tert-butyl 4-(benzylamino)-2-(4-
carbamoyl-2-methyl-1H-indol-1-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate
+
1
(115) (120 mg, yield: 78%). LRMS (M+H ) m/z: calcd. 513.2; found 513.2. HNMR (300 MHz,
CDCl ): δ 8.13 (d, J = 8.1 Hz, 1H, Ph of indole), 7.49 (d, J= 7.5 Hz, 1H, Ph of indole), 7.35-7.30
3
(m, 5H, Ph of NHBn), 7.09 (t, J = 7.8 Hz, 1H, Ph of indole), 6.83 (s, 1H, 3-H of indole), 4.74 (s,
2H, CH Ph), 4.34 (s, 2H, NCH ), 3.76 (t, J = 5.7 Hz, 2H, NCH CH ), 2.90 (t, J = 5.7 Hz, 2H,
2
2
2
2
NCH CH ), 2.63 (s, 3H, Me), 1.51 (s, 9H, C(CH ) ).
2
2
3 3
Step 3: 1-(4-(Benzylamino)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-
o
4
-carboxamide (65). To a 0 C solution of the aforementioned crude Boc-protected intermediate
(
115) (11.5 g, 22.5 mmol) in methanol (500 mL) was bubbled hydrogen chloride slowly for 30
minutes. The resulting solution was concentrated in vacuo, the residue was dissolved in DCM
200 mL) and neutralized with ammonium hydroxide, the separated organic layer was dried over
(
sodium sulfate, concentrated in vacuo and purified by column chromatography (silica gel, DCM,
methanol) to give the desired final product (65) (8.0 g, yield: 86%, purity: 99.8%) as solid.
+
1
LRMS (M+H ) m/z: calcd. 413.2; found 413.1. HNMR (400 MHz, CD OD): δ 7.70 (d, J = 8.0
3
Hz, 1H, Ph of indole), 7.45 (d, J = 7.6 Hz, 1H, Ph of indole), 7.31-7.30 (m, 4H, Ph of NHBn),
7
.26-7.22 (m, 1H, Ph of NHBn), 6.96 (t, J = 7.6 Hz, 1H, Ph of indole), 6.77 (s, 1H, 3-H of
indole), 4.71 (s, 2H, CH Ph), 3.78 (s, 2H, NCH ), 3.16 (t, J = 6.0 Hz, 2H, NCH CH ), 2.77 (t, J =
2
2
2
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.0 Hz, 2H, NCH CH ), 2.43 (s, 3H, Me). CNMR (400 MHz, CD OD): δ 174.10, 161.77,
2
2
3
61.61, 156.15, 140.97, 140.36, 138.96, 129.58, 128.59, 127.95, 127.85, 125.79, 122.04, 121.87,
17.37, 110.11, 105.31, 45.29, 43.40, 42.87, 31.89, 15.51.
10
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-(4-(Benzylamino)-5,6,7,8-tetrahydroquinazolin-2-yl)-2-methyl-1H-indole-4-carboxamide
(69): a two-step procedure similar to the aforementioned was followed to couple intermediates
(
74h) and (93p) followed by oxidation using Pd(OAc) catalyzed oxidation with acetaldehyde
2
+
oxime to yield the desired molecule (69) (purity: 98%). LRMS (M+H ) m/z: calcd. 412.2; found
1
412.2. HNMR (400 MHz, CD OD): δ 7.63 (d, J = 8.4 Hz, 1H, Ph of indole), 7.46 (d, J = 7.6 Hz,
3
1
1
H, Ph of indole), 7.44-7.31 (m, 5H, Ph of Bn), 6.96 (t, J = 7.6 Hz, 1H, Ph of indole), 6.76 (s,
H, 3-H of indole), 4.72 (s, 2H, CH Ph), 2.74 (t, J = 5.6 Hz, 2H, CH ), 2.54 (t, J = 6.0 Hz, 2H,
2
2
CH ), 2.42 (s, 3H, Me), 1.94-1.92 (m, 4H, (CH ) ).
2
2 2
1
-[4-(Benzylamino)-5H,7H,8H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-
carboxamide (71): a two-step procedure similar to the aforementioned was followed to couple
intermediate (74k) with intermediate (93p) and then Pd(OAc)2 catalyzed oxidation with
+
acetaldehyde oxime to yield the desired molecule (71) (purity: 98.5%). LRMS (M+H ) m/z:
1
calcd. 414.2; found 414.1. HNMR (400 MHz, CD OD): δ 7.73 (d, J = 8.4 Hz, 1H, Ph), 7.46 (d,
3
J = 7.6 Hz, 1H, Ph), 7.34-7.29 (m, 4H, Ph), 7.27-7.24 (m, 1H, Ph), 6.97 (t, J = 7.6 Hz, 1H, Ph),
6
.78 (s, 1H, 3-H of indole), 4.71 (s, 2H, CH Ph), 4.64 (s, 2H, OCH ), 4.05 (t, J = 5.6 Hz, 2H,
2 2
13
OCH CH ), 2.82 (t, J = 5.6 Hz, 2H, OCH CH ), 2.45 (s, 3H, Me). CNMR (400 MHz, CD OD):
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δ 174.06, 160.71, 160.45, 156.45, 140.82, 140.42, 138.91, 129.58, 128.62, 127.95, 127.83,
25.75, 122.06, 121.91, 117.57, 109.72, 105.48, 65.68, 64.08, 45.24, 31.78, 15.72.
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Biology:
The ATPase assay is performed according to the following protocol: compounds were diluted in
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DMSO with a three-fold ten-point serial dilution starting at 10 M. The assay was done in 384-
well plate with each row as a single dilution series with duplicate of each compound
concentration points. In 5 L total volume, 20 nM p97 hexameric enzyme and 20 M ATP were
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added to start the reaction. The plate was sealed and incubated at 37 C for 15 minutes after
mixing thoroughly in an orbital shaker. Compound dilution, ATP and enzymes addition were
conducted with automated liquid handling using the Freedom Evo (Tecan Systems Inc., San Jose
CA). ADP Glo reagents 1 and 2 were added according to manufacturer’s protocol (Promega,
Madison, WI). The luminescence was measured by Envision plate reader as the end point of the
reaction. The IC50 of each compound was derived by fitting the luminescence values to a 4
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parameter sigmoidal curve.
A549 and other tumor cell lines were cultured according to ATCC guidelines. Cells were
cultured in black or white, clear-bottomed, tissue culture-treated 384-well plates. Cells were
treated with 10-point dose titration of the compound in well duplicates. After 72-hour treatment,
Cell Titer Glo (Promega, Madison, WI) was added to the white plates to measure cell viability.
Luminescence values were fit to a 4 parameter sigmoidal curve to determine IC concentrations.
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plates for 5 minutes following a 6-hour treatment with compound. Cells were then washed in
PBS and processed for immunofluorescence. Cells were blocked in 1 x phosphate buffered saline
(PBS) with 1% BSA, 0.3% Triton-X100 and Hoechst (1:10,000) for 1 hour and then incubated in
primary antibodies at 4 degrees Celsius for 16 hours. Primary antibodies used are as follows:
anti-Lys48 ubiquitin at 1:20,000 (05-1307, Millipore, Billerica, MA), anti-CHOP at 1:2,000 (SC-
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351, Santa Cruz, Biotechnology Inc., Santa Cruz, CA) and p62/SQSTM1 at 1:2,000 (SC-28359,
Santa Cruz, Biotechnology Inc., Santa Cruz, CA). Cells were washed 3 times in PBS and
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secondary antibodies were added for 2 hours at 25 C. Cells were washed 4 times in PBS and
imaged with an automated wide field fluorescence microscope. Automated image analysis was
written in Matlab (Mathworks, Natick, MA). Cellular intensities for each marker were measured.
Fluorescence intensity values were fit to a 4 parameter sigmoidal curve to determine IC50
concentrations for each marker.
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All mice were maintained in the Cleave Biosciences animal vivarium, and all in vivo
experiments were performed in compliance with applicable regulations and institutional
guidelines and had been approved by the Cleave Biosciences IACUC.
Abbreviations used: VCP, valosin-containing protein; CDC48, cell division cycle 48; AAA,
ATPases associated with diverse cellular activities; UPS, ubiquitin-proteasome system; ER,
endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; UPR, unfolded
protein response; HTS, high-throughput screening; SAR, structure and activity relationship;
p97i, p97 inhibition; CTG, cell titer Glo; CHOP, CCAAT/enhacncer-binding homologous
protein; p62, sequestosome 1; PARP, poly (ADP-ribose) polymerase; SGF, simulated gastric
fluids; SIF, simulated intestinal fluids; MLM, mice liver microsome; cLogP, calculated LogP;
PSA, molecular polar surface area; LipE, lipophilic efficiency; i.v., intravenous administration;
p.o., oral administration; PK, pharmacokinetics; PD, pharmacodynamics; F%, absolute
bioavailability.
Acknowledgment: We thank Professor Ray Deshaies at California Institute of Technology,
Assistant Professor Tsui-Fen Chou at Harbor-ULCA and Dr. Frank J. Schoenen and Dr. Kelin
Li at University of Kansas for sharing their SAR knowledge on discovery of ML240; Special
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