Synthesis and structure-activity relationship studies of N -benzyl-2-phenylpyrimidin-4-amine derivatives as potent usp1/uaf1 deubiquitinase inhibitors with anticancer activity against nonsmall cell lung cancer

Article abstract of DOI:10.1021/jm5010495

Deregulation of ubiquitin conjugation or deconjugation has been implicated in the pathogenesis of many human diseases including cancer. The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1), in association with UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been shown as a promising anticancer target. To further evaluate USP1/UAF1 as a therapeutic target, we conducted a quantitative high throughput screen of >400000 compounds and subsequent medicinal chemistry optimization of small molecules that inhibit the deubiquitinating activity of USP1/UAF1. Ultimately, these efforts led to the identification of ML323 (70) and related N-benzyl-2-phenylpyrimidin-4-amine derivatives, which possess nanomolar USP1/UAF1 inhibitory potency. Moreover, we demonstrate a strong correlation between compound IC₅₀ values for USP1/UAF1 inhibition and activity in nonsmall cell lung cancer cells, specifically increased monoubiquitinated PCNA (Ub-PCNA) levels and decreased cell survival. Our results establish the druggability of the USP1/UAF1 deubiquitinase complex and its potential as a molecular target for anticancer therapies.

Full text of DOI:10.1021/jm5010495

Article
pubs.acs.org/jmc
Open Access on 09/17/2015
Synthesis and Structure−Activity Relationship Studies of N‑Benzyl-2-
phenylpyrimidin-4-amine Derivatives as Potent USP1/UAF1
Deubiquitinase Inhibitors with Anticancer Activity against Nonsmall
Cell Lung Cancer
Thomas S. Dexheimer,†,§ Andrew S. Rosenthal,†,§ Diane K. Luci,† Qin Liang,‡ Mark A. Villamil,‡
Junjun Chen,‡ Hongmao Sun,† Edward H. Kerns,† Anton Simeonov,† Ajit Jadhav,† Zhihao Zhuang,*,‡
and David J. Maloney*,†
†National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville,
Maryland 20850, United States
‡Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, Delaware 19716, United States
S
* Supporting Information
ABSTRACT: Deregulation of ubiquitin conjugation or deconjugation has been implicated in the pathogenesis of many human
diseases including cancer. The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1), in association with UAF1 (USP1-
associated factor 1), is a known regulator of DNA damage response and has been shown as a promising anticancer target. To
further evaluate USP1/UAF1 as a therapeutic target, we conducted a quantitative high throughput screen of >400000
compounds and subsequent medicinal chemistry optimization of small molecules that inhibit the deubiquitinating activity of
USP1/UAF1. Ultimately, these efforts led to the identification of ML323 (70) and related N-benzyl-2-phenylpyrimidin-4-amine
derivatives, which possess nanomolar USP1/UAF1 inhibitory potency. Moreover, we demonstrate a strong correlation between
compound IC50 values for USP1/UAF1 inhibition and activity in nonsmall cell lung cancer cells, specifically increased
monoubiquitinated PCNA (Ub-PCNA) levels and decreased cell survival. Our results establish the druggability of the USP1/
UAF1 deubiquitinase complex and its potential as a molecular target for anticancer therapies.
to serve multiple nonproteolytic functions.3−6 Similar to other
types of post-translational modifications, ubiquitination is a
INTRODUCTION
■
Ubiquitin is a small, highly conserved protein of 76 amino acids
reversible process counter-regulated by enzymes known as
that is post-translationally conjugated to substrate proteins,
deubiquitinases (DUBs), which catalyze the removal of
including itself, via a three-step enzymatic reaction. The initial
covalent attachment primarily occurs between the C-terminal
ubiquitin from modified proteins.7 More importantly, dysfunc-
tion in ubiquitin-dependent signaling pathways has been linked
to various human diseases, suggesting inhibition of ubiquitin
pathway components as novel therapeutic targets for drug
discovery.
Pharmacological intervention within the ubiquitin−protea-
some system has been well established by the successful use of
glycine of ubiquitin and the ε-amino group of lysine residue(s)
of the target protein.1 Additional ubiquitin molecules can be
ligated to one of the seven internal lysines of ubiquitin,
resulting in diverse ubiquitin chain topologies. The biological
outcome of ubiquitination is determined by both the length and
linkage topology.2 For example, Lys48-linked polyubiquitin
chains are almost exclusively associated with allocating proteins
for proteasome-dependent degradation, while monoubiquitina-
tion or chains linked through different lysines have been shown
Received: July 11, 2014
© XXXX American Chemical Society
A
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proteasome inhibitors for anticancer treatment.8 Nevertheless,
the ubiquitin−proteasome system offers additional opportu-
nities for therapeutic intervention that could afford increased
specificity and the possibility for improved clinical efficacy,
which proteasome inhibitors are currently lacking.9 The most
apparent targets include enzymes involved in ubiquitin
conjugation and deconjugation (i.e., ubiquitin ligases and
DUBs), processes upstream of proteasome-mediated protein
degradation. Among the DUBs, ubiquitin-specific protease 1
(USP1) has become an attractive anticancer target because of
its involvement in regulating DNA damage response pathways.
USP1 associates with UAF1 (USP1-associated factor 1),
resulting in the heterodimeric USP1/UAF1 complex that is
required for deubiquitinase activity.10 The USP1/UAF1
complex has been shown to regulate the tolerance of DNA
damage induced by DNA cross-linking agents through
deubiquitination of PCNA (proliferating cell nuclear antigen)11
and FANCD2 (Fanconi anemia complementation group D2),12
which are proteins that function in translesion synthesis and the
Fanconi anemia pathway, respectively. Williams et al. also
recently reported that USP1/UAF1 deubiquitinates and
prevents proteasomal degradation of ID (inhibitor of DNA-
binding) proteins,13 which have been shown to activate
multiple pathways involved in tumor progression, including
preservation of the cancer stem cell phenotype.14 Moreover,
evidence for aberrant overexpression of USP1 in several tumor
types suggests that inhibitors of USP1 are likely to provide a
therapeutic benefit.13,15
Several small-molecule inhibitors of USP1/UAF1 have been
identified by high-throughput screening. We initially identified
the first USP1/UAF1 inhibitors, pimozide and GW7647, from a
collection of bioactive molecules.16 Although GW7647 and
pimozide display reasonable selectivity over related proteases
and demonstrate cellular inhibition of human USP1, both
compounds show moderate PubChem promiscuity with a 9.5%
and 11.4% hit rate, respectively, and have annotated activity
against unrelated targets.17−19 Since then, Mistry et al. reported
C527 and related analogues, which exhibit submicromolar
inhibition (IC50 = 0.88 μM) of USP1/UAF1 activity, promote
ID1 degradation, and cause cytotoxicity in several leukemic cell
lines.20 However, C527 displays limited selectivity for USP1/
UAF1 in vitro. We recently described the discovery and
biological activity of ML323 (70) (Figure 1), a novel small-
molecule inhibitor of USP1/UAF1. Compound 70 was found
to exhibit selective, nanomolar inhibition of USP1/UAF1
deubiquitinase activity, leading to an increase in monoubiquiti-
nated forms of PCNA and FANCD2 and synergistic interaction
with cisplatin in human tumor cell lines.21 Herein, we describe
the medicinal chemistry efforts that led to the selection of 70 as
a chemical probe and provide biological activity and in vitro
ADME data for additional analogues.
Figure 1. Structures of previously identified USP1/UAF1 inhibitors
and ML323.
was then reacted with a variety of amines to provide analogues
2−21 (Table 1).23 Notably, compound 1 could also be
synthesized in this manner using thiophen-2-ylmethanamine in
step d.
The synthesis of analogues 22−53 and 71−75 were all
achieved in a similar manner using (4-(pyridin-3-yl)phenyl)-
methanamine for the amino side chain but differed in the
dichloropyridimide-based scaffold starting material as shown in
Scheme 1B. The preparation of analogues 22−36 involved
addition of (4-(pyridin-3-yl)phenyl)methanamine to 2,4-
dichloroquinazoline to obtain the desired chloropyridimide
intermediate (iv). Suzuki coupling of iv with various boronic
acids using Pd(PPh3)4 in a microwave reactor for 30 min at 150
°C gave analogues 22−33. For subsequent analogues, we
changed to a silica bound DPP-palladium catalyst in order to
alleviate any palladium contamination concerns. The synthesis
of compounds 34−36 was conducted using the same sequence.
However, instead of a Suzuki coupling, intermediate iv was
heated in a sealed tube for 24 h at 100 °C with an excess of
requisite amine. The synthetic route for compounds 37−53
(Table 3) was performed as described above starting from
other commercially available 2,4-dichloropyrimidine hetero-
cycles, and 2-isopropylphenyl boronic acid was utilized in the
cross coupling reaction.
Compounds 54−69 were synthesized using 2,4-dichloro-5-
methylpyrimidine (v) as the starting material along with various
commercially available heterocyclic and aromatic methan-
amines, followed by a Suzuki coupling with 2-isopropylphenyl
boronic acid (Scheme 2). The tetrazole analogue (68) was
obtained via reaction of the 4-CN-Ph moiety with sodium azide
in DMF. Compounds 62−67 were synthesized by reacting
commercially available tert-butyl 4-(aminomethyl)piperidine-1-
carboxylate (for 62−65), tert-butyl-3-(aminomethyl)-
pyrrolidine-1-carboxylate (for 67), or tert-butyl 3-
(aminomethyl)azetidine-1-carboxylate (for 66), with 2,4-
dichloro-5-methylpymidine, followed by the aforementioned
Suzuki coupling conditions. The elevated temperatures used in
CHEMISTRY
■
To initiate our medicinal chemistry efforts, we began by
resynthesizing the HTS hit molecule (1), which was prepared
as described in Scheme 1A/B.21 The synthesis of related
analogues (2−21) commenced with an EDC-mediated amide
coupling between 2-aminobenzamide (i) and 2-9-
(trifluoromethyl)benzoic acid as shown in Scheme 1A.22 The
resulting diamide was heated to reflux with sodium hydroxide
for 3 h to provide the cyclized quinazoline scaffold, which upon
treatment with POCl3 gave the key intermediate 4-chloro-2-(2-
(trifluoromethyl)phenyl)quinazoline (ii). This intermediate
B
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a
Scheme 1. Synthesis of Analogues 1−53 and 71−75
a
Reagents and conditions: (a) 2-(trifluoromethyl)benzoic acid, EDC (1.2 equiv), HOBt (1.2 equiv), Et3N (3.0 equiv), CH2Cl2, 72 h; (b) 5% NaOH,
reflux 3 h; (c) N,N-dimethylaniline, POCl3, toluene, reflux 1 h; (d) RNH2, i-Pr2NEt, DMF, 18 h, 60 °C; (e) for analogue 1, [thiophen-2-
ylmethanamine]; for analogues 22−53 and 71−75, [(4-(pyridin-3-yl)phenyl)methanamine], Et3N, CHCl3, 18 h, 60 °C; (f) for analogues 22−33,
37−53, and 71−75, R′B(OH)2 (3 equiv), 2 M Na2CO3 (4 equiv), DPP-Pd silica bound (0.3 equiv), or Pd(PPh3)4 (0.2 equiv), DME, MW, 150 °C,
30−45 min; (g) for analogues 34−36, R′NH2 (3.0 equiv), THF, sealed tube 100 °C, 24 h.
a
Scheme 2. Synthesis of Analogues 54−69
a
Reagents and conditions: (a) 4-R-amine (1.1 equiv), Et3N (3.0 equiv), DMF, 100 °C 18 h; (b) 2-isopropylphenyl boronic acid (3.0 equiv), 2 M
NaHCO3 (4.0 equiv), DPP-Pd silica bound Silicycle, DME, MW, 150 °C, 30 min; (c) for R = 4-CN-Ph, NaN3 (8.0 equiv), NH4Cl (8.0 equiv), DMF
130 °C, 30 min; (d) t-butyl 4-(aminomethyl)-R-1-carboxylate (1.1 equiv), Et3N (3.0 equiv), DMF, 100 °C 18 h; (e) 2-isoproylphenyl boronic acid
(3.0 equiv), 2 M NaHCO3 (4.0 equiv), DPP-Pd silica bound Silicycle, DME, MW, 150 °C, 30 min; (f) for analogues 63−64, RBr (1.2 equiv),
Cs2CO3, dicyclohexyl(2′,4′,6′-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, RuPhos palladacycle, toluene, sealed tube, 100 °C, 18 h; (g) for
analogues 65−67, RBr (1.2 equiv), NaOtBu, Pd2(dba)3 (0.02 equiv), DavePhos (0.02 equiv) xylenes, sealed tube, 100 °C, 1 h; (h) for analogue 62,
oxetan-3-one, sodium triacetoxyborohydride (10 equiv), DCE, 2 h.
the Suzuki coupling resulted in concomitant removal of the N-
Boc group to provide intermediates viii. Buchwald-Hartwig
conditions for the cross coupling of the requisite amine and the
heteroaryl bromides gave the desired products 63−67.24,25
Initially, we utilized the DavePhos ligand, however, this was met
with limited success. Subsequent analysis of other ligands
revealed that the RuPhos palladacycle proved optimal for
compounds 63−64, whereas compounds 65−67 worked best
with DavePhos. Oxetane analogue (62) was obtained via a
reductive amination of intermediate viii (n = 2) and
commercially available oxetan-3-one, with sodium triacetox-
yborohydride.
Compounds 70 (ML323, Table 4) and 76−84 (Table 5),
were synthesized starting with 4-amino-benzonitrile as a TFA
salt, trimethylsilyl azide, and t-butyl nitrite at 0 °C to give the
desired azide in 98% yield (Scheme 3).26 The resulting azide
was dissolved in a mixture of DMSO/H2O, treated with copper
sulfate, ethynyltrimethylsilane, sodium ascorbate, and heated
for 24 h at 80 °C to give the desired 1,2,3-triazole. For the
synthesis of the deuterated benzyl amine derivatives, we initially
tried using the H-Cube flow-reactor with D2O but incomplete
reduction and deuterium enrichment was observed. As such,
the reduction was then carried out with lithium aluminum
deuteride (LAD) to give the desired product in moderate yield
(40%).27 Our initial attempts for the nitrile reduction on the H-
C
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a
Scheme 3. Synthesis of Analogues 70 and 76−84
a
(a) TFA (1.0 equiv), rt, 5 min then t-butyl nitrite (1.5 equiv), azidotrimethylsilane (1.4 equiv), 0 °C, 30 min, 98% yield; (b) ethynyltrimethylsilane
(3.0 equiv), sodium ascorbate (0.8 equiv), Cu(II)SO4 (0.07 equiv), DMSO/H2O (ratio 4/1) 80 °C 24 h; (c) TFA (1 equiv), acetonitrile, reflux, 1.5
h (57%); (d) H-Cube Pro, 10% Pd/C, 50 °C, 40 bar, TFA (1.2 equiv), MeOH/DMF (ratio 10/1) (98%); (e) lithium aluminum deuteride (LAD)
(3.0 equiv), THF, 0 °C, 1.5 h (40%); (f) 2,4-dichloro-5-methylpyrimidine, Et3N (3 equiv), DMF, 100 °C, 18 h; (g) (2-isopropylphenyl)boronic acid
(3.0 equiv), 2 M NaHCO3 (4.0 equiv), DPP-Pd silica bound Silicycle, DME, 150 °C, 30 min, 35−70% yield.
Cube with a 70 mm Catcart (10% Pd/C) at 50 °C and 40 bar
returned just trace amounts of product. However, the addition
of TFA and changing to a MeOH/DMF mixture resulted in
complete conversion to the amine (by LC-MS analysis). Given
the water solubility of the 4-triazole-benzyl amine intermediates
(x), it was used directly in the next step without further
purification or characterization. The resulting mixture was
heated for 18 h with 2,4-dichloro-5-methylpyrimidine and Et3N
to give derivative xi in moderate yields of (∼40%) over the two
steps. Compound xi was then coupled to the 2-isopropylphenyl
boronic acid as described previously to give the final compound
70. Compounds 76−84 were synthesized as described above,
however, several of the required boronic acids were not
commercially available and had to be synthesized separately.
For the deuterated analogues 80−82 and 84, the requisite d6
and d7-2-isopropylphenyl boronic acids were prepared in four
steps starting from 2-bromoacetophenone (see Supporting
Information for details).
RESULTS AND DISCUSSION
■
After resynthesis of the thiophene-containing HTS hit (1) and
confirmation of its activity in both the primary Ub-rhodamine
qHTS assay and orthogonal gel-based diubiquitin assay, we
decided to focus our initial SAR exploration efforts around the
thiophene moiety (northern portion). Despite the modest
activity against USP1/UAF1 (Table 1; IC50 = 7.9 μM), we were
a
Table 1. USP1-UAF1 Inhibition of Analogues (1−21)
The synthesis of the oxetane containing analogues 72 and 77
involved preparation of 3-(2-bromophenyl)oxetane (xii) using
a method described by Jacobsen et al. for a comparable phenyl
oxetane derivative.28 Formation of the (2-(oxetan-3-yl)phenyl)-
boronic acid (xiii) was achieved in situ via treatment of xii with
N-butyllithium and triisopropyl borate in THF at −78 °C and
subsequently used as-is with vi or xi to provide compounds 72
and 77, respectively (Scheme 4).
a
Scheme 4. Synthesis of Analogues 72 and 77
a
IC50 values represent the half-maximal (50%) inhibitory concen-
tration as determined in the HTS assay (n ≥ 3).
encouraged by the selectivity of this compound, with no
significant activity against a small panel of DUBs and
appreciable inhibition of only one of 451 kinases.21 Initial
changes to the thiophene functionality were generally well
tolerated, with only slight changes in potency being observed.
Replacing the thiophene moiety with a simple phenyl ring (3)
led to a modest improvement in potency (3.1 μM), however,
most substituted (electron-withdrawing and electron-donating
a
(a) N-butyllithium (2.0 equiv), triisopropyl borate (2.0 equiv) THF,
−78 °C, 18 h; (b), 2 M Na2CO3 (4.0 equiv), DPP-Pd silica bound
Silicycle 0.26 mmol/g (20 mol %), DME, MW, 150 °C, 30 min, 35−
50% yield.
D
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groups) phenyl rings (analogues 4−10) demonstrated fairly flat
SAR. We were encouraged that substitution at the 4-position
with a phenyl ring (e.g., 4-phenyl benzyl amine 12) was well
tolerated with a comparable IC50 value of 3.7 μM. This allowed
for a variety of different analogues to be synthesized with
representative examples (analogues 13−21) shown in Table 1.
Potencies approached 1 μM with both the 4-pyridine (16) and
3-pyridine (17) with IC50 values of 1.9 and 1.1 μM,
respectively. Given the comparable potency of these two
analogues and the propensity of 4-pyridine derivatives to
interact with CYPs, we decided to proceed with the 3-pyridine
group for the next round of analogues which were aimed at
modifying the 2-CF3-phenyl group and to investigate core
scaffold changes.
over the original HTS hit compound 1 from 4.7 to 0.18 μM
(26-fold), we decided to turn our attention toward modification
of the quinazoline core.
Initial data from analogues in our qHTS collection suggested
that replacement of the quinazoline core with a pyrimidine
would be tolerated. This change would be beneficial in that it
would reduce the molecular weight and lipophilicity of the lead
compound. Gratifyingly, this modification was in fact tolerated
and resulted in a compound with comparable potency (37,
Table 3). Introduction of a 5-methyl group (38) resulted in a
a
Table 3. USP1-UAF1 Inhibition of Analogues (37−53)
Exploration of the SAR around the 2-CF3-phenyl group
indicated that substitution at the 2-position was greatly favored
compared to the 3- and 4-positions (Table 2). This finding is
a
Table 2. USP1-UAF1 Inhibition of Analogues (22−36)
compd
R
IC50 (μM)
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
3-CF3-Ph
4-CF3-Ph
2-NO2-Ph
2-OMe-Ph
2-Me-Ph
2-Et-Ph
inactive
inactive
7.8 2.4
0.94 0.35
1.1 0.4
0.78 0.17
0.18 0.06
1.4 0.3
6.5 3.0
2.1 1.0
1.6 0.6
inactive
2-iPr-Ph
2-CH(F)2-Ph
2-F-Ph
2-Cl-Ph
2-Br-Ph
cyclopentyl
morpholine
pyrrolidine
imidazole
inactive
inactive
inactive
a
IC50 values represent the half-maximal (50%) inhibitory concen-
a
IC50 values represent the half maximal (50%) inhibitory concen-
tration as determined in the HTS assay (n ≥ 3); inactive denotes an
IC50 > 57 μM.
tration as determined in the HTS assay (n ≥ 3).
∼2-fold increase in potency with an IC50 value of 70 nM.
Interestingly, moving the methyl group to the 6-position (39)
resulted in a 3-fold decrease in potency (210 nM). The 5,6-
dimethyl derivative (40) was also well tolerated as was the
cyclopentylpyrimidine analogue 45, with IC50 values of 120 and
160 nM, respectively. Incorporation of other heteroaromatic
core scaffolds (41−44 and 46−48) provided compounds with
good potency, with the most potent being the furan derivative
48. Other groups such as OMe (49), F (50), NH2 (51), NMe2
(52), and SMe (53) provided good potency with IC50 values of
70, 110, 310, 190, and 110 nM, respectively. However, as stated
above, our interest in these structural modifications was to
improve or maintain potency while reducing molecular weight.
Therefore, we decided to continue our SAR explorations with
the 5-methyl-pyridimine (38) as the core scaffold given the
potent inhibition (70 nM) and reduced size.
exemplified by the inactivity of the 3-CF3-phenyl (22) and 4-
CF3-phenyl (23) derivatives. Given these results, we decided to
focus on exploring SAR at the 2-position by replacing the CF3
group with various electron-donating/withdrawing groups as
well as varying the steric bulk in this portion of the molecule.
Incorporation of the electron-withdrawing group; e.g., 2-NO2
(24) led to a 7-fold loss in activity, whereas the electron-
donating group, e.g., 2-OMe (25), provided comparable activity
(IC50 = 0.94 μM). Replacement with the simple alkyl
substituents (R = 2-Me, 26 or R = 2-Et, 27) also provided
comparable activity to the 2-CF3 group. Our biggest potency
improvement (6-fold) occurred when we replaced the 2-CF3
with an isopropyl group (28), which had an IC50 of 180 nM.
Changing the methyl groups of the isopropyl moiety to fluoro
groups (29) led to an appreciable decrease in potency. Other
electron-withdrawing groups were prepared (analogues 30−
32), yet none of these had improved activity. Finally,
modification of the phenyl ring to a cyclopentyl group (33)
or nitrogen containing heterocycles (34−36) resulted in
inactive compounds. Having already improved the potency
Having established the 5-methyl pyrimidine core and the 2-
isopropyl group as optimal, we then decided to return to
exploration of the “northern portion” SAR. Despite the potency
of the terminal 3-pyridine group, some initial ADME data
suggested that this group may be a metabolic liability.
E
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a
Replacement of the 4-(3-pyridine)-benzyl amine with a simple
3-methyl-phenyl (54), 3-pyridine (55), or thiophene (56)
generally showed good potency in the Ub-Rho assay (130,
1300, and 270 nM, respectively), however, none of these had
comparable potency to 38 in the orthogonal diubiquitin assay
(data not shown). In an attempt to increase the hydrophilicity
of the compound, a branched hydroxymethyl group (57) was
introduced; however, this led to a large decrease in potency.
Replacement of the phenyl group with an N-Me-piperidine
(58) led to complete loss of activity. Given these data, we
returned our focus to the two ring analogues (59−70). Adding
a fluoro (59) or methyl (60) group, α to the pyridine nitrogen,
was well tolerated (59, IC50 = 80 nM; 60, IC50 = 50 nM), which
could help alleviate potential CYP interactions if necessary.
However, these changes increase lipophilicity, which is
something we were trying to avoid if possible. Looking to
increase the sp3 character of this side chain, we replaced the
central phenyl group with a piperidine (analogues 62−65),
which generally resulted in comparable potency with exception
of oxetane derivative 62. However, the corresponding four-
membered (66) and five-membered (67) rings resulted in a
loss of potency with IC50 values of 21.7 and 0.8 μM,
respectively. These data seem to indicate a size preference for
six-membered ring with the potency rank order being phenyl ≈
piperidine > pyrollidine ≫ azetidine. In an attempt to attenuate
the potential metabolic liability of the 3-pyridine moiety, several
other nitrogen containing heterocycles were prepared and
tested (analogues 68−70). The imidazole derivative (69)
demonstrated moderate potency (300 nM), whereas the
tetrazole (68) resulted in a significant decrease in potency.
The latter result is unsurprising given the pKa differences in
these heterocycles and the general preference for basic moieties
in this position (e.g., pyridine). However, we were particularly
intrigued by the 1,2,3-triazole derivative (70, IC50 = 76 nM),
given its improved metabolic stability, cell permeability, and
activity in other biological assays (vide infra).21
Table 4. USP1-UAF1 Inhibition of Analogues (54−70)
a
IC50 values represent the half-maximal (50%) inhibitory concen-
tration as determined in the HTS assay (n ≥ 3); inactive denotes an
IC50 > 57 μM.
a
Table 5. USP1-UAF1 Inhibition of Analogues (38, 70−84)
Compound 70 demonstrated modest metabolic stability
(T1/2 = 15 min) as shown in Table 5, and Met ID studies (see
Supporting Information, Figure S1) indicated that the two
major metabolites involving oxidative removal of the N-benzyl
group and hydroxylation of the isopropyl group. As such, in an
effort to improve the half-life we first focused on the benzylic
position and synthesized gem-dimethyl analogues 79 and 82
and deuterated analogues 83 and 84. Direct hydrogen
abstraction is thought to be the rate-limiting step for CYP-
mediated hydroxylation of aliphatic compounds, and C−H
bond cleavage is approximately 6−10 times faster than the
corresponding C−D bond (kinetic isotope effect).29,30 Thus,
replacement of metabolically labile hydrogen atoms with
deuterium has been an attractive concept in drug discovery,
as the change should have a negligible effect on the
physicochemical properties yet improve metabolic stability.10
Accordingly, we sought to utilize this strategy at both the
benzylic position and 2-isopropyl moiety in effort to obviate
metabolic instability. We thought this approach was particularly
attractive for the 2-isopropyl group because our SAR efforts
revealed this to be critical for activity and not easily replaced.
Unfortunately, despite thorough investigation of various
deuterated analogues (80−84), we did not notice any
appreciable improvement in the metabolic half-life of this
series. Analogue 79, which possesses the gem-dimethyl group,
did show a moderate improvement in our studies, however, it
remained below the desired stability of >30 min. In an attempt
b
compd
R1
R2
IC50 (μM)
T1/2 (min)
38
71
72
73
74
75
70
76
77
78
79
80
81
82
83
84
CH(CH3)2
cyclopropane
oxetane
H
0.07 0.03
0.18 0.07
inactive
5.8
6.2
>30
ND
ND
ND
15
H
H
CH2OH
H
10.5 1.9
2.0 0.8
CH(OH)CH3
C(O)CH3
CH(CH3)2
cyclobutane
oxetane
H
H
8.7 2.1
H
0.076 0.014
0.18 0.09
inactive
H
5.1
>30
21
H
CHF2
H
0.82 0.25
0.59 0.12
0.14 0.04
0.10 0.04
0.76 0.14
0.12 0.05
0.19 0.04
CH(CH3)2
CH(CD3)2
CD(CD3)2
CD(CD3)2
CH(CH3)2
CD(CD3)2
CH3
H
22
7.9
9.5
10
H
CH3
D
12
D
13
a
IC50 values represent the half-maximal (50%) inhibitory concen-
tration as determined in the HTS assay (n ≥ 3); inactive denotes an
IC50 > 57 μM. Represents the half-life in minutes of the compounds
b
in the presence of rat liver microsomes (with NADPH).
F
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Figure 2. Disruption of USP1/UAF1-catalyzed deubiquitination of PCNA in H1299 cells. (A) H1299 cells were treated with the indicated
compounds at 20 μM for 4 h. Whole cell extracts were separated on 4−12% gradient SDS-PAGE and subjected to Western blotting with antibodies
against PCNA and GAPDH (loading control). (B) Fold changes in monoubiquitinated PCNA (Ub-PCNA) were calculated by normalizing to
unmodified PCNA and GAPDH. The data represent the mean SEM of two biological replicates. (C) Correlation between mean IC50 values
obtained in the HTS assay and fold change in Ub-PCNA in H1299 cells (Pearson r = −0.988; P < 0.0001).
Figure 3. Effects of USP1/UAF1 inhibition on cell survival in H1299 cells. (A) Representative colony formation assays (CFA) in H1299 cells after
treatment with indicated compounds. Cells were grown in the absence or presence of 5 μM of compound for 7 days and stained with crystal violet.
(B) Correlation between mean IC50 values obtained in the HTS assay and percent survival at 5 μM in H1299 cells (Pearson r = 0.967; P = 0.0004).
(C) Correlation between fold change in Ub-PCNA (see Figure 2A,B) and percent survival at 5 μM in H1299 cells (Pearson r = −0.954; P = 0.0008).
(D) Survival curve using CFA in H1299 cells treated with increasing concentrations of 70 or 25 μM of 77.
to address the undesired aliphatic hydroxylation at isopropyl
group, we synthesized the cyclopropyl (71), cyclobutyl (76),
oxetane (72 and 77), and difluoro (78) derivatives. While the
cyclopropyl (71) and cyclobutyl (76) analogues demonstrated
potent inhibition (IC50 = 180 nM), the metabolic stability was
not improved, with T1/2 = 6.2 and 5.1 min, respectively.
Replacement of lipophilic moieties with an oxetane group has
been shown to improve the metabolic stability and solubility of
the parent compound.31 Interestingly, this result was realized in
our case, as both oxetane derivatives (72 and 77) had favorable
metabolic stability (T1/2 > 30 min) and solubility (data not
shown), however, they had lost all activity toward USP1/UAF1.
While disappointed by this result, we decided to use compound
77 as our inactive control in subsequent biological studies,
making it a vital analogue. The preference for having lipophilic
moieties at the 2-position of the phenyl ring was further
solidified with the testing of the alcohol (73−74) and ketone
(75) analogues, which exhibited potencies in the micromolar
range.
required for USP1/UAF1 inhibition. Thus, we sought to
further evaluate the cellular activity of a number of selected
compounds. We initially assessed the ability of the compounds
to modulate USP1/UAF1 activity in cells by monitoring the
level of monoubiquitinated PCNA (Ub-PCNA). Upon treat-
ment of H1299 nonsmall cell lung cancer cells with select
compounds (1, 64, 70, and 77), we observed a dose-dependent
increase in Ub-PCNA compared to both the untreated control
and the inactive oxetane analogue (77) (see Supporting
Information, Figure S2). Importantly, an increase in Ub-
PCNA was observed at concentrations as low as 1 μM for top
compounds (e.g., 64 and 70). To ensure that a robust increase
in Ub-PCNA was observed, additional compounds were tested
at a fixed concentration of 20 μM. As shown in Figure 2A,B,
several compounds significantly increased the level of Ub-
PCNA. More importantly, we observed a linear correlation
between the logarithm of IC50 values obtained in the HTS assay
and fold change in Ub-PCNA (R2 = 0.98, Figure 2C). In
addition to validating the reliability of the HTS assay, these
results further support the on-target effects of this compound
Our detailed SAR explorations around compound 1 led to a
sufficient understanding of the structural features that are
G
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a
Table 6. In vitro ADME Profile
PBS buffer (pH 7.4)
solubility (μM)
Log D
(pH 7.4)
microsomal stability
(T1/2 in min)
Caco-2 (A→B) Papp
efflux
ratio
PBS buffer (pH 7.4)
stability (48 h) (%)
mouse plasma
stability (2 h) (%)
compd
(10−6 cm/s)
70
86
1.97
15
RLM
26
HLM
15
MLM
22.98
0.9
99
99
a
Aqueous solubility (PBS buffer), mouse liver microsome (MLM) human liver microsome stability (HLM), Caco-2, and plasma stability studies
were conducted at Pharmaron Inc. The Log D data was conducted by Analiza Inc. using a miniaturized shake-flask methodology. The rat liver
microsome (RLM) studies were conducted at NCATS. The microsomal stability data represents the stability in the presence of NADPH. Compound
70 showed no degradation without NADPH present over a 1 h period.
series in preventing the USP1/UAF1-catalyzed deubiquitina-
tion of PCNA in human cells.21
which is likely a result of the marginal phase I metabolic
stability discussed above.
We next examined the effects of these compounds on cell
survival using a colony formation assay. As shown in Figure 3A,
several of the compounds markedly inhibited the colony-
forming capacity of H1299 cells relative to DMSO-treated
control. Five compounds (48, 49, 64, 69, and 70) caused
greater than 70% reduction of colony formation at 5 μM,
whereas the original HTS hit (1) and the inactive oxetane
analogue (77) had minimal or no effect on cell survival,
respectively. A direct correlation between IC50 values for
USP1/UAF1 inhibition and cell survival was identified (R2 =
0.93, Figure 3B). As anticipated, the fold change in Ub-PCNA
also correlated with cell survival (R2 = 0.91, Figure 3C),
suggesting PCNA monoubiquitination as a candidate pharma-
codynamic biomarker predictive of efficacy. Compound 70 was
further evaluated in dose−response and demonstrated to have
an EC50 of 3.0 μM, while the inactive analogue 77 had no effect
on cell survival up to 25 μM (Figure 3D). These results suggest
that USP1/UAF1 inhibitors could show promise as a
monotherapy despite being initially envisioned as a combina-
tion therapy with DNA damaging agents.16,21,32
In Vitro ADME Profile. Having already evaluated the
selectivity of this molecule against numerous DUBs, human
proteases, and kinases,21 we next wanted to determine ancillary
pharmacology against GPCR and ion channel and transporter
targets. To do so, we tested compound 70 at 10 μM in the
CEREP LeadProfilingScreen2, which contains 80 different
GRCRs, ion channels, and transporters. Once again, compound
70 demonstrated an encouraging selectivity profile, with only 7
targets showing inhibition of >50% at 10 μM (5-HT2A,2B,2C, A3,
Ca2+ channel, MT1, and PPARγ) (see Supporting Information,
Figure S4). A selection of the in vitro ADME attributes of
compound 70 is highlighted in Table 6. Notably, compound 70
possesses good kinetic solubility in PBS buffer (pH 7.4) of 86
μM, and the experimental Log D (pH 7.4) of 1.97 is well within
the ideal range for orally bioavailable drug-like molecules. This
value was obtained by Analiza Inc. using their “scaled-down
shake flask lipophilicity” method. As anticipated, compound 70
is stable in PBS buffer, assay buffer, pH 2, pH 9 (see Supporting
Information, Figure S3, and mouse plasma (Table 6).
Compound 70 also exhibits excellent Caco-2 permeability
[Papp = 23 (10−6 cm/s)] and shows no signs of efflux (ratio =
0.9), which seems to indicate the compound is not subject to
the action of transporters, such as Pgp. One remaining liability
is the less than optimal microsomal stability, with an observed
T1/2 of 15 min (rat and mouse liver microsomes) and 26 min
(human liver microsome). While our efforts to improve this
liability have thus far been met with limited success, we fully
expect that through further medicinal chemistry optimization
this metabolic liability can be attenuated. Finally, preliminary in
vivo PK studies of this compound indicate favorable
bioavailability (>80%) yet rapid clearance (>70 mL/min/kg),
CONCLUSION
■
DUBs have been implicated in cancer and other human
diseases and thus have become increasingly regarded as
prospective targets for therapeutic intervention.33−35 Here, we
summarize the medicinal chemistry optimization efforts of a
series of N-benzyl-2-phenylpyrimidin-4-amine based USP1/
UAF1 deubiquitinase inhibitors. We succeeded in generating
several inhibitors with nanomolar potency, which corresponds
to ≥100-fold improved activity compared to the original HTS
hit (1). In addition, we demonstrate activity in cells with
selected compounds as single agents, specifically increasing the
level of monoubiquitinated-PCNA and decreasing cell survival
in nonsmall cell lung cancer cells (Figure 2 and 3). It has
recently been demonstrated that small-molecule inhibitors of
USP1/UAF1 are cytotoxic to both established leukemic cell
lines and patient-derived leukemic cells.20 The data presented
here also supports the use of USP1/UAF1 inhibitors as a
monotherapy in nonsmall cell lung cancer (Figure 3). In
addition to further medicinal chemistry optimization to
improve ADME properties, we are currently attempting to
identify cancer subtypes or patient populations that may benefit
from USP1/UAF1-targeted therapy.
EXPERIMENTAL SECTION
■
General Chemistry Methods. All air or moisture sensitive
reactions were performed under positive pressure of nitrogen with
oven-dried glassware. Chemical reagents and anhydrous solvents were
obtained from commercial sources and used as-is. Preparative
purification was performed on a Waters semipreparative HPLC. The
column used was a Phenomenex Luna C18 (5 μm, 30 mm × 75 mm)
at a flow rate of 45 mL/min. The mobile phase consisted of
acetonitrile and water (each containing 0.1% trifluoroacetic acid). A
gradient of 10−50% acetonitrile over 8 min was used during the
purification. Fraction collection was triggered by UV detection (220
nm). Analytical analysis for purity was determined by two different
methods denoted as final QC methods 1 and 2. Method 1: Analysis
was performed on an Agilent 1290 Infinity series HPLC UHPLC long
gradient equivalent 4−100% acetonitrile (0.05% trifluoroacetic acid) in
water over 3.5 min run time of 4 min with a flow rate of 0.8 mL/min.
A Phenomenex Kinetex 1.7 μm C18 column (2.1 mm × 100 mm) was
used at a temperature of 50 °C. Method 2: Analysis was performed on
an Agilent 1260 with a 7 min gradient of 4−100% acetonitrile
(containing 0.025% trifluoroacetic acid) in water (containing 0.05%
trifluoroacetic acid) over 8 min run time at a flow rate of 1 mL/min. A
Phenomenex Luna C18 column (3 μm, 3 mm × 75 mm) was used at a
temperature of 50 °C. Purity determination was performed using an
Agilent diode array detector for both method 1 and method 2. Mass
determination was performed using an Agilent 6130 mass
spectrometer with electrospray ionization in the positive mode. All
of the analogues for assay have purity greater than 95% based on both
1
analytical methods. H and 13C NMR spectra were recorded on a
Varian 400 (100) MHz spectrometer. High resolution mass
H
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spectrometry was recorded on Agilent 6210 time-of-flight LC-MS
system.
2-(2-Isopropylphenyl)-5-methyl-N-((1-(pyridin-3-yl)piperidin-4-
yl)methyl)pyrimidin-4-amine (64). The synthetic method for
compound 48 was followed and 2,4-dichloro-5-methylpyrimidine
was substituted for 2,4-dichlorofuro[3,2-d]pyrimidine and tert-butyl
4-(aminomethyl)piperidine-1-carboxylate for (4-(pyridin-3-yl)-
phenyl)methanamine to give the desired product. The cross coupling
step removed the tert-butyl carbamate to give 2-(2-isopropylphenyl)-5-
methyl-N-(piperidin-4-ylmethyl)pyrimidin-4-amine, which was puri-
fied on silica gel with CH2Cl2/MeOH(saturated with NH3). LC-MS
retention time (method 1): 2.538 min. This compound was used in
the next step with no further characterization. To degassed xylene (5
mL), 2-(2-isopropylphenyl)-5-methyl-N-(piperidin-4-ylmethyl)-
pyrimidin-4-amine (0.52 g, 1.60 mmol), 2′-(dicyclohexylphosphino)-
N,N-dimethylbiphenyl-2-amine (DavePhos) (0.01 g, 0.02 mmol),
sodium tert-butoxide (0.22 g, 1.4 mmol), Pd2(dba)3 (0.02g, 0.02
mmol), and 3-bromopyridine (0.30 g, 1.92 mmol) were added and
heated in a sealed tube to 100 °C for 18 h. The reaction material was
loaded directly onto silica gel and purified using (0−10% MeOH/
N-(Thiophen-2-ylmethyl)-2-(2-(trifluoromethyl)phenyl)-
quinazolin-4-amine (1).21 2,4-Dichloroquinazoline (0.50 g, 2.51
mmol), thiophen-2-ylmethanamine (0.34 g, 3.0 mmol), and triethyl-
amine (Et3N) (1.0 mL, 7.54 mmol) was stirred overnight in CH2Cl2
(6.0 mL) at room temperature. The reaction mixture was poured into
water and extracted (3×) with CH2Cl2, and the organic layers were
combined, washed (1×) with brine, dried over Na2SO4, filtered, and
concentrated to provide 2-chloro-N-(thiophen-2-ylmethyl)quinazolin-
4-amine, which was used without further purification in the next
reaction. LC-MS retention time (method 1) = 3.334 min.
A 5 mL microwave reaction vessel was charged with a mixture of the
above-mentioned 2-chloro-N-(thiophen-2-ylmethyl)quinazolin-4-
amine (0.09 g, 0.31 mmol), 2-(trifluoromethyl)phenylboronic acid
(0.18 g, 0.96 mmol), Pd(PPh3)4 (0.06 g, 0.05 mmol), sodium
carbonate (0.40 M in water, 1.2 mL, 0.48 mmol), and acetonitrile (1.2
mL). The vessel was sealed and heated, with stirring, at 150 °C for 10
min via microwave irradiation. The organic portion was concentrated
under reduced pressure, and the residue was purified by silica gel
chromatography using (80% hexanes, 20% ethyl acetate) to give the
1
DCM). H NMR (400 MHz, DMSO-d6) δ 8.67−8.79 (m, 1 H), 8.37
(d, J = 2.74 Hz, 1 H), 8.23 (s, 1 H), 8.08 (d, J = 5.09 Hz, 1 H), 7.72−
7.83 (m, 1 H), 7.53−7.62 (m, 3 H), 7.45−7.52 (m, 1 H), 7.34−7.43
(m, 1 H), 3.81−3.94 (m, 2 H), 3.19−3.28 (m, 1 H), 2.80 (t, J = 11.74
Hz, 2 H), 2.18 (s, 3 H), 1.89 (dd, J = 3.72, and 7.14 Hz, 1 H), 1.78 (d,
J = 12.13 Hz, 2 H), 1.21−1.32 (m, 3 H), 1.20 (s, 3 H), and 1.18 (s, 3
H). 13C NMR (151 MHz, DMSO-d6) δ 161.99, 159.42, 148.29,
147.84, 141.13, 131.89, 129.83, 127.98, 126.91, 126.36, 118.30, 116.32,
114.19, 47.12, 46.61, 35.19, 29.51, 28.96, 24.40, 13.87. LC-MS
retention time (method 1), 2.683 min; (method 2), 3.717 min.
HRMS: m/z (M + H)+ (calculated for C25H32N5 402.2653) found,
402.2655.
N-(4-(1H-Imidazol-1-yl)benzyl)-2-(2-isopropylphenyl)-5-methyl-
pyrimidin-4-amine (69). 1-(4-(Chloromethyl)phenyl)-1H-imidazole
(0.50 g, 2.60 mmol) and potassium phthalimide (0.58 g, 3.1 mmol)
were heated in DMF (2.5 mL) to 120 °C for 18 h. The reaction
mixture was cooled to 0 °C, poured into water, and the solids were
filtered and washed with water to give 2-(4-(1H-imidazol-1-yl)benzyl)-
isoindoline-1,3-dione. The dried 2-(4-(1H-imidazol-1-yl)benzyl)-
isoindoline-1,3-dione (0.40 g, 1.32 mmol) was heated to 95 °C in a
mixture of EtOH and hydrazine (4:1) for 1 h, the mixture was cooled,
and the solids filtered. The solids were washed with EtOH to give (4-
(1H-imidazol-1-yl)phenyl)methanamine. LC-MS retention time
(method 1): 2.233 min. This material was used as-is in the synthesis
of N-(4-(1H-imidazol-1-yl)benzyl)-2-chloro-5-methylpyrimidin-4-
amine.
1
desired product. H NMR (400 MHz, DMSO-d6) δ 8.47−8.37 (m, 1
H), 8.03−7.75 (m, 7 H), 7.71 (ddd, J = 1.25, 7.10, and 8.34 Hz, 1 H),
7.38 (dd, J = 1.27, and 5.12 Hz, 1 H), 7.07 (dd, J = 1.22, and 3.43 Hz,
1 H), 6.95 (dd, J = 3.46, and 5.09 Hz, 1 H) and 5.03 (d, J = 5.70 Hz, 2
H). 13C NMR (101 MHz, DMSO-d6) δ 160.09, 159.72, 158.66,
158.33, 140.73, 135.43, 132.43, 131.94, 130.96, 128.01, 127.70, 127.40,
126.24, 125.67, 123.93, 122.95, 118.24, 115.30, 112.89, and 40.60. LC-
MS retention time (method 2) = 4.808 min and (method 1) = 3.128
min. HRMS: m/z (M + H) (calculated for C20H15F3N3S 386.0933)
found, 386.0942.
2-(2-Isopropylphenyl)-N-(4-(pyridin-3-yl)benzyl)furo[3,2-d]-
pyrimidin-4-amine (48). 2,4-Dichlorofuro[3,2-d]pyrimidine (0.10 g,
0.53 mmol), (4-(pyridin-3-yl)phenyl)methanamine (0.10 g, 0.53
mmol), and triethylamine (0.22 mL, 1.59 mmol) were heated in
chloroform to 50 °C for 18 h. The mixture was poured into saturated
NaHCO3 and extracted with chloroform (2×). The organic layers
were combined, dried over Na2SO4, filtered, and concentrated to give
an oil which was used in the next step without further purification. LC-
MS retention time (method 1) = 2.692 min. 2-Chloro-N-(4-(pyridin-
3-yl)benzyl)furo[3,2-d]pyrimidin-4-amine (72 mg, 0.21 mmol), (2-
isopropylphenyl)boronic acid (42 mg, 0.26 mmol), Pd(PPh3)4 (25 mg,
0.02 mmol), and sodium carbonate (2 M, 0.21 mL, 0.43 mmol) in 1,2-
dimethoxyethane (2 mL) was sealed in a microwave tube and heated
to 150 °C for 30 min in a Biotage microwave reactor. The reaction was
filtered through a pad of Celite and washed with ethyl acetate,
concentrated, and purified on reversed phase to give the desired
(4-(1H-Imidazol-1-yl)phenyl)methanamine 0.20 g, 1.17 mmol),
2,4-dichloro-5-methylpyrimidine (0.19 g, 1.17 mmol), and Et3N (0.33
mL, 2.34 mmol) in DMF (4 mL) were heated to 100 °C for 18 h. The
reaction mixture was monitored by LC-MS; when complete, the
mixture was poured into water and extracted with ethyl acetate (3×),
and the organic layers were combined, dried over Na2SO4, filtered, and
concentrated to give the desired intermediate. LC-MS retention time
(method 1): 2.460 min. The cross coupling used the method as
described in compound 48, with a yield of 40%. 1H NMR (400 MHz,
DMSO-d6) δ 9.21 (d, J = 2.52 Hz, 1 H), 9.14 (s, 1 H), 8.29 (d, J = 1.26
Hz, 1 H), 8.07 (t, J = 1.64 Hz, 1 H), 7.73−7.64 (m, 3 H), 7.57−7.39
(m, 5 H), 7.33 (ddd, J = 1.44, 6.97, and 7.67 Hz, 1 H), 4.81 (d, J =
5.97 Hz, 2 H), 3.10 (td, J = 7.44, 13.64, and 14.23 Hz, 1 H), 2.22 (d, J
= 1.02 Hz, 3 H), and 1.00 (d, J = 6.80 Hz, 6 H). 13C NMR (151 MHz,
DMSO-d6) δ 162.03, 159.83, 147.80, 142.20, 139.50, 135.11, 134.63,
131.75, 129.85, 128.75, 126.73, 126.26, 123.10, 122.07, 120.73, 118.26,
116.28, 114.36, 44.07, 29.31, 24.17, and 13.86. LC-MS retention time
(method 2): 3.428 min. HRMS: m/z (M + H)+ (calculated for
C24H26N5 384.2183) found, 384.2169.
1
compound. H NMR (400 MHz, DMSO-d6) δ 9.46 (br s, 1 H), 8.95
(d, J = 1.96 Hz, 1 H), 8.63 (d, J = 5.09 Hz, 1 H), 8.47 (s, 1 H), 8.23 (d,
J = 7.83 Hz, 1 H), 7.73 (d, J = 8.22 Hz, 2 H), 7.62 (dd, J = 5.09, and
7.83 Hz, 1 H), 7.40−7.53 (m, 5 H), 7.24−7.33 (m, 1 H), 7.12 (d, J =
1.96 Hz, 1 H), 4.86 (d, J = 5.87 Hz, 2 H), 3.31 (ddd, J = 6.85, 7.04,
and 13.50 Hz, 1 H), 1.05 (s, 3 H), and 1.03 (s, 3 H). LC-MS retention
time (method 1): 2.706 min; (method 2): 4.214 min. HRMS: m/z (M
+ H)+ (calculated for C27H26N4O 421.2023) found, 421.2011.
2-(2-Isopropylphenyl)-5-methoxy-N-(4-(pyridin-3-yl)benzyl)-
pyrimidin-4-amine (49). Follow the synthesis for compound 48 and
substitute 2,4-dichloro-5-methoxypyrimidine for 2,4-dichlorofuro[3,2-
d]pyrimidine to give the desired product. 1H NMR (400 MHz,
DMSO-d6) δ 9.51−9.67 (m, 1 H), 8.88−8.97 (m, 1 H), 8.63 (d, J =
4.70 Hz, 1 H), 8.16−8.25 (m, 1 H), 8.11 (s, 1 H), 7.71 (d, J = 8.22 Hz,
2 H), 7.32−7.64 (m, 7 H), 4.78 (d, J = 5.87 Hz, 2 H), 3.97−4.08 (m, 3
H), 2.98−3.10 (tddd, J = 6.46, 6.60, 6.73, and 13.52 Hz, 1 H), 1.02 (s,
3 H), and 1.01 (s, 3 H). 13C NMR (151 MHz, DMSO-d6) δ 155.72,
155.29, 147.79, 146.91, 146.03, 139.07, 138.69, 136.71, 136.44, 135.48,
131.79, 131.63, 129.87, 128.21, 127.46, 126.66, 126.22, 125.19, 57.72,
43.90, 29.42, and 24.12. LC-MS retention time (method 1), 2.738
min; (method 2), 1.489 min. HRMS: m/z (M + H)+ (calculated for
C26H27N4O 411.2179) found, 411.2181.
4-(1H-1,2,3-Triazol-1-yl)benzonitrile TFA (x).21 4-Aminobenzoni-
trile (1.00 g, 8.46 mmol) and trifluoroacetic acid (TFA) (0.65 mL,
8.46 mmol) in acetonitrile (70 mL) were stirred at room temperature
for 5 min. The reaction mixture was cooled to 0 °C in a salt ice bath
before the dropwise addition of tert-butyl nitrite (1.51 mL, 12.70
mmol), followed by azidotrimethylsilane (1.35 mL, 10.16 mmol). This
reaction mixture was stirred for 30 min at 0 °C and allowed to warm to
I
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room temperature (rt) before pouring into ethyl acetate (50 mL) and
water (75 mL). The water layer was extracted (2×) with ethyl acetate,
and the organic layers were combine and washed (1×) with brine. The
combined organic layer was dried over Na2SO4, filtered, and
concentrated under reduced pressure to give 1.22 g of the product
as a reddish-brown solid. The compound was used as-is in the next
reaction. LC-MS retention time (method 2:3 min) = 3.331 min.
4-Azidobenzonitrile (1.22 g, 8.33 mmol), sodium ascorbate (1.32 g,
6.66 mmol), copper(II) sulfate (93 mg, 0.06 mmol), and
ethynyltrimethylsilane (6.20 mL, 50.0 mmol) was heated in DMSO/
water (80 mL/40 mL) to 80 °C in a sealed tube for 24 h. The reaction
was allowed to cool to rt, poured into ethyl acetate, and washed (3×)
with 100 mL of water. The combined organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure to give a
pale-yellow residue. The residue was taken up in acetonitrile (16 mL)
and TFA (0.64 mL, 8.33 mmol) and heated to reflux for 3 h. After this
time, the reaction was cooled to rt and poured in to ethyl acetate (30
mL), washed (2×) with saturated sodium bicarbonate, dried over
Na2SO4, filtered, and concentrated and placed on a reverse phase flash
system for purification (gradient 20−100% acetonitrile w/0.1% TFA in
N-(4-(1H-1,2,3-Triazol-1-yl)benzyl)-5-methyl-2-(2-(oxetan-3-yl)-
phenyl)pyrimidin-4-amine TFA (77).21 3-(2-Bromophenyl)oxetane
was synthesized using the method described by Jacobsen et. al,23
except diethyl 2-(2-bromophenyl)malonate was used as the starting
material instead of diethyl 2-(2-(benzyloxy)phenyl)malonate. This
synthetic sequence was carried out without further purification/
characterization of the intermediates.
In an oven-dried 50 mL round-bottom flask, the above-mentioned
3-(2-bromophenyl)oxetane (0.14 g, 0.66 mmol) and THF (4 mL) was
cooled to −78 °C. The dropwise addition of N-butyllithium (0.85 mL,
1.36 mmol) by syringe occurred over a 15 min period. After this time,
the reaction mixture was allowed to stir at −78 °C for an additional 2
h. The contents were transferred via cannula to a second 50 mL oven-
dried round-bottom flask containing a stirred solution of tripropyl
borate (0.29 mL, 1.32 mmol) and THF (2.0 mL) and cooled to −78
°C. Once transferred (by cannula), the mixture was allowed to stir at
−78 °C for 10 min, after which time the ice bath was removed and the
reaction mixture stirred overnight. One N HCl was used to adjust the
pH to ∼1, and the acidic mixture was stirred for 45 min, poured into
water, and extracted with diethyl ether (3×). The organic layers were
combined, dried over MgSO4, concentrated, and once the solvent was
evaporated, a ∼1 M solution of the crude (2-(oxetan-3-yl)phenyl)-
boronic acid in 1,2-DME was made and used in next step with no
further purification. In a sealed tube N-(4-(1H-1,2,3-triazol-1-
yl)benzyl)-2-chloro-5-methylpyrimidin-4-amine (0.10 g, 0.33 mmol)
was combined with (2-oxetainphenyl)boronic acid as a 1 M solution in
DME sodium carbonate (2.0 M in water, 0.33 mL, 0.67 mmol) and
DPP-Pd Silicycle 0.26 mmol/g (0.20 g) in DME (2.0 mL), and heated
at 150 °C for 30 min in a Biotage Initiator microwave reactor. The
resulting mixture was filtered over Celite and purified by reversed-
phase HPLC (gradient 20−100% acetonitrile w/0.1% TFA in water
1
water w/0.1% TFA). H NMR (400 MHz, DMSO-d6) δ 8.98 (d, J =
1.26 Hz, 1 H), and 8.22−8.00 (m, 5 H). 13C NMR (101 MHz, CDCl3)
δ 139.81, 135.13, 133.94, 133.14, 121.64, 120.71, 120.69, 117.68, and
112.40; m/z (M + H)+ = 171.1. LC-MS retention time (method 2:3
min): 2.671 min.
N-(4-(1H-1,2,3-Triazol-1-yl)benzyl)-2-chloro-5-methylpyrimidin-
4-amine TFA (xi).21 4-(1H-1,2,3-Triazol-1-yl)benzonitrile (1.2 g, 7.05
mmol) and TFA (0.60 mL, 7.80 mmol) was dissolved in methanol
(100 mL)/DMF (10 mL) and passed through a H-Cube Pro flow
reactor using a 10% Pd/C 70 mm Catcart at 40 bar and 50 °C. Once
the reaction was complete by LC-MS, the MeOH was concentrated
and the crude used in the next reaction sequence. LC-MS retention
time (method 2:3 min) = 1.386 min (m/z (M + H)+ = 174.2). (4-(1H-
1,2,3-Triazol-1-yl)phenyl)methanamine, TFA (7.05 mmol), 2,4-
dichloro-5-methylpyrimidine (1.16 g, 7.05 mmol), and triethylamine
(3.0 mL, 21.3 mmol) was heated overnight to 100 °C in DMF (25
mL). The completed reaction was poured into water (30 mL) and
extracted with ethyl acetate. The ethyl acetate layer was washed (2×)
with water (1×) with saturated sodium bicarbonate, dried over
Na2SO4, filtered, and concentrated. The residue was purified on a
reverse phase flash system (gradient 10−100% acetonitrile w/0.1%
1
w/0.1% TFA) to give the desired product 77. H NMR (400 MHz,
DMSO-d6) δ ppm 9.36−9.47 (m, 1 H), 8.80 (s, 1 H), 8.35 (s, 1 H),
8.29 (d, J = 7.83 Hz, 1 H), 7.97 (s, 1 H), 7.90 (d, J = 8.22 Hz, 2 H),
7.68 (d, J = 8.61 Hz, 3 H), 7.52 (s, 2 H), 5.12 (br s, 1 H), 4.93−5.00
(m, 2 H), 4.41−4.50 (m, 2 H), 3.61 (dd, J = 4.30, and 10.56 Hz, 2 H),
and 2.22 (s, 3 H). 13C NMR (151 MHz, CDCl3) δ 161.40, 153.48,
144.27, 138.36, 137.52, 136.20, 134.57, 134.28, 129.62, 128.71, 128.52,
128.14, 126.14, 121.96, 120.88, 115.47, 109.98, 62.49, 51.61, 44.94,
39.85, and 14.15. LC-MS: method 2 (short), retention time: 2.526
min. HRMS: m/z (M + H) (calculated for C23H23N6O 399.1928)
found, 399.1924.
1
TFA in water w/0.1% TFA) to give 0.19 g of desired product. H
NMR (400 MHz, DMSO-d6) δ 8.76 (d, J = 1.17 Hz, 1 H), 7.97−7.90
(m, 2H), 7.86−7.80 (m, 3 H), 7.54−7.48 (m, 2 H), 4.63 (d, J = 5.98
Hz, 2 H), and 2.18−1.75 (m, 3 H). 13C NMR (101 MHz, DMSO-d6)
δ 162.60, 157.80, 154.97, 140.20, 135.90, 134.78, 129.01, 123.61,
120.61, 113.64, 43.54, and 13.50. LC-MS retention time (method 2:3
min) = 2.770 min; m/z (M + H)+ = 301.1.
N-(2-(4-(1H-1,2,3-Triazol-1-yl)phenyl)propan-2-yl)-2-(2-isopropyl-
phenyl)-5-methylpyrimidin-4-amine TFA (79). 2-Chloro-5-methyl-
N-(2-(4-nitrophenyl)propan-2-yl)pyrimidin-4-amine, TFA. 2-(4-
Nitrophenyl)propan-2-amine, HCl (0.60 g, 2.77 mmol), 2,4-
dichloro-5-methylpyrimidine (0.45 g, 2.77 mmol), and Et3N (1.15
mL, 8.31 mmol) in DMF (8.2 mL) was sealed and heated in a
microwave vial to 150 °C for 1 h. The reaction was cooled and poured
into EtOAc and washed with water (2×), dried over Na2SO4, filtered,
and concentrated. The residue was purified using reverse phase over
30 min and gradient of 10−100 water/CH3CN 0.01% TFA to give the
desired compound in a 49% yield. LC-MS retention time (method 1):
3.478 min.
2-(2-Isopropylphenyl)-5-methyl-N-(2-(4-nitrophenyl)propan-2-
yl)pyrimidin-4-amine, TFA. 2-Chloro-5-methyl-N-(2-(4-nitrophenyl)-
propan-2-yl)pyrimidin-4-amine, TFA (0.19 g, 0.45 mmol), (2-
isopropylphenyl)boronic acid (0.22 g, 1.26 mmol), sodium carbonate
(2 M, 0.90 mL, 1.81 mmol), and DPP-Pd silica bound (0.20 g, 0.26
mmol/g) in dimetoxyethane (3 mL) was sealed and heated in a
microwave reactor to 150 °C for 45 min. The reaction was filtered and
concentrated immediately placed on a reverse phase column to give
75% of the desired intermediate, which was used without further
characterization. LC-MS retention time (method 1): 3.144 min.
N-(2-(4-Aminophenyl)propan-2-yl)-2-(2-isopropylphenyl)-5-
methylpyrimidin-4-amine. 2-(2-Isopropylphenyl)-5-methyl-N-(2-(4-
nitrophenyl)propan-2-yl)pyrimidin-4-amine, TFA (0.28 g, 0.55
mmol), acetic acid (0,37 mL, 6.54 mmol), and zinc (0.18 g, 2.73
mmol) was stirred in CH3OH (5.50 mL) for 1 h. The reaction mixture
was filtered through a pad of Celite, rinsed with CH3OH,
N-(4-(1H-1,2,3-Triazol-1-yl)benzyl)-5-methyl-2-(2-
isopropylphenyl)pyrimidin-4-amine TFA (70). N-(4-(1H-1,2,3-Tria-
zol-1-yl)benzyl)-2-chloro-5-methylpyrimidin-4-amine (0.19 g, 0.63
mmol) was combined with (2-isopropylphenyl)boronic acid (0.31 g,
1.90 mmol), sodium carbonate (2.0 M in water, 1.23 mL, 2.53 mmol),
and DPP-Pd Silicycle 0.26 mmol/g (0.30 g) in DMF (4.50 mL). The
reaction was sealed and heated at 150 °C for 30 min in a Biotage
Initiator microwave reactor. The resulting mixture was filtered over
Celite and purified by HPLC (gradient 20−100% acetonitrile w/0.1%
TFA in water w/0.1% TFA) to give, after lyophilization, N-(4-(1H-
1,2,3-triazol-1-yl)benzyl)-5-methyl-2-(2-isopropylphenyl)-pyrimidin-4-
1
amine (70) as a TFA salt (0.01 g, 0.02 mmol, 30%). H NMR (400
MHz, CDCl3) δ 8.82 (dd, J = 3.4, and 7.9 Hz, 1 H), 8.02−7.93 (m, 2
H), 7.76 (d, J = 1.4 Hz, 1 H), 7.64−7.56 (m, 2 H), 7.51−7.33 (m, 5
H), 7.25−7.17 (m, 1 H), 4.88 (d, J = 5.9 Hz, 2 H), 3.19 (p, J = 6.8 Hz,
1H), 2.22 (s, 3 H), 1.10 (d, J = 1.2 Hz, 3 H) and 1.09−1.07 (m, 3 H).
13C NMR (100 MHz, CDCl3) δ 162.02, 160.18, 148.00, 140.63,
138.03, 136.14, 134.35, 134.28, 131.89, 130.34, 129.25, 129.00, 126.61,
126.04, 122.04, 121.99, 120.74, 113.81, 44.62, 29.43, 23.95, 13.71 and
13.67. LC-MS retention time (method 1) = 2.938 min and (method 2)
= 1.756 min. HRMS: (ESI) m/z (M + H)+ (calculated for C23H25N6
385.2135) found, 385.2146.
J
dx.doi.org/10.1021/jm5010495 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
concentrated, and used as in with no further purification or
characterization. LC-MS retention time (method 1): 2.670 min. N-
(2-(4-Aminophenyl)propan-2-yl)-2-(2-isopropylphenyl)-5-methylpyri-
midin-4-amine (0.12 g, 0.33 mmol) and trifluoroacetic acid (TFA)
(0.04 mL, 0.33 mmol) in acetonitrile (2 mL) was stirred at room
temperature for 5 min. The reaction mixture was cooled to 0 °C in an
ice bath before the dropwise addition of tert-butyl nitrite (0.06 mL,
0.50 mmol) followed by azidotrimethylsilane (0.05 mL, 0.40 mmol).
This mixture was stirred for 30 min at 0 °C, allowed to warm to room
temperature (rt), and poured into ethyl acetate (10 mL) and water (15
mL). The water layer was extracted with ethyl acetate (2×) and
organic layers were combined and washed with brine (1×), dried over
Na2SO4, filtered, and concentrated under reduced pressure to give to
give the N-(2-(4-azidophenyl)propan-2-yl)-2-(2-isopropylphenyl)-5-
methylpyrimidin-4-amine, which was used as-is in the next reaction
without further purification. LC-MS retention time (method 1): 3.331
min. N-(2-(4-Azidophenyl)propan-2-yl)-2-(2-isopropylphenyl)-5-
methylpyrimidin-4-amine (33 mg, 0.09 mmol), ethynyltrimethylsilane
(0.13 mL, 1.03 mmol), sodium ascorbate (14 mg, 0.07 mmol), and
copper(II)sulfate (1 mg, 0.006 mmol) in DMSO/H2O (1 mL/0.5 mL)
was sealed in a tube and heated to 80 °C for 24 h. the cooled reaction
mixture was poured into water and extracted with ethyl acetate (3×),
dried over Na2SO4, filtered, concentrated, and taken up in acetonitrile
(3 mL) and TFA (0.11 mL, 0.92 mmol), heated to reflux for 3 h, then
the reaction was cooled and concentrated under reduced pressure.
This material was purified with reverse phase to give compound 79. 1H
NMR (400 MHz, DMSO-d6) δ 8.75 (d, J = 1.17 Hz, 1H), 8.23 (s,
1H), 7.94 (d, J = 1.13 Hz, 1H), 7.81−7.73 (m, 2H), 7.55−7.47 (m,
2H), 7.40 (t, J = 7.60 Hz, 1H), 7.33−7.26 (m, 1H), 7.16 (t, J = 7.54
Hz, 1H), 7.02 (d, J = 7.64 Hz, 1H), 3.05−3.17 (m, 1 H), 2.33 (d, J =
1.06 Hz, 3H), 1.78 (s, 6H), and 0.72 (d, J = 6.80 Hz, 6H). LC-MS
retention time (method 2): 4.378 min. HRMS: m/z (M + H)+:
(calculated for C25H29N6 413.2448) found, 413.2447.
AUTHOR INFORMATION
■
Corresponding Authors
*For D.J.M.: phone, 301-217-4381; fax, 301-217-5736; E-mail,
maloneyd@mail.nih.gov.
*For Z.Z.: phone, 302-831-8940; E-mail, zzhuang@udel.edu.
Author Contributions
§These authors (T.S.D. and A.S.R.) contributed equally to this
work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by U.S. National Institutes of
Health (NIH) grants R03DA030552 and R01GM097468 to
Z.Z. T.S.D., A.S.R., D.K.L., E.H.K., A.S., A.J., and D.J.M. were
supported by the intramural research program of the National
Center for Advancing Translational Sciences and the Molecular
Libraries Initiative of the National Institutes of Health
Roadmap for Medical Research (U54MH084681). We thank
Sam Michael and Richard Jones for automation support, Paul
Shinn Misha Itkin, and Danielle van Leer for the assistance with
compound management, William Leister, Heather Baker,
Elizabeth Fernandez, and Christopher Leclair for analytical
chemistry and purification support, and Kimloan Nguyen for in
vitro ADME data.
ABBREVIATIONS USED
■
DUBs, deubiquitinases; USP1, ubiquitin-specific protease 1;
UAF1, USP1-associated factor 1; PCNA, proliferating cell
nuclear antigen; FANCD2, Fanconi anemia group comple-
mentation group D2; IS, inhibitor of DNA binding; HTS, high-
throughput screening; ADME, absorption, distribution, metab-
olism, elimination; POCl3, phosphorus oxychloride; HOBT,
hydroxybenzotriazole; DMF, dimethylformamide; DME, dime-
thoxyethane; DPP, diphenylphosphine; MW, microwave; DCE,
dichloroethane; DMSO, dimethyl sulfoxide; LAD, lithium
aluminum deuteride; TFA, trifluoroacetic acid; SAR, struc-
ture−activity relationship; qHTS, quantitative high-throughput
screening; CYP, cytochrome P450; Ub-Rho, ubiquitin rhod-
amine; NADPH, nicotinamide adenine dinucleotide phosphate;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RLM, rat
liver microsome; MLM, mouse liver microsome; HLM, human
liver microsome; CFA, colony formation assay
Biological Methods. USP1/UAF1 Quantitative High-Through-
put Screening Assay. USP1/UAF1 activity was monitored in a
fluorometric ubiquitin−rhodamine110 assay as previously de-
scribed.16,21
Western Blot. H1299 cells were seeded in 12-well plates at a density
of 1 × 105 per well and allowed to attach overnight. Cells were treated
with 20 μM of the indicated compounds or with an equal volume of
DMSO for 4 h. Total protein extracts were prepared in RIPA lysis
buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1%
sodium deoxycholate, 0.1% SDS) containing protease and phosphatase
inhibitor cocktail (Thermo Scientific). The concentration of protein
lysates was determined using the Bio-Rad DC protein assay (Bio-Rad).
Cell extracts were separated on 4−12% gradient SDS-PAGE and
transferred to nitrocellulose. The membranes were blotted with PCNA
antibody (PC10, Santa Cruz) or GAPDH antibody (D16H11, Cell
Signaling), followed by incubation with HRP-conjugated antimouse or
antirabbit secondary antibody, respectively. Blots were imaged using
the Bio-Rad ChemiDocTM XRS gel imager and quantified using
ImageQuant TL (GE).
Colony Formation Assay. H1299 cells were seeded in 6-well plates
at a density of 200 cells per well and allowed to attach overnight. Cells
were treated with 5 μM of the indicated compounds or with an equal
volume of DMSO. Seven days after compound treatment, plates were
fixed with 20% methanol and stained with 0.1% crystal violet. Plates
were images with the Typhoon FLA 7000 (GE), and colonies were
counted using ImageQuant TL (GE). Cell survival was expressed in
percentage with DMSO-treated cells set as 100%.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Additional supplemental figures, experimental procedures, and
spectroscopic data (1H NMR, LC-MS and HRMS) for
representative compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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