Discovery of NCT-501, a Potent and Selective Theophylline-Based Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1)

Article abstract of DOI:10.1021/acs.jmedchem.5b00577

Aldehyde dehydrogenases (ALDHs) metabolize reactive aldehydes and possess important physiological and toxicological functions in areas such as CNS, metabolic disorders, and cancers. Increased ALDH (e.g., ALDH1A1) gene expression and catalytic activity are vital biomarkers in a number of malignancies and cancer stem cells, highlighting the need for the identification and development of small molecule ALDH inhibitors. A new series of theophylline-based analogs as potent ALDH1A1 inhibitors is described. The optimization of hits identified from a quantitative high throughput screening (qHTS) campaign led to analogs with improved potency and early ADME properties. This chemotype exhibits highly selective inhibition against ALDH1A1 over ALDH3A1, ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the pharmacokinetic evaluation of selected analog 64 (NCT-501) is also highlighted.

Full text of DOI:10.1021/acs.jmedchem.5b00577

Article
pubs.acs.org/jmc
Discovery of NCT-501, a Potent and Selective Theophylline-Based
Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1)
Shyh-Ming Yang,*,† Adam Yasgar,† Bettina Miller,‡ Madhu Lal-Nag,† Kyle Brimacombe,† Xin Hu,†
Hongmao Sun,† Amy Wang,† Xin Xu,† Kimloan Nguyen,† Udo Oppermann,§,∥ Marc Ferrer,†
Vasilis Vasiliou,‡,⊥ Anton Simeonov,† Ajit Jadhav,† 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 Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz
Medical Campus, Aurora, Colorado 80045, United States
§Botnar Research Center, NIHR Oxford Biomedical Research Unit, Oxford OX3 7LD, U.K.
∥Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.
⊥Department of Environmental Health Sciences, Yale School of Public Health, 60 College Street, New Haven, Connecticut 06510,
United States
S
* Supporting Information
ABSTRACT: Aldehyde dehydrogenases (ALDHs) metabo-
lize reactive aldehydes and possess important physiological and
toxicological functions in areas such as CNS, metabolic
disorders, and cancers. Increased ALDH (e.g., ALDH1A1)
gene expression and catalytic activity are vital biomarkers in a
number of malignancies and cancer stem cells, highlighting the
need for the identification and development of small molecule
ALDH inhibitors. A new series of theophylline-based analogs
as potent ALDH1A1 inhibitors is described. The optimization
of hits identified from a quantitative high throughput screening
(qHTS) campaign led to analogs with improved potency and
early ADME properties. This chemotype exhibits highly
selective inhibition against ALDH1A1 over ALDH3A1,
ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the
pharmacokinetic evaluation of selected analog 64 (NCT-501) is also highlighted.
implicated in obesity11,12 and inflammation,13 suggesting that
INTRODUCTION
■
inhibition of this enzyme may offer new therapeutic options for
The human genome encodes 19 aldehyde dehydrogenase
obese patients and patients with Crohn’s disease.13 These data
(ALDH) enzymes that metabolize reactive aldehydes to their
corresponding carboxylic acid derivatives.1 Unbalanced bio-
suggest that discovery of small molecule ALDH inhibitors is a
prudent approach for identifying potential cancer and/or CSC-
logical activity of ALDHs and specific contribution to their
metabolism pathway have been associated in a variety of disease
states, including alcoholic liver disease, Sjogren−Larsson
directed therapeutics as well as a better understanding of the
physiological and pathophysiological actions of the ALDHs.14,15
̈
Among reported ALDH inhibitors,16 CVT-10216 (1)
represents an attractive small molecule that effectively inhibits
both ALDH1A (1.3 μM) and ALDH2 (0.029 μM) (Figure
1).17 This reversible inhibitor was designed and optimized
based on the interaction of daidzin (2) with human
ALDH2.18,19 As a result of the potent ALDH2 inhibition, 1
has been developed for the treatment of alcoholism and has
also demonstrated activity in the reduction of cocaine-seeking
behavior.20 More recently, indolinedione-based analogs (e.g.,
3)21 and substituted tricyclic pyrimidinone 422 reported by
syndrome (SLS), type 2 hyperprolinemia, hyperammonemia,
Parkinson’s disease, and cancers.2−5 It is well established that
overexpression of certain ALDHs, especially ALDH1A1, in a
number of malignancies and cancer stem cells (CSCs)
correlates with poor prognosis and tumor aggressiveness and
that ALDH1A1 is linked to drug resistance in traditional cancer
chemotherapy.6,7 Although the majority of the research
community has considered ALDH1A1 as a marker of cancer
stem cells and a predictor of the prognosis, this enzyme also
plays an important role in the biology of tumors and cancer
stem cells.8,9 Initial evidence toward this end has been
established using nonspecific ALDH inhibitors and siRNA
silencing techniques.10 More recently, ALDH1A1 has also been
Received: April 13, 2015
© XXXX American Chemical Society
A
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Figure 1. Representative small molecule ALDH1A1 inhibitors and theophylline-based qHTS hits.
Hurley and co-workers exhibit significant hALDH1A1 inhib-
itory activity (0.02 and 4.6 μM for 3 and 4, respectively) and
were reported to be selective inhibitors against other ALDH
isozymes such as ADLH2 and ALDH3A1. The 3-carbonyl
functionality of these indolinedione analogs appears to form
reversible covalent adducts with catalytic cysteine residue,
particularly with ALDH3A1, and additional mechanism of
action studies suggest that these molecules are substrate
competitive inhibitors.21,22 Furthermore, the inhibition of
ALDH1A1 activity by compound 4 resulted in disruption of
ovarian cancer spheroid formation and cell viability. Despite the
favorable activity of these compounds, to the best of our
knowledge a systematic and thorough medicinal chemistry
effort had not been reported on ALDH1A1 inhibitors. As such,
our efforts toward identifying novel, more druglike small
molecule of ALDH1A1 inhibitors for cancer and CSCs research
began with a qHTS23 campaign (PubChem assay identifier
1030: http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=
1030, last modified March 15, 2010) that ultimately led to
identification of the theophylline-based chemotype, exemplified
as hits 5 (0.084 μM) and 6 (0.17 μM), along with other active
analogs (Figure 1).24,25 Furthermore, we aimed to fill the need
for optimized druglike ALDH1A1 inhibitors with improved
ADME/PK properties required for evaluating the fundamental
biological roles (e.g., cancer and CSCs) of ALDH1A1 in vivo.
These theophylline-based hits (e.g., 5 and 6) are structurally
distinct from existing ALDH inhibitors and appear quite
specific with minimal activity across 570 assays screened in
PubChem, which suggests the theophylline core is a suitable
scaffold for further investigation.26,27 Herein, we report the
systematic medicinal chemistry optimization and structure−
activity relationships (SARs) of theophylline-based analogs as
potent and selective ALDH1A1 inhibitors.
with representative bromides provided intermediates 8−10 in
which the Cl functionality is poised for derivatization of the C8
substitution. For instance, analog 11 was readily prepared by
utilizing a substitution protocol with ethoxycarbonylpiperazine
under microwave irradiation. Analogs 12−13 were synthesized
similarly by Cl displacement with methyl 4-hydroxybenzoate
followed by hydrolysis and methyl amide formation. The key
intermediates 14−18 with diverse C8 substitutions that focused
on the variation of ring size (four-membered vs six-membered
rings) and linkers (O or NH) were generated in a similar
manner after Boc deprotection. With these intermediates in
hand, the piperidine moiety was further reacted with halo-
substituted heteroarenes to give 19 and 20, acylated or
sulfonated with substituted carbonyl chloride or sulfonyl
chloride, respectively, to furnish analogs 26−38 (see Table
1). These transformations allowed for efficient SAR inves-
tigations around ethyl carbamate portion of qHTS hits. Further
utilizing intermediate 16 by acylation with cyclopropanecar-
bonyl chloride followed by removal of the benzyl group under
palladium-catalyzed hydrogenation conditions gave intermedi-
ate 22. This is then poised for derivatization of the N7 position
by alkylation or a Mitsunobu reaction with the requisite halides
or hydroxyl material, respectively, to provide analogs 39−56
(see Table 2).
The piperazine-type analogs, including resynthesized hits 5
and 6, were prepared in a different manner as highlighted in
Scheme 2. The hydroxyl intermediate 24 was generated via a
two-step sequence involving acylation of 23 with hydroxyacetic
acid followed by cyclization under basic conditions.29 Further
alkylation with various alkyl and benzyl bromides followed by
oxidation with Dess−Martin periodinane gave aldehyde 25.
Subsequent reductive amination with corresponding piperazine
derivatives using sodium triacetoxyborohydride as the reducing
agent afforded qHTS hits 5 and 6 and desired analogs 57−67
(see Table 3).
CHEMISTRY
■
The synthetic routes that allow access to the majority of
designed molecules with modification of two arms (N7 and
C8) of theophylline core are described in Scheme 1.28
Alkylation of commercially available 8-chlorotheophylline 7
RESULTS AND DISCUSSION
■
Structure−Activity Relationships (SARs). The inhibitory
activity of these analogs was tested using human ALDH1A1
B
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a
Scheme 1. Synthetic Routes Assessing Theophylline-Based ALDH1A1 Inhibitors
a
Reagents and conditions: (a) R1-Br, K2CO3, DMF, rt to 60 °C, 1−24 h, 8 (98%), 9 (95%), 10 (99%); (b) 1-ethoxycarbonylpiperazine, DMSO,
microwave, 160 °C, 30 min, 11 (58%); (c) methyl 4-hydroxybenzoate, K2CO3, DMF, microwave, 160 °C, 1 h, 66−83%; (d) 1.5 N LiOH(aq), THF/
MeOH, 50 °C, 2−3 h, 48−70%; (e) MeNH2 (2 M in THF), HATU, DMF, (i-Pr)2NEt, rt, 2 h, 46−59%; (f) N-Boc-4-hydroxypiperidine or N-Boc-4-
hydroxyazetidine (for 14−16 and 18), NaH, DMF, rt, 0.5−2 h, 81−96%; or N-Boc-4-aminopiperidine (for 17), DMSO, microwave, 160 °C, 45 min,
66%; (g) 4 M HCl in dioxane, rt, 3−24 h, 90−99%; (h) 2-chloro-4-methylpyrimidine or 2-bromo-5-methylthiazole, K2CO3, DMF, microwave, 160
°C, 1 h; (i) corresponding substituted carbonyl chloride, Et3N, CH2Cl2, rt, 0.5−1 h, for 26−27, 29−34, and 36−38; or ethyl isocyanate, Et3N,
CH2Cl2, rt, 0.5 h, for 28; or cyclopropanesulfonyl chloride, Et3N, CH2Cl2, rt, 0.5 h, for 35; (j) 16, cyclopropanecarbonyl chloride, Et3N, CH2Cl2, rt,
0.5 h, 21 (96%); (k) Pd(OH)2 (20 mol %), EtOAc/EtOH, 70 °C, 24 h, 22 (75%); (l) corresponding alkyl or benzyl bromide or chloride, K2CO3,
t
t
DMF, 50−-60 °C, 3−24 h, for 39−52; or corresponding hydroxyl compounds, BuO2CNNCO2 Bu, PPh3, THF, rt or 60 °C, 1−3 h, for 53−56.
(hALDH1A1). Importantly, the resynthesized material of 5 and
6 had comparable potency to that obtained in the primary
qHTS assay (Table 1). As part of our medicinal chemistry
optimization campaign, we also characterized all synthesized
compounds for early ADME (eADME) data such as micro-
somal stability, PAMPA permeability, and aqueous solubility
(pH 7.4) to concurrently develop structure−property relation-
ships. Thus, we immediately realized that our hit compounds
possessed poor rat liver microsomal (RLM) stability (5, t1/2 = 5
min) which would need to be improved along with retaining
the desired potency and selectivity profile. Initial structural
modifications were primarily focused on the R2 portion as
shown in Table 1. With the 3-methylbenzyl R1 substitution held
constant, direct attachment of the piperazine substituent (11)
or rearranging the nitrogen atom from piperazine to amino-
piperidine substituent (26) resulted in ∼14-fold loss of
potency. While the hydroxypiperidine analog exhibited
reasonable inhibitory activity (27, 213 nM), removal of the
ethyl carbamate functionality significantly dropped the potency
to the micromolar range (14, 13.7 μM), indicating further
investigation of this position was required. Subsequently,
changing the ethyl carbamate to ethylurea (28) or short
chain amides (29 and 30) minimally affected the potency.
However, increasing the size of side chain, such as 31 and 32,
resulted in a loss of potency. In comparison with linear amides
such as 31, the branched amides (e.g., 33 and 34), particularly
cyclopropylamide, showed significant improvement in potency
(34, 84 nM). Switching the cyclopropylamide to sulfonamide
(35) caused a 3-fold potency loss. Other attempts to
structurally mimic ethyl carbamate with simple methylheter-
oarenes, such as methylpyrimidine (19) and methylthiazole
(20), were unsuccessful and led to inhibitory activities in the
micromolar range (3.8−19.2 μM). The isopentyl-substituted
analogs again demonstrated that the cyclopropylamide
contributed significantly to the potency (37, 87 nM vs 36,
138 nM). Furthermore, reducing the ring size of piperidine to
four-membered azetidine also caused a 6-fold potency drop (37
vs 38). Finally, the phenoxy moiety was found to be a suitable
replacement of 4-hydroxypiperidine and improved the inhib-
ition dramatically (12 and 13, 57−89 nM). In comparison to
the lead analogs (such as 5, 6, 36, and 37) with aqueous
solubility greater than 62 μg/mL, the phenoxy analogs
exhibited poor solubility (<1 and 6.1 μg/mL) for analogs 12
and 13, respectively. As such, despite the potent inhibitory
C
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Table 1. SAR of R2 Portion of Theophylline-Based
ALDH1A1 Inhibitors
Table 2. SAR of R1 Portion of Theophylline-Based
ALDH1A1 Inhibitors
a
Values with standard deviation (SD) represent the average of three to
six runs from one to two experiments. The IC50 values are shown in
b
nM unless otherwise specified. RLM represents rat liver microsomal
stability conducted at NCATS in the presence of NADPH.
a
potency, these compounds were deprioritized for further
development. Beyond the demonstrated potency increase by
cyclopropylamide modification, no further improvement in
RLM stability was observed. Other key SAR findings taken
from Table 1 include the following: (1) the 3-methylbenzyl
Values with standard deviation (SD) represent the average of three to
six runs from one to two experiments. The IC50 values are shown in
nM unless otherwise specified. RLM represents rat liver microsomal
stability conducted at NCATS in the presence of NADPH. IC50 value
was given based on resynthesized material.
b
c
D
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a
Scheme 2. Synthesis of Piperazine-Substituted, Theophylline-Based ALDH1A1 Inhibitors
a
Reagents and conditions: (a) 2-hydroxyacetic acid, neat, 100 °C, 3 h, 49%; (b) NaOH(aq), 100 °C, 2 h, 24 (82%); (c) R1-Br, K2CO3, DMF, rt to 60
°C, 3−24 h; (d) Dess−Martin periodinane, CH2Cl2, 0 °C to rt, 1−2 h; (e) piperazine or homopiperazine derivatives (or its HCl salt), NaBH(OAc)3,
Et3N, rt, 4 h.
Table 3. Combination of Favored R1 Substitution and
Given that the cyclopropylamide moiety improved inhibition
of ALDH1A1, we maintained this motif on the second set of
Piperazine-Type R2 Substitution
analogs aimed at addressing the relationships between R1
substitution and RLM instability from newly identified leads
34 and 37. Analogs with various substituted aryl and alkyl R1
substitutions were synthesized and screened (Table 2).
Removal of R1 group (22) led to a significant loss in activity,
while benzyl (21) or phenylethyl (39) only slightly diminished
potency in a comparison with 34. Importantly, these analogs
exhibit improved RLM stability, leading us to suspect the 3-Me
group as key factor in the observed RLM instability. This was
further supported by the finding that the RLM stability is
greatly influenced by substitution pattern of phenyl ring. For
instance, electron-withdrawing groups, such as CF3 (41), Cl
(42), or F (45), are preferred, giving marked improvement in
RLM stability (t1/2 ≥ 20 min), though the IC50 values are
decreased compared to the Me substitution (34). The electron-
donating group, exemplified by OMe (40), is undesirable in
both potency and RLM stability. Interestingly, moving these
substitutions to 2-position, such as 43 and 46 vs 42 and 45,
respectively, gained potency back while improving RLM
stability. However, the 2-F substitution did not overcome the
undesired RLM issue caused by 3-Me substitution, as analog 47
had a RLM stability of only 4 min. Replacing the phenyl ring
with pyridine produced analogs with improved RLM stability
but with significantly lower potency (48, 12.5 μM). Finally, the
bicyclic naphthylene substituent also produced potent analog
49 (91 nM) but again resulted in a RLM stability issue.
In parallel, investigation of alkyl type R1 substitution was
conducted (Table 2). Compared to lead compound 37, the
shorter alkyl chain (50) increased RLM stability, albeit with
significant loss of potency to 3.9 μM, while the opposite results
were obtained with longer alkyl chain analog 51 (81 nM; RLM,
a
Values with standard deviation (SD) represent the average of three to
six runs from one to two experiments. The IC50 values are shown in
3 min), leading us to focus on modifying the isopentyl portion.
Changing to a more hydrophobic dimethylbutyl side chain (52)
showed comparable potency (76 nM) and RLM stability (12
min) to 37. Further tethering the dimethyl groups of the
isopentyl moiety led to analogs with either improved RLM
stability (example, 53, RLM, 25 min) or retained potency (54,
30 nM) but not both. Surprisingly, the oxetane analog (56)
completely lost inhibitory activity. The data obtained for R1
modification suggest that hydrophobic interactions with
enzyme in this region are preferred. This is consistent with
b
nM unless otherwise specified. RLM represents rat liver microsomal
stability conducted at NCATS in the presence of NADPH.
group seemed responsible for RLM instability as suspected
(e.g., 5 vs 6; 12 vs 13; 34 vs 37), and (2) the methyl piperazine
analogs are slightly more potent than 4-hydroxypiperidine
analogs, e.g., 5 (40 nM) and 6 (77 nM) vs 27 (213 nM) and 36
(138 nM), respectively.
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a
Table 4. Selectivity against Other ALDH Isozymes and Dehydrogenases
IC50 (μM)
hALDH3A1
compd
hALDH1A1
hALDH1B1
hALDH2
HPGD
HSD17β4
5
0.040
0.077
0.057
0.089
0.213
0.084
0.138
0.087
0.033
0.065
0.040
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
24.3
>57
24.3
1.5
2.4
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
>57
6
>57
6.1
12
13
27
34
36
37
57
58
64
2.4
4.9
3.9
>57
>57
>57
>57
>57
>57
15.3
>57
2.4
7.7
>57
>57
a
Compounds noted as >57 μM represent a very weak or no inhibition [efficacy of ≤50% of full inhibition at highest tested concentration (57 μM)].
reported cocrystallized structure of a theophylline-based analog
that R1 substitution points toward the space surrounded by
greasy residues Phe171 and Phe466.27
ALDH3A1 and ALDH2 inhibitions (12 and 13). Finally,
removal of 3-methyl group resulted in enhanced selectivity
(e.g., 58 vs 57). These data, along with the above-mentioned
SAR and structure−property relationship (SPR) explorations,
suggested that compound 64 should be selected for further
evaluation and characterization. Accordingly, compound 64 was
profiled for off-target kinase activity using DiscoveRx
KINOMEscan screening platform. The results indicated 64 is
a clean inhibitor with no significant hits (i.e., inhibition of
≥55% at 10 μM) observed against more than 450 human
kinases (Supporting Information Figure S1). To further
evaluate any potential off-target activity of 64, we profiled it
against the gpcrMAX panel which contains 168 GPCRs
covering 60 receptor families and is run in both agonist and
antagonist mode. As with the kinase profile, compound 64
demonstrated very little activity in either agonist or antagonist
mode (Supporting Information Figure S2).
Mechanism Studies. During the preparation of this
manuscript, Hurley and co-workers published an X-ray crystal
structure of a similar theophylline-based analog with
ALDH1A1.27 This structure along with kinetic studies indicated
that these analogs are substrate noncompetitive inhibitors,
which bind near the solvent exposed exit of substrate-binding
site. Independent kinetic studies in our laboratory corroborated
these findings (data not shown). This mode differs from the
previously reported indolinedione and tricyclic pyrimidinone
type of inhibitors (e.g., 3 and 4) which both demonstrated
substrate competitive inhibition.21,22 To further investigate the
mechanism of action, a rapid dilution experiment using 64 was
conducted to determine the reversibility of the inhibition. The
reaction time courses were collected after incubation with 64 at
an IC90 concentration (red triangle, Figure 2) and rapid dilution
of enzymatic reaction. The diluted sample rapidly recovered
enzymatic activity (orange box), similar to the low-compound
incubated control (blue triangle) or in the absence (green circle
) of inhibitor, indicating that compound 64 demonstrates
reversible inhibition of ALDH1A1.
Piperazine containing analogs generally gave better potency
(e.g., 5 and 6 vs 27 and 36, respectively), so we decided to
pursue another set of compounds containing this moiety while
varying the R1 substitutions as shown in Table 3. A direct
comparison of piperazine containing analogs 57, 58, 61, 62, 63,
and 64 vs 4-hydroxypiperidine analogs 34, 21, 43, 45, 51, and
37, respectively, confirmed our expectation to further improve
potency. 2-Fluorobenzyl substitution was the exception and
resulted in a 2-fold loss of potency (60, 390 nM vs 46, 153
nM). As seen with analogs 34 and 51, the 3-methylbenzyl (57)
and isohexyl (63) substitutions exhibited excellent inhibition
but suffered low RLM stability. Ring expansion of piperazine to
homopiperazine (64 vs 67) not only caused an 8-fold loss of
potency but also decreased RLM stability significantly. These
data, together with the result from ring constrained analog 38,
indicate that six-membered rings, such as piperazine or
piperidine, are preferred. Overall, the isopentyl R1 substitution
and piperazine amide type of R2 substitution appear to be the
optimized combination in terms of potency and eADME (e.g.,
RLM stability), exemplified as compound 64 (NCT-501) that
is suitable for further evaluation.
Selectivity Evaluation. With all analogs examined against
ALDH1A1, a set of potent analogs with representative R1
substitutions (3-methylbenzyl and isopentyl) and variations in
R2 substitutions were selected and screened against ALDH
isozymes (examples ALDH1B1, ALDH3A1, and ALDH2) and
other dehydrogenases such as 15-hydroxyprostaglandin dehy-
drogenase (HPGD) and type 4 hydroxysteroid dehydrogenase
(HSD17β4).30 In general, ALDH1A1 shares nearly 70%
sequence identity to mitochondrial ALDH2 and ALDH1B1
isozymes, less than 50% sequence identity to ALDH3A1, and
significant less similarity to HPGD or HSD17β4. As the results
compiled in Table 4, these theophylline-based analogs exhibited
no inhibition toward ALDH1B1, HPGD, or HSD17β4. Some
low inhibitory activities in the micromolar range against
ALDH3A1 and/or ALDH2 were observed and were seemingly
dependent on the R1 or R2 substitution pattern. For instance,
the alkyl type isopentyl R1 substitution, such as analogs 6, 36,
and 64, typically showed better selectivity over other ALDH
isozymes and other dehydrogenases, while the 3-methylbenzyl
exhibited some degree of inhibition toward ALDH3A1 and
ALDH2 (examples 5, 27, and 57). In contrast, the phenoxy
type R2 substitution seemed to play a vital role in picking up
Pharmacokinetics and in Vitro ADME Profiles. After
demonstrating desirable potency and selectivity with a better
understanding of the mechanism of action, compound 64,
which possessed favorable kinetic aqueous solubility (>60 μg/
mL) and RLM stability (>30 min), was further evaluated for its
pharmacokinetics (PK) in CD1 mice. The compound was
dosed at 2 mg/kg, 10 mg/kg, and 30 mg/kg for intravenous
(iv), oral (po), and intraperitoneal (ip) administration,
respectively. Brain and liver tissues are also collected and
F
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low oral bioavailability seems to be attributed to the rapid
clearance via first pass metabolism as mentioned above.
CONCLUSION
■
The overexpression of specific ALDH isozymes in certain CSCs
suggests ALDHs as potential targets for cancer and CSC-
directed therapeutics. In addition, it has been shown that
selective targeting of ALDH isozymes prevents spheroids
formation in vitro and the size of xenograft tumors formed in
vivo.33 In this study, we performed a systematic medicinal
chemistry optimization of theophylline-based qHTS hits that
ultimately led to potent ALDH1A1 inhibitors with improved
eADME properties. This chemotype demonstrated a high
degree of selectivity over other ALDH isozymes (ALDH1B1,
ALDH3A1, and ALDH2) and other dehydrogenases (HPGD
and HSD17β4). A rapid dilution experiment suggested the
selected analog 64 is a reversible inhibitor. The PK study
demonstrated that analog 64 had reasonable drug exposure via
ip administration and should be suitable for in vivo proof of
concept animal studies. However, further improvement of the
half-life and oral bioavailability would be beneficial if this
compound is to be dosed po. Current studies are focused on
evaluating this class of molecules in cell-based studies (e.g.,
patient-derived CSCs) and other disease-relevant models,
which could benefit from selective pharmacological
ALDH1A1 inhibition. As the combined activity of two or
more ALDH isoforms is likely to be associated with a specific
cancer, the feasibility of using a combination of isoform
selective inhibitors or a dual inhibitor as a clinical prognostic
application should also be considered.15
Figure 2. Rapid dilution assay. The experiment was conducted using
64 as the inhibitor in the presence of ALDH1A1 (20 nM), substrate
(propionaldehyde, 80 μM), and cofactor (NAD+, 1 mM) in 1536-well
format.
analyzed after po administration to further evaluate the
feasibility of potential long-term treatment for metabolic
indications. The PK result compiled in Table 5 revealed that
analog 64 has more desirable plasma exposure for IP (AUC24h
= 5670 h·ng/mL; 30 mg/kg) than po (AUC24h = 484 h·ng/mL;
10 mg/kg) along with 100% and 29% bioavailability,
respectively. (see Supporting Information Table S1 and Figures
S4 and S5 for more details). Reasonable volume of distribution
(Vss = 1.8 L/kg) but short half-life (t1/2 < 1 h) and high
clearance level (CL = 98 mL min−1 kg−1) indicate that 64 is
well absorbed and distributed but rapidly metabolized and/or
excreted. This is consistent with the finding of short t1/2 of CD1
mouse liver microsomal stability (17 min, multipoint format).31
Carefully analyzing the in vitro metabolism of 64 (see
Supporting Information) and the plasma sample from PK
studies by ultraperformance LC MS/MS suggested the
hydroxylation at the terminal of isopentyl side chain and the
hydrolysis of cyclopropylamide moiety are the major
metabolites. In addition, the high clearance presumably also
attributed partially from its low plasma protein binding (68.6%
bound) which increases the fraction of unbound drug making it
more susceptible to being metabolized and/or excreted. Tissue
analysis revealed that 64 is distributed at a higher drug
concentration in liver (L/P = 3.88) where the ALDH activity
expresses the highest level.32 This finding also supported that
the phase I modification in liver seems to be the major issue for
elimination, since 64 possessed reasonable in vitro stability in
CD1 mouse plasma (>60 min). Notably, despite the low drug
exposure in brain, brain/plasma ratio (B/P = 0.54) suggests
that 64 does penetrate the blood−brain barrier. As such,
potential CNS related effects would need to be considered
particularly for a long-term treatment. Finally, the permeability
measured in Caco-2 cell line (efflux ratio of Papp(B−A) (10−6 cm/
s)/Papp(A−B) (10−6 cm/s) = 19.58/19.28 = 1.02) also indicates
that 64 exhibits excellent permeability and does not seem to be
a substrate of transporters, such as P-gp. Given the favorable
solubility and Caco-2 permeability data for this chemotype, the
EXPERIMENTAL SECTION
■
General Methods for Chemistry. 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
instrument. 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% trifluoro-
acetic acid). A gradient from 10% to 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 instrument. UHPLC long gradient equivalent from 4% to 100%
acetonitrile (0.05% trifluoroacetic acid) in water over 3 min run time
of 4.5 min with a flow rate of 0.8 mL/min. A Phenomenex Luna C18
column (3 μm, 3 mm × 75 mm) was used at a temperature of 50 °C.
Method 2. Analysis was performed on an Agilent 1260 with a 7 min
gradient from 4% to 100% acetonitrile (containing 0.025% trifluoro-
acetic 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
Table 5. Pharmacokinetic Results of 64 in CD1 Mice
admin (dosage)
sample
Cmax (ng/mL)
T1/2 (h)
AUC24h (h·ng/mL)
Vss (L/kg)
CL (mL min−1 kg−1
98
)
F (%)
a
b
iv (2 mg/kg)
plasma
plasma
plasma
brain
2070
0.5
0.7
0.4
0.4
0.4
332
1.8
a
ip (30 mg/kg)
8080
696
5670
484
100
29
a
po (10 mg/kg)
po (10 mg/kg)
po (10 mg/kg)
a
a
c
1130
2690
261
d
liver
1880
a
b
c
n = 3. The compound (64) was formulated as solution in 30% HPβCD in saline. Cmax = C0 (t = 0) for iv administration. Brain/plasma (B/P) ratio
d
of 0.54. Liver/plasma (L/P) ratio of 3.88.
G
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Journal of Medicinal Chemistry
Article
(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 analogs for assay have purity greater
Step 3. To a mixture of 4-((1,3-dimethyl-7-(3-methylbenzyl)-2,6-
dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)oxy)benzoic acid (42 mg, 0.1
mmol) and HATU (95 mg, 0.25 mmol) were added DMF (1 mL),
methanamine (2 M in THF, 1 mL, 2 mmol), and then Hunig’s base
(0.087 mL, 0.5 mmol). The mixture was stirred at rt for 2 h. The
mixture was filtered through a filter and submitted for purification by
semipreparative HPLC to give 4-((1,3-dimethyl-7-(3-methylbenzyl)-
2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)oxy)-N-methylbenzamide,
TFA (12, 25 mg, 0.046 mmol, 46% yield). 1H NMR (400 MHz,
DMSO-d6) δ 8.45 (q, J = 4.5 Hz, 1H), 7.93−7.82 (m, 2H), 7.40−7.33
(m, 2H), 7.22 (t, J = 7.5 Hz, 1H), 7.18−7.04 (m, 3H), 5.39 (s, 2H),
3.28 (s, 3H), 3.22 (s, 3H), 2.77 (d, J = 4.5 Hz, 3H), 2.24 (s, 3H). LC−
MS (method 2): tR = 4.87 min, m/z (M + H)+ = 434. HRMS
calculated for C23H24N5O4 (M + H)+: 434.1823. Found: 434.1834.
Preparation of 1,3-Dimethyl-7-(3-methylbenzyl)-8-(piperi-
din-4-yloxy)-1H-purine-2,6(3H,7H)-dione, HCl (14). Step 1. To a
solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (403 mg, 2
mmol) in DMF (4 mL) under N2 at rt was added NaH (72 mg, 3
mmol). After 5 min stirring, 8-chloro-1,3-dimethyl-7-(3-methylben-
zyl)-1H-purine-2,6(3H,7H)-dione (8, 638 mg, 2 mmol) was added.
The mixture was then stirred at rt for 30 min and was poured into
EtOAc/H2O (10 mL/10 mL). The aqueous layer was extracted with
EtOAc (5 mL × 2). The combined organic layer was dried (Na2SO4)
and filtered. After removal of solvent, the crude product was purified
by silica gel chromatography using 20−60−80% EtOAc/hexane as the
eluent to give tert-butyl 4-((1,3-dimethyl-7-(3-methylbenzyl)-2,6-
dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)oxy)piperidine-1-carboxylate
(857 mg, 1.77 mmol, 89% yield). 1H NMR (400 MHz, chloroform-d)
δ 7.23−7.11 (m, 3H), 7.08 (ddq, J = 7.3, 1.6, 0.8 Hz, 1H), 5.25 (s,
2H), 5.17 (dt, J = 7.4, 3.7 Hz, 1H), 3.57 (ddd, J = 13.6, 7.6, 3.9 Hz,
2H), 3.49 (s, 3H), 3.40 (s, 3H), 3.39−3.30 (m, 2H), 2.31 (d, J = 0.8
Hz, 3H), 1.97 (m, 2H), 1.79 (m, 2H), 1.47 (s, 9H).
1
than 95% based on both analytical methods. H NMR spectra were
recorded on Varian 400 MHz spectrometers. High resolution mass
spectrometry results were recorded on Agilent 6210 time-of-flight LC/
MS system.
Representative Synthetic Procedures. Preparation of 8-
Chloro-1,3-dimethyl-7-(3-methylbenzyl)-1H-purine-2,6-
(3H,7H)-dione (8). To a mixture of 8-chloro-1,3-dimethyl-1H-purine-
2,6(3H,7H)-dione (7, 2.15 g, 10 mmol) and K2CO3 (2.07 g, 15 mmol)
was added N,N-dimethylformamide (12 mL). The mixture was stirred
at rt for 5 min, and 1-(bromomethyl)-3-methylbenzene (2.22 g, 12
mmol) was added. The mixture was stirred at rt for 30 min and then
heated at 60 °C for 2 h. The mixture was then dropped into vigorously
stirred H2O (250 mL). The resulting solid was filtered, washed with
H2O (30 mL × 2), hexane (5 mL × 2), and then dried to give 8-
chloro-1,3-dimethyl-7-(3-methylbenzyl)-1H-purine-2,6(3H,7H)-dione
(8, 3.12 g, 9.79 mmol, 98% yield). 1H NMR (400 MHz, chloroform-d)
δ 7.23 (dd, J = 8.2, 7.4 Hz, 1H), 7.17−7.09 (m, 3H), 5.51 (s, 2H), 3.55
(s, 3H), 3.40 (s, 3H), 2.33 (s, 3H). LC−MS (method 1): tR = 3.40
min, m/z (M + H)+ = 319.
Preparation of Ethyl 4-(1,3-Dimethyl-7-(3-methylbenzyl)-
2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperazine-1-car-
boxylate, TFA (11). In a microwave tube was placed 8-chloro-1,3-
dimethyl-7-(3-methylbenzyl)-1H-purine-2,6(3H,7H)-dione (8, 159
mg, 0.5 mmol) and ethyl piperazine-1-carboxylate (237 mg, 1.5
mmol), and to the mixture was added DMSO (1 mL). The mixture
was sealed and heated at 160 °C under microwave irradiation for 30
min. The crude mixture was filtered through a filter and submitted for
purification by semipreparative HPLC to give ethyl 4-(1,3-dimethyl-7-
(3-methylbenzyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-
piperazine-1-carboxylate, TFA (11, 160 mg, 0.289 mmol, 58% yield).
1H NMR (400 MHz, DMSO-d6) δ 7.19 (t, J = 7.6 Hz, 1H), 7.11−6.98
Step 2. To a solution of tert-butyl 4-((1,3-dimethyl-7-(3-methyl-
benzyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)oxy)piperidine-1-
carboxylate (945 mg, 1.95 mmol) in 1,4-dioxane (2 mL) was added
HCl (4 M in 1,4-dioxane, 4 mL). The mixture was stirred at rt for
overnight, and the mixture was concentrated to remove all the solvent.
Then the product was dried in vacuo to give 1,3-dimethyl-7-(3-
methylbenzyl)-8-(piperidin-4-yloxy)-1H-purine-2,6(3H,7H)-dione,
HCl (14, 796 mg, 1.90 mmol, 97% yield). The material was used
without further purification. Some material was submitted for
purification by semipreparative HPLC to give TFA salt of 14 for
(m, 2H), 6.93 (d, J = 7.6 Hz, 1H), 5.34 (s, 2H), 4.02 (q, J = 7.1 Hz,
2H), 3.39 (m, 7H), 3.17 (s, 3H), 3.13−3.01 (m, 4H), 2.24 (s, 3H),
1.16 (t, J = 7.1 Hz, 3H). LC−MS (method 2): tR = 5.44 min, m/z (M
+ H)+ = 441. HRMS calculated for C22H29N6O4 (M + H)+: 441.2245.
Found: 441.2262.
Preparation of 4-((1,3-Dimethyl-7-(3-methylbenzyl)-2,6-
dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)oxy)-N-methylbenza-
mide, TFA (12). Step 1. In a microwave tube was placed 8-chloro-1,3-
dimethyl-7-(3-methylbenzyl)-1H-purine-2,6(3H,7H)-dione (8, 319
mg, 1 mmol), methyl 4-hydroxybenzoate (183 mg, 1.2 mmol), and
K2CO3 (207 mg, 1.5 mmol). Then, DMF (3 mL) was added. The
mixture was sealed and heated at 160 °C under microwave irradiation
for 1 h. The mixture was poured into stirred H2O (60 mL), and the
solid was filtered. The solid was then dissolved in CH2Cl2 and was
purified by silica gel chromatography using 50−100% EtOAc/hexane
as the eluent to give methyl 4-((1,3-dimethyl-7-(3-methylbenzyl)-2,6-
dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)oxy)benzoate (361 mg, 0.83
1
screening. H NMR (TFA salt, 400 MHz, DMSO-d6) δ 8.50 (s, 2H),
7.27−7.14 (m, 1H), 7.13−7.00 (m, 3H), 5.30−5.13 (m, 3H), 3.36 (s,
3H), 3.20 (s, 3H), 3.17−3.04 (m, 4H), 2.26 (s, 3H), 2.12 (m, 2H),
1.92 (m, 2H). LC−MS (method 1): tR = 2.70 min, m/z (M + H)+ =
384.
Preparation of 1,3-Dimethyl-7-(3-methylbenzyl)-8-((1-(4-
methylpyrimidin-2-yl)piperidin-4-yl)oxy)-1H-purine-2,6-
(3H,7H)-dione, TFA (19). In a microwave tube was placed 1,3-
dimethyl-7-(3-methylbenzyl)-8-(piperidin-4-yloxy)-1H-purine-2,6-
(3H,7H)-dione, HCl (14, 63 mg, 0.15 mmol), 2-chloro-4-methylpyr-
imidine (38.6 mg, 0.3 mmol), and K2CO3 (83 mg, 0.6 mmol). Then,
DMF (1 mL) was added sequentially. The mixture was sealed and
heated at 160 °C under microwave irradiation for 1 h. The mixture was
filtered and submitted for purification by semipreparative HPLC to
give 1,3-dimethyl-7-(3-methylbenzyl)-8-((1-(4-methylpyrimidin-2-yl)-
piperidin-4-yl)oxy)-1H-purine-2,6(3H,7H)-dione, TFA (19, 41.3 mg,
0.07 mmol, 47% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J =
5.0 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.11 (t, J = 1.7 Hz, 1H), 7.05 (dt,
J = 7.0, 1.4 Hz, 2H), 6.52 (dd, J = 5.0, 1.3 Hz, 1H), 5.26−5.22 (m,
1H), 5.21 (s, 2H), 3.77 (m, 4H), 3.37 (s, 3H), 3.20 (s, 3H), 2.27 (s,
3H), 2.19 (s, 3H), 2.02−1.87 (m, 2H), 1.77−1.62 (m, 2H). LC−MS
(method 2): tR = 5.15 min, m/z (M + H)+ = 476. HRMS calculated for
C25H30N7O3 (M + H)+: 476.2405. Found: 476.2424.
1
mmol, 83% yield). H NMR (400 MHz, chloroform-d) δ 8.14−8.03
(m, 2H), 7.35−7.28 (m, 2H), 7.23−7.20 (m, 3H), 7.13−7.07 (m, 1H),
5.43 (s, 2H), 3.93 (s, 3H), 3.45 (s, 3H), 3.42 (s, 3H), 2.30 (d, J = 0.8
Hz, 3H). LC−MS (method 2): tR = 6.03 min, m/z (M + H)+ = 435.
Step 2. To a solution of methyl 4-((1,3-dimethyl-7-(3-methylben-
zyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)oxy)benzoate (148
mg, 0.34 mmol) in THF/MeOH (4 mL/0.5 mL) was added LiOH(aq)
(1.5 N, 2 mL). The mixture was stirred at 50 °C for 3 h, and HCl(aq)
(1 N, 4 mL) was added. Then hexane (10 mL) was added and the
resulting solid was filtered, washed with H2O (2 mL × 2), 5% Et2O/
hexane (2 mL × 2), and then dried to give 4-((1,3-dimethyl-7-(3-
methylbenzyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)oxy)benzoic
1
acid (69 mg, 0.164 mmol, 48% yield). H NMR (400 MHz, DMSO-
Preparation of 7-Benzyl-8-((1-(cyclopropanecarbonyl)-
piperidin-4-yl)oxy)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione
(21). To a solution of 7-benzyl-1,3-dimethyl-8-(piperidin-4-yloxy)-1H-
purine-2,6(3H,7H)-dione, HCl (16, 0.61 g, 1.5 mmol) in CH2Cl2 (5
mL) was added Et3N (1.25 mL, 9 mmol) and then cyclo-
d6) δ 13.03 (s, 1H), 8.04−7.94 (m, 2H), 7.44−7.34 (m, 2H), 7.22 (t, J
= 7.6 Hz, 1H), 7.15−7.03 (m, 3H), 5.39 (s, 2H), 3.29 (s, 3H), 3.22 (s,
3H), 2.24 (s, 3H). LC−MS (method 2): tR = 5.08 min, m/z (M + H)+
= 421.
H
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Journal of Medicinal Chemistry
Article
propanecarbonyl chloride (0.31 g, 3 mmol) dropwise. The mixture was
stirred at rt for 30 min and then poured into EtOAc/H2O/Na2CO3(aq)
(50 mL/25 mL/25 mL). The organic layer was washed with H2O (50
mL), dried (Na2SO4), and filtered. After removal of solvent, the
product was dried to give 7-benzyl-8-((1-(cyclopropanecarbonyl)-
piperidin-4-yl)oxy)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (21,
630 mg, 96%). This material was used for the next step without
further purification. Small amount of product was submitted for
purification by semipreparative HPLC to give TFA salt of 21 for
screening. 1H NMR (TFA salt, 400 MHz, DMSO-d6) δ 7.46−7.11 (m,
5H), 5.26 (s, 2H), 5.20 (dt, J = 7.0, 3.4 Hz, 1H), 3.67 (s, 2H), 3.48 (s,
2H), 3.36 (s, 3H), 3.20 (s, 3H), 1.96 (m, 3H), 1.68 (m, 2H), 0.76−
0.60 (m, 4H). LC−MS (method 1): tR = 3.24 min, m/z (M + H)+ =
438. HRMS calculated for C23H27N5O4Na (M + Na)+: 460.1955.
Found: 460.1977.
purification by semipreparative HPLC to give 8-((1-
(cyclopropanecarbonyl)piperidin-4-yl)oxy)-1,3-dimethyl-7-(3-methyl-
benzyl)-1H-purine-2,6(3H,7H)-dione, TFA (34, 25.5 mg, 0.045 mmol,
1
45% yield). H NMR (400 MHz, DMSO-d6) δ 7.20 (t, J = 7.5 Hz,
1H), 7.14−7.03 (m, 3H), 5.22 (s, 2H), 5.19 (dd, J = 6.8, 3.4 Hz, 1H),
3.66−3.40 (m, 4H), 3.36 (s, 3H), 3.20 (s, 3H), 2.24 (s, 3H), 1.96 (m,
3H), 1.69 (m, 2H), 0.78−0.59 (m, 4H). LC−MS (method 2): tR
=
5.15 min, m/z (M + H)+ = 452. HRMS calculated for C24H29N5O4Na
(M + Na)+: 474.2112. Found: 474.2123.
Preparation of 8-((1-(Cyclopropanecarbonyl)piperidin-4-yl)-
oxy)-1,3-dimethyl-7-phenethyl-1H-purine-2,6(3H,7H)-dione,
TFA (39). To a mixture of 8-((1-(cyclopropanecarbonyl)piperidin-4-
yl)oxy)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (22, 20.8 mg, 0.06
mmol) and K2CO3 (16.6 mg, 0.12 mmol) was added N,N-
dimethylformamide (1 mL). The mixture was stirred at rt for 5 min,
and (2-bromoethyl)benzene (33.3 mg, 0.18 mmol) was added. The
mixture was heated at 50 °C for 3 h. The mixture was filtered through
a filter and submitted for purification by semipreparative HPLC to give
8-((1-(cyclopropanecarbonyl)piperidin-4-yl)oxy)-1,3-dimethyl-7-phe-
nethyl-1H-purine-2,6(3H,7H)-dione, TFA (39, 8.9 mg, 0.016 mmol,
Preparation of 8-((1-(Cyclopropanecarbonyl)piperidin-4-yl)-
oxy)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (22). In a two-
neck flask was placed 7-benzyl-8-((1-(cyclopropanecarbonyl)piperidin-
4-yl)oxy)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (21, 1.75 g, 4
mmol) and Pd(OH)2 (0.56 g, 0.8 mmol). Then, EtOH/EtOAc (40
mL/20 mL) was added. The air was removed and refilled with H2 (3
times). The mixture was heated to 70 °C for 24 h under H2 balloon.
After cooling to rt, the mixture was filtered through Celite and eluted
with CH2Cl2. The filtrate was concentrated and the solid was triturated
with 2% EtOAc/hexane and then dried in vacuo to give 8-((1-
(cyclopropanecarbonyl)piperidin-4-yl)oxy)-1,3-dimethyl-1H-purine-
1
26% yield). H NMR (400 MHz, DMSO-d6) δ 7.27−7.13 (m, 3H),
7.09−7.03 (m, 2H), 4.96 (dt, J = 7.1, 3.5 Hz, 1H), 4.22 (t, J = 6.7 Hz,
2H), 3.43 (br s, 4H), 3.35 (s, 3H), 3.23 (s, 3H), 2.99 (t, J = 6.7 Hz,
2H), 1.95 (tt, J = 7.3, 5.1 Hz, 1H), 1.78 (m, 2H), 1.45 (m, 2H), 0.70
(t, J = 6.4 Hz, 4H). LC−MS (method 2): tR = 5.08 min, m/z (M +
H)+ = 452. HRMS calculated for C24H30N5O4 (M + H)+: 452.2292.
Found: 452.2275.
1
2,6(3H,7H)-dione (22, 1.04 g, 3 mmol, 75% yield). H NMR (400
P r e p a r a t i o n o f 7 - ( 2 - C y c l o h e x y l e t h y l ) - 8 - ( ( 1 -
(cyclopropanecarbonyl)piperidin-4-yl)oxy)-1,3-dimethyl-1H-
purine-2,6(3H,7H)-dione, TFA (54). In a microwave tube were
placed 8-((1-(cyclopropanecarbonyl)piperidin-4-yl)oxy)-1,3-dimethyl-
1H-purine-2,6(3H,7H)-dione (22, 20.84 mg, 0.06 mmol) and 2-
cyclohexylethanol (23.08 mg, 0.180 mmol). The tube was sealed, and
the air was removed and then refilled with N2. Then, (E)-di-tert-butyl
diazene-1,2-dicarboxylate (41.4 mg, 0.180 mmol) and Ph3P (47.2 mg,
0.180 mmol) in THF (1 mL) was added, and the mixture was stirred
at rt for 3 h. The mixture was concentrated, redissolved in DMF,
filtered through a filter, and then submitted for purification by
semipreparative HPLC to give 7-(2-cyclohexylethyl)-8-((1-
(cyclopropanecarbonyl)piperidin-4-yl)oxy)-1,3-dimethyl-1H-purine-
MHz, chloroform-d) δ 11.15 (s, 1H), 5.26 (dt, J = 7.2, 3.6 Hz, 1H),
3.90 (br s, 2H), 3.75−3.57 (m, 2H), 3.55 (s, 3H), 3.43 (s, 3H), 2.08
(br s, 2H), 1.90 (br s, 2H), 1.81−1.73 (m, 1H), 1.00 (dt, J = 4.7, 3.1
Hz, 2H), 0.79 (dt, J = 8.0, 3.3 Hz, 2H). LC−MS (method 2): tR = 3.51
min, m/z (M + H)+ = 348. HRMS calculated for C16H22N5O4 (M +
H)+: 348.1666. Found: 348.1677.
Preparation of 7-Isopentyl-1,3-dimethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purine-8-carbaldehyde (25g). Step 1. To a
mixture of 8-(hydroxymethyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-
dione (24, 1.05 g, 5 mmol) and K2CO3 (2.073 g, 15 mmol) was
added N,N-dimethylformamide (15 mL). The mixture was stirred at rt
for 5 min, and 1-bromo-3-methylbutane (2.398 mL, 20 mmol) was
added. The mixture was heated at 60 °C for overnight and was poured
into EtOAc/H2O (80 mL/80 mL). The organic layer was washed with
H2O (80 mL), dried (Na2SO4), and then filtered. After removal of
solvent, the product was purified by silica gel chromatography using
80−100% EtOAc/hexane as the eluent to give 8-(hydroxymethyl)-7-
isopentyl-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (1.21 g, 4.32
1
2,6(3H,7H)-dione, TFA (54, 14.7 mg, 0.026 mmol, 43% yield). H
NMR (400 MHz, DMSO-d6) δ 5.20 (dt, J = 7.4, 3.7 Hz, 1H), 4.04 (t, J
= 7.0 Hz, 2H), 3.95−3.39 (m, 4H), 3.36 (s, 3H), 3.19 (s, 3H), 2.19−
1.45 (m, 12H), 1.25−1.02 (m, 4H), 0.89 (m, 2H), 0.76−0.63 (m, 4H).
LC−MS (method 2): tR = 5.99 min, m/z (M + H)+ = 458. HRMS
calculated for C24H36N5O4 (M + H)+: 458.2762. Found: 458.2772.
Preparation of Ethyl 4-((1,3-Dimethyl-7-(3-methylbenzyl)-
2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)methyl)piperazine-
1-carboxylate (5). To 1,3-dimethyl-7-(3-methylbenzyl)-2,6-dioxo-
2,3,6,7-tetrahydro-1H-purine-8-carbaldehyde (25a, 62.5 mg, 0.2
mmol) was added a solution of ethyl piperazine-1-carboxylate (95
mg, 0.600 mmol) in CH2Cl2 (2 mL) at rt. The mixture was stirred for
3−5 min, and sodium triacetoxyborohydride (212 mg, 1.000 mmol)
was added. The mixture was stirred at rt for 1.5 h and was poured into
CH2Cl2/Na2CO3(aq) (3 mL/3 mL). The aqueous layer was extracted
with CH2Cl2 (3 mL × 2). The combined organic layer was dried
(Na2SO4) and filtered. After removal of solvent, the crude product was
purified by silica gel chromatography using 50−80% EtOAc/hexane as
the eluent to give ethyl 4-((1,3-dimethyl-7-(3-methylbenzyl)-2,6-
dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)methyl)piperazine-1-carboxy-
late (87 mg, 0.191 mmol, 96% yield). 1H NMR (400 MHz,
chloroform-d) δ 7.24−7.15 (m, 1H), 7.12−7.04 (m, 1H), 6.92 (ddd,
J = 7.6, 1.9, 0.9 Hz, 2H), 5.73 (s, 2H), 4.13 (q, J = 7.1 Hz, 2H), 3.59 (s,
3H), 3.54 (s, 2H), 3.41 (s, 7H), 2.39 (t, J = 5.2 Hz, 4H), 2.31 (s, 3H),
1.25 (t, J = 7.1 Hz, 3H). LC−MS (method 1): tR = 2.80 min, m/z (M
+ H)+ = 455.
1
mmol, 86% yield). H NMR (400 MHz, chloroform-d) δ 4.75 (dd, J
= 6.0, 0.9 Hz, 2H), 4.39−4.23 (m, 2H), 3.56 (d, J = 0.9 Hz, 3H), 3.41
(d, J = 0.8 Hz, 3H), 2.71 (td, J = 6.1, 3.1 Hz, 1H), 1.80−1.62 (m, 3H),
1.06−0.92 (m, 6H). LC−MS (method 1): tR = 2.76 min, m/z (M +
H)+ = 281.
Step 2. To a solution of 8-(hydroxymethyl)-7-isopentyl-1,3-
dimethyl-1H-purine-2,6(3H,7H)-dione (1550 mg, 5.53 mmol) in
DCM (15 mL) was added Dess−Martin periodinane (3283 mg,
7.74 mmol) at 0 °C. After 10 min, the mixture was stirred at rt for 2 h.
The mixture was concentrated and the residue was purified by silica gel
chromatography using 30−60% EtOAc/hexane as the eluent to give 7-
isopentyl-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purine-8-car-
1
baldehyde (25g, 1160 mg, 4.17 mmol, 75% yield). H NMR (400
MHz, chloroform-d) δ 9.91 (s, 1H), 4.94−4.61 (m, 2H), 3.62 (s, 3H),
3.44 (s, 3H), 1.87−1.61 (m, 3H), 1.00 (d, J = 6.2 Hz, 6H). LC−MS
(method 1): tR = 3.30 min, m/z (M + H)+ = 279.
Preparation of 8-((1-(Cyclopropanecarbonyl)piperidin-4-yl)-
oxy)-1,3-dimethyl-7-(3-methylbenzyl)-1H-purine-2,6(3H,7H)-
dione, TFA (34). To a solution of 1,3-dimethyl-7-(3-methylbenzyl)-8-
(piperidin-4-yloxy)-1H-purine-2,6(3H,7H)-dione, HCl (14, 42 mg, 0.1
mmol) in CH2Cl2 (2 mL) were added Et3N (0.07 mL, 0.5 mmol) and
then cyclopropanecarbonyl chloride (21 mg, 0.2 mmol) dropwise. The
mixture was stirred at rt for 30 min. MeOH (0.2 mL) was added and
stirred for another 30 min. The mixture was concentrated, dissolved in
DMSO (2 mL), filtered through a filter, and then submitted for
Preparation of 8-((4-(Cyclopropanecarbonyl)piperazin-1-yl)-
methyl)-7-isopentyl-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione,
HCl (64). To a mixture of 7-isopentyl-1,3-dimethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purine-8-carbaldehyde (25g, 195 mg, 0.7 mmol) and
cyclopropyl(piperazin-1-yl)methanone, HCl (267 mg, 1.400 mmol)
I
DOI: 10.1021/acs.jmedchem.5b00577
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
were added CH2Cl2 (6 mL) and then Et3N (0.293 mL, 2.100 mmol) at
rt. The mixture was stirred for 3−5 min, and sodium triacetoxybor-
ohydride (297 mg, 1.400 mmol) was added. The mixture was stirred at
rt for 4 h and was poured into CH2Cl2/Na2CO3(aq) (5 mL/5 mL). The
aqueous layer was extracted with CH2Cl2 (5 mL × 2). The combined
organic layer was dried (Na2SO4) and filtered. After removal of
solvent, the crude product was purified by silica gel chromatography
using 0−5% MeOH/EtOAc as the eluent to give 8-((4-
(cyclopropanecarbonyl)piperazin-1-yl)methyl)-7-isopentyl-1,3-di-
methyl-1H-purine-2,6(3H,7H)-dione (251 mg, 0.603 mmol, 86%
short to medium half-life), and carbamazepine and antipyrine (for long
half-life). The assay incubation system consisted of 0.5 mg/mL
microsomal protein, 1.0 μM drug concentration, and NADPH
regeneration system (containing 0.650 mM NADP+, 1.65 mM glucose
6-phosphate, 1.65 mM MgCl2, and 0.2 unit/mL G6PDH) in 100 mM
phosphate buffer at pH 7.4. The incubation was carried out at 37 °C
for 15 min. The reaction was quenched by adding 550 μL of
acetonitrile (∼1:2 ratio) containing 0.28 μM albendazole (internal
standard). After a 20 min centrifugation at 3000 rpm, 30 μL of the
supernatant was transferred to an analysis plate and was diluted 5-fold
using 1:2 v/v acetonitrile/water before the samples were analyzed by
LC/MS−MS.
1
yield). H NMR (400 MHz, DMSO-d6) δ 4.30 (dd, J = 9.7, 5.6 Hz,
2H), 3.70 (s, 2H), 3.63 (s, 2H), 3.43 (s, 2H), 3.39 (s, 3H), 3.21 (s,
3H), 2.41 (m, 4H), 1.93 (m, 1H), 1.68 (m, 3H), 0.94 (d, J = 5.9 Hz,
6H), 0.68 (tt, J = 7.9, 2.9 Hz, 4H); 13C NMR (101 MHz, chloroform-
d) δ 171.91, 154.78, 151.50, 148.67, 147.53, 107.79, 54.48, 53.22,
52.89, 45.14, 44.50, 41.80, 39.73, 29.67, 27.86, 26.15, 22.39, 10.80, 7.40
(four peaks shown on piperazine ring). LC−MS (method 1): tR = 2.82
min, m/z (M + H)+ = 417. HRMS calculated for C21H32N6O3Na (M +
Na)+: 439.2428. Found: 439.2443. This material was converted to its
HCl salt for PK study. To a solution of 8-((4-(cyclopropanecarbonyl)-
piperazin-1-yl)methyl)-7-isopentyl-1,3-dimethyl-1H-purine-2,6-
(3H,7H)-dione (250 mg, 0.600 mmol) in 1,4-dioxane (6 mL) at rt was
added HCl solution (4 M in 1,4-dioxane, 0.3 mL, 1.2 mmol, 2 equiv).
The mixture was stirred at rt for 5 min, and hexane (15 mL) was
added and stirred for 15 min. The solid was filtered and washed with
hexane (5 mL × 3) and dried in vacuo to give 8-((4-
(cyclopropanecarbonyl)piperazin-1-yl)methyl)-7-isopentyl-1,3-di-
methyl-1H-purine-2,6(3H,7H)-dione, HCl (64, 270 mg, 0.596 mmol,
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional supplemental figures, tables, and detailed exper-
imental procedures and spectroscopic data (1H NMR, LC−MS,
HRMS) for screening compounds; a xlsx file containing
molecular formula strings. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acs.jmedchem.5b00577.
AUTHOR INFORMATION
Corresponding Authors
■
*S.-M.Y.: e-mail, yangs9@mail.nih.gov; phone, 301-217-4685.
*D.J.M. (for major correspondence and request of material): e-
mail, maloneyd@mail.nih.gov; phone, 301-217-4381; fax, 301-
217-5736.
1
99% yield) as a white solid. Mp 187−189 °C; H NMR (400 MHz,
DMSO-d6) δ 4.36 (d, J = 7.6 Hz, 2H), 3.55 (s, 2H), 3.43 (s, 3H), 3.23
(s, 3H), 2.07−1.89 (m, 1H), 1.64 (m, 3H), 0.93 (d, J = 6.2 Hz, 6H),
0.73 (d, J = 7.7 Hz, 4H) (The protons of piperazine ring (8 H) are
very broad between 4.6 and 2.8 ppm).
Notes
The authors declare no competing financial interest.
Biological Methods. Protein Expression and Activity
Measurement. Human ALDH1A1, ALDH1B1, and ALDH3A1
were expressed and purified as described elsewhere.34−36 Human
ALDH2 was purchased from Abcam (Cambridge, MA).
ACKNOWLEDGMENTS
■
S.-M.Y., A.Y., M.L.-N., K.N., K.B., A.W., X.X., M.F., A.S., A.J.,
and D.J.M. were supported 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 (Grant
U54MH084681). The authors thank Sam Michael and Richard
Jones for automation support; Paul Shinn, Danielle van Leer,
Crystal McKnight, and Misha Itkin for the assistance with
compound management; William Leister, Heather Baker,
Elizabeth Fernandez, Burchelle Blackman, and Christopher
Leclair for analytical chemistry and purification support; and
Emma Hughes for preparing plasma samples for bioanalytical
characterization. This research was also supported in part by
National Institutes of Health Grants AA022057 (V.V.) and
AA021724 (V.V.).
General Protocol for ALDH Enzymatic Assays. Briefly, an
amount of 3 μL of enzyme (final concentration 20, 50, 20, and 5 nM
for ALDH1A1, ALDH1B1, ALDH2, and ALDH3A1, respectively) or
assay buffer (100 mM HEPES, pH 7.5, with 0.01% Tween 20) was
dispensed into a 1536-well solid-bottom black plate (Greiner Bio One,
Monroe, NC) followed by pin-tool transfer (23 nL) of candidate
inhibitors (final concentration range 968 pM to 57.2 μM) and control
(Bay 11-7085, final concentration range from 1.31 nM to 2.86 μM).
Samples were incubated (rt, protected from light) for 15 min followed
by a 1 μL substrate addition of NAD+ and propionaldehyde (final
concentrations of 1 mM and 80 μM, respectively, for ALDH1A1,
ALDH1B1, and ALDH2; or NAD+ and benzaldehyde at 1 mM and
200 μM, respectively, for ALDH3A1). Plates were centrifuged at 1000
rpm for 15 s, then read in kinetic mode on a ViewLux high-throughput
CCD imager (PerkinElmer) equipped with standard UV fluorescence
optics (340 nm excitation, 450 nm emission) for 10 min (ALDH1A1,
ALDH1B1, ALDH2) or 4 min (ALDH3A1). The change in
fluorescence intensity over the 4 or 10 min reaction period was
normalized against no-inhibitor and no-enzyme controls, and the
resulting percent inhibition data were fitted for biological activity.
Disulfiram was used as internal standard with comparable activity to
reported potency.16
ABBREVIATIONS USED
■
ADME, absorption, distribution, metabolism, and excretion;
ALDH, aldehyde dehydrogenase; CSC, cancer stem cell;
eADME, early absorption, distribution, metabolism, and
excretion; HPbCD, hydroypropyl β-cyclodextrin; HPGD, 15-
hydroxyprostaglandin dehydrogenase; HSD17β4, type 4
hydroxysteroid dehydrogenase; qHTS, quantitative high
throughput screening; RLM, rat liver microsomes; SAR,
structure−activity relationship
Dehydrogenase Selectivity Assays. The inhibitory activity
against dehydrogenases HPGD and HSD17β4 were measured
according protocols described previously.30
High-Throughput RLM Measurement. Microsomal stability of
test articles was determined in a HTS format with the single time point
and in the 96-well plates, and automated sample preparation with
Tecan EVO 200 robot and LC/MS/MS (Waters Xevo TQ-S)
instrument were used to measure the percentage of compound
remaining after incubation. This allows the calculation of the in vitro
half-life (t1/2). Six standard controls were tested in each run: buspirone
and propranolol (for short half-life), loperamide and diclofenac (for
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