Tumor-Activated Benzothiazole Inhibitors of Stearoyl-CoA Desaturase

Article abstract of DOI:10.1021/acs.jmedchem.0c00899

A series of N-acyl benzothiazoles shows selective and potent cytotoxicity against cancer cell lines expressing cytochrome P450 4F11. A prodrug form is metabolized by cancer cells into an active inhibitor of stearoyl-CoA desaturase (SCD). Substantial variation on the acyl portion of the inhibitors allowed the identification of (R)-27, which balanced potency, solubility, and lipophilicity to allow proof-of-concept studies in mice. The prodrugs were activated inside the tumor, where they can arrest tumor growth. Together, these observations offer promise that a tumor-activated prodrug strategy might exploit the essentiality of SCD for tumor growth, while avoiding toxicity associated with systemic SCD inhibition.

Full text of DOI:10.1021/acs.jmedchem.0c00899

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Tumor-Activated Benzothiazole Inhibitors of Stearoyl-CoA
Desaturase
Noelle S. Williams,* Stephen Gonzales, Jacinth Naidoo, Giomar Rivera-Cancel, Sukesh Voruganti,
Prema Mallipeddi, Panayotis C. Theodoropoulos, Sophie Geboers, Hong Chen, Francisco Ortiz,
Bruce Posner, Deepak Nijhawan,* and Joseph M. Ready*
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ABSTRACT: A series of N-acyl benzothiazoles shows selective
and potent cytotoxicity against cancer cell lines expressing
cytochrome P450 4F11. A prodrug form is metabolized by cancer
cells into an active inhibitor of stearoyl-CoA desaturase (SCD).
Substantial variation on the acyl portion of the inhibitors allowed
the identification of (R)-27, which balanced potency, solubility,
and lipophilicity to allow proof-of-concept studies in mice. The prodrugs were activated inside the tumor, where they can arrest
tumor growth. Together, these observations offer promise that a tumor-activated prodrug strategy might exploit the essentiality of
SCD for tumor growth, while avoiding toxicity associated with systemic SCD inhibition.
INTRODUCTION
■
Selective toxicity is a hallmark of modern chemotherapies.
Targeted anticancer drugs frequently show toxicity toward
cancers with specific mutations or gene expression changes,
even if among the same tissue subtype. They are frequently
discovered using target-based screens against specific enzymes
or receptors identified through genetic interrogation of tumor
types. By contrast, historical chemotherapies such as
vinblastine, Taxol, and others, were discovered using unbiased
phenotypic screening. These drugs are not selective and kill
most cancer cell types regardless of genotype. Combining these
approachesusing phenotypic screening to identify selective
cytotoxinscould provide new lead compounds for cancer
treatment and uncover new vulnerabilities within various
cancers.1
To identify selective cytotoxins in an unbiased manner,
members of the Cancer Target Discovery and Development
(CTD2) consortium completed a high throughput toxicity
screen using 12 distinct nonsmall cell lung cancer (NSCLC)
cell lines.2 Each cell line was treated with approximately
200,000 small molecules at a concentration of 2.5 μM for 4
days. Cell viability was assessed using CellTiterGlo(R) and
Figure 1. HTS hits (1−2) and reported inhibitors of SCD (3−8).
potentially selective compounds were identified using secon-
dary dose-response assays. We recently described two chemical
series that emerged from this screen, represented by the N-acyl
instance, compound 1 is toxic to H2122 cells with IC50 = 50
nM, whereas the IC50 against H2009 cells is 22 μM.
aminobenzothiazole 1 and the oxalic acid diamide 2
(oxalamides) (Figure 1).3,4 Dose−response experiments
Received: June 3, 2020
Published: August 5, 2020
demonstrated that benzothiazole 1 displayed IC50 values <1
μM against four NSCLC lines (H2122, H460, HCC44,
HCC95) but >5 μM against 8 other NSCLC lines (H2009,
H1395, H1819, H1993, HCC366, H2073, HCC4017, H1155)
and one immortalized human bronchial epithelial cell line. For
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The enzyme stearoyl CoA desaturase (SCD) was identified
as a covalent binding partner of the benzothiazoles and
oxalamides.5 This enzyme catalyzes the dehydrogenation of
saturated fatty acids to form cis-Δ9 unsaturated fatty acids.6
Inhibition of SCD can be toxic to cancer cells because
unsaturated fatty acids are required for the maintenance of
membrane fluidity and the synthesis of new membranes. For
example, a recent functional genomic screen of 66 genes
involved in lipid biosynthesis identified SCD as essential in
breast and prostate cancer cells.7 Similarly, SCD and
unsaturated fatty acid levels were elevated in patient samples
from clear cell renal cell carcinoma tumors, and the levels of
both enzyme and lipids were positively correlated with tumor
growth rates.8
therefore, to identify tumor-activated SCD inhibitors that
could minimize skin toxicity because sebocytes do not express
CYP4F11 or CYP4F12 to appreciable degrees. Here we
describe our efforts to optimize the benzothiazole series into
bioavailable, tumor-targeted SCD inhibitors (Figure 1).
RESULTS AND DISCUSSION
■
Optimization of Benzothiazoles. We first explored
changes to the benzamide portion of compound 1 using
toxicity toward H2122 cells as a readout of potency and
toxicity toward H2009 cells as a measure of selectivity. At this
stage in the project, the mechanism of action of the
benzothiazoles was unknown, so our objectives were to
understand structure−activity relationships and to develop
tools for target identification. Removing the amine from 1
compromised activity against H2122 cells by approximately
10-fold (9) as did replacing it with a methoxy or fluoro group
(10, 11, 12). A nitro group maintained activity (13), although
selectivity versus H2009 cells decreased (Table 1).
Several experimental results indicate that SCD represents
the functional target of both the benzothiazoles and
oxalamides3,4 (e.g., 1, 2). (1) The benzothiazoles and the
oxalamides both bind SCD irreversibly. Thus, derivatives
bearing an alkyne moiety could form covalent adducts with
SCD. These adducts could be visualized by click-conjugation
to an azide-bearing dye followed by denaturing gel electro-
phoresis and fluorescent imaging. Alternatively, click-conjuga-
tion to biotin-azide facilitated immunoprecipitation of a drug-
SCD complex, which was identified using mass spectrometry.
(2) Binding affinity of the benzothiazoles and oxalamides
toward SCD was found to correlate to cytotoxicity in H2122
cells, indicating the biological relevance of SCD binding. (3)
Both compound classes inhibited SCD enzymatic activity at
concentrations similar to their cytotoxic IC50’s. (4) The
toxicity of these compounds could be rescued by supplemen-
tation with unsaturated fatty acids, which removes the
requirement for de novo synthesis of unsaturated fatty acids.
Several classes of SCD inhibitors have been described
previously in the context of drug discovery efforts focused on
cancer and metabolic disorders (Figure 1).9 Representative
examples include urea 3 (Abbott),10 pyridazine 4 (Abbott),11
quinazalinone 5 (CV Therapeutics),12 isoxazole 6 (Merck),13
thiazolylpyridinone 7 (Novartis/Xenon),14 and pyridazine 8
(Xenon).15 These inhibitors were originally of interest as
potential treatments for metabolic disorders, with Merck’s
inhibitor 6 advancing to phase 2 clinical trials,13 and Aramchol,
an amide derived from cholic acid and arachidic acid,
advancing to phase 3 trials for nonalcoholic steatohepatitis.16
More recently, the anticancer activity of SCD inhibitors has
been documented in both cell culture and rodent models of
human cancer.10b,c,11b,12b However, clinical development of
SCD inhibitors has been hampered by on-target toxicity.17 In
particular, inhibition of SCD in the skin causes atrophy of
sebocytes. In turn, this toxicity leads to dry eye, dry skin, and
hypothermia due to loss of sebum, which is comprised of fatty
acid esters. Similarly, the global SCD-knockout animal suffers
from alopecia, closed eyes, and suffers deficiencies in thermal
regulation.18 By contrast, the liver-specific SCD-knockout
mouse displayed no obvious phenotype, indicating that SCD
is not essential in the liver.19
Benzamides featuring a polar linker at C4 attached to a
lipophilic group were highly potent against H2122 cells. For
example, compounds 14 and 15 feature a benzyl ether and
diaryl amine, respectively. Single-digit nM potency was
achieved with a benzophenone (16) while retaining >100×
selectivity relative to H2009 cells. The benzophenone was
originally introduced as a potential photo-cross-linker.
Similarly, an aryl azide (17), a second potential photo-cross-
linker, displayed activity comparable to the initial hit 1 (IC50
0.12 vs 0.06 μM).
Benzophenone analog 16 was highly potent, but it was too
lipophilic for in vivo use: IP administration at 10 mg/kg
resulted in low plasma Cmax and AUC values (Table 2).
Subsequent investigation revealed that the compounds
accumulated in the fat pad of mice in the peritoneal space.
In particular, Cmax in the fat was 90 μM compared to 0.3 μM in
plasma and 0.9 μM in the lungs. This property was reflected in
the high calculated log P value of 16 (5.0). To identify more
polar compounds, we replaced the benzamide with a
nicotinamide group (19), which reduced activity but did not
abrogate it entirely. Returning to the benzamide substructure,
various amides were installed at the 4-position. The piperazine
and morpholine amides (20, 21) showed weak activity. A
piperidine (22) and benzyl amide (23) maintained activity, but
were nonselective.
As an alternative strategy to decrease log P, we synthesized a
series of compounds that retained the diaryl amide of 16 but
added polarity in the linker or in one of the aryl rings. For
example, alcohol 24 and ketones 25 and 26 had IC50’s ∼ 20
nM. Finally, amine (R)-27 killed H2122 cells with an IC50 of
11 nM and 100-fold selectivity toward H2009 cells. Its
enantiomer (S)-27 was approximately seven-fold less active.
Amine rac-27 showed marginally improved plasma pharmaco-
kinetic properties in CD-1 mice relative to 16. Following a 10
mg/kg IP injection, Cmax and AUC were increased fivefold.
More encouragingly, Cmax and AUC in the lungs increased
more than 50-fold relative to 16. For comparison, the pyridyl
alcohol rac-24 showed better plasma PK than rac-27 in mice,
but lower lung exposure. The high lung exposure of rac-27
translated into high tumor exposure as well, supporting in vivo
evaluation of this analog (see below). Intratumoral compound
levels of 7 μM were detected 7 h following a 20 mg/kg IP dose
of rac-27 to nod-SCID mice bearing tumor xenografts derived
from H2122 cells.
The benzothiazoles and oxalamides are only toxic to
approximately 30% of NSCLC lines because they are activated
by CYP4F11 and CYP4F12.3 These cytochrome P450
isoforms are differentially expressed across various cancer cell
lines, with high expression in sensitive lines and low expression
in insensitive cell lines.20 Thus, compounds 1 and 2 are
prodrugs that are converted to active SCD inhibitors in H2122
cells but not H2009 cells, for example. The potential exists,
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both inactive against both cell lines. Similarly, the sulfonamide
Table 1. Modification of the Benzamide
30 was not toxic to either H2122 or H2009 cells (Figure 2).
Figure 2. Inactive analogs.
Exploration of the benzothiazole ring revealed steep
structure−activity relationships (Table 3). For instance, the
methoxy group was not required for activity or selectivity, but
other changes were deleterious. In several direct comparisons,
the 6-methoxy benzothiazoles displayed similar activity and
selectivity as the unsubstituted comparators including: 4-OMe
(10 vs 33), benzophenone (16 vs 36), 3-Me (18 vs 34), and 3-
Py (19 vs 35). Most analogs retained the methoxy group
Table 3. Modification of the Benzo-Azole
Table 2. Pharmacokinetic Properties in CD-1 Female Mice
Following a 10 mg/kg IP Dose
plasma
fat
lung
Cmax
AUC
Cmax
AUC
Cmax
AUC
Cmpd
16
(rac)-27
(rac)-24
(μM)
(μM*h)
(μM)
(μM*h)
(μM)
(μM*h)
0.3
1.5
11
0.6
3.0
9.6
90
94
120
ND
77
70
0.9
61
26
2.6
160
17
An aryl amide appeared to be required for activity. The
phenyl acetamide (28) and the dihydrocinnamide (29) were
a
IC50 versus H1819 cell line.
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because at this stage in the project, the mechanism of action
was not understood; we planned to replace the methoxy group
with an alkyne as a click handle to facilitate target
identification.
cells with t1/2 = 19 h, whereas its half-life in the presence
H1155, an insensitive cell line, was similar to media alone (t1/2
> 48 h; Figure 4). It was also stable in the presence of H2122
A series of three benzo-azoles was synthesized for
comparison. Among these, the benzothiazole (36) was the
most active and selective, followed by the benzimidazole (37),
while the benzoxazole was essentially inactive (38). Similarly,
the unsubstituted thiazole 39 and phenyl-thiazoles 40 and 41
lost substantial activity relative to the benzothiazole. Taken
together, these results supported continued focus on the
benzothiazole ring system.
We next investigated substitution on the benzothiazole. A 5-
amino group was not tolerated on the benzothiazole ring (42),
but a 5-methoxy-substituted benzothiazole (43) was similar to
the 6-substituted analog 13. Replacing the 6-methoxy group
with a propyl group lost both potency and selectivity for
H2122 (44) while a bromopropyl group rendered the analog
inactive (45). Likewise, brominating the 5 position (46) cost
nearly 100-fold in terms of activity against H2122 cells relative
to the nonbrominated analog 16. Replacing the 6-alkoxy group
with an ester (47) substantially decreased activity. On the
other hand, reversing the central amide was well-tolerated, and
48 was both potent and selective.
Figure 4. Conversion of prodrug (R)-27 to active form (R)-53 in
H2122 or H1155 NSCLC cells. Cells were incubated with 100 nM
(R)-27 and culture contents evaluated for (R)-27 and (R)-53 levels
by LC−MS/MS.
cells that had been additionally treated with HET0016, an
inhibitor of the CYP4F family (t1/2 > 48 h).23 In the H2122
cell culture, we observed a major metabolite consistent with a
demethylation event. To test this hypothesis, we synthesized
(R)-53 (Table 4), the putative metabolite from (R)-27 and
confirmed that it had the same retention time, mass, and MS/
MS fragmentation as the metabolite formed from (R)-27.
Phenol (R)-53 was not formed in H1155 cells (Figure 4), nor
was it formed in H2122 cells in the presence of HET0016
(Figure S1).
Finally, we synthesized several analogs featuring an alkyne
handle to facilitate click conjugation (Figure 3). Propargyl
Taken together, the data indicate that (R)-27 represents a
prodrug that is converted to its active form (R)-53 by
CYP4F11, which is present in sensitive (e.g., H2122), but not
insensitive (e.g., H1155) NSLCL cell lines. Thus, while (R)-27
showed potent but selective toxicity toward H2122, (R)-53
demonstrated potent toxicity toward both H2122 and H1155
cells with IC50 < 10 nM (Table 4, entries 1, 4). Moreover,
cotreatment with HET0016 increased the IC50 of the prodrug
(R)-27 by > 10-fold while having no impact on the potency of
the drug form (R)-53, consistent with the CYP4F11
metabolism of (R)-27 (entry 3). Additionally, overexpression
of CYP4F11 sensitized H1155 to (R)-27 (entry 5). By
contrast, the drug form (R)-53 does not require CYP
activation; accordingly, it shows similar toxicity toward both
H2122 and H1155 cell lines (entries 1, 4). For both the drug
and prodrug forms, toxicity was rescued by the addition of the
unsaturated fatty acid oleate, supporting the proposal that
toxicity arises from the inhibition of SCD (entry 2).
The unsubstituted benzothiazole (R)-54 and related analogs
showed a selectivity pattern similar to that observed with the
methoxy-substituted SCD inhibitors (Table 4). For example,
amine (R)-54 was toxic to H2122 cells, whereas H1155 cells
were insensitive to this analog (entries 1, 4). Moreover, toxicity
to H2122 cells was rescued by supplemental oleate, confirming
that SCD was the functional target of (R)-54 (entry 2). These
observations raised the hypothesis that the unsubstituted
benzothiazoles could undergo oxidation to the same drug form
as was generated by the demethylation of (R)-27. Indeed,
monitoring of the H2122 cell culture in the presence of (R)-54
revealed the formation of (R)-53 as judged by HPLC retention
time and MS/MS signature (Figure 5). Provocatively, however,
Figure 3. Click probes.
ether 49 introduced an alkyne on the benzothiazole while 51
featured the propargyl ether on the benzophenone subunit.
Carbamate 50 and amide 52 include an alkyne on the amide
portion. Alkynes 49, 51, and 52 maintained potent activity
against H2122 cells, but only 51 and 52 retained high
selectivity. Carbamate 50 showed limited solubility. As
described previously, 51 was found to covalently modify
SCD in H2122 cells.3 Moreover, competition studies showed
that binding to SCD by 51 could be competed by active but
not by inactive analogs. Finally, we confirmed that 27 inhibited
SCD enzymatic activity with IC50 = 54 nM.3,21
Benzothiazoles are CYP4F11-Activated Prodrugs.
Among a panel of 12 NSCLC cell lines, amine 27 was
selectively toxic to 4 cell lines with IC50 = 22−120 nM. The
IC50 values against the other 8 NSCLC lines were >10 μM.22
The sensitivity of various NSCLC cell lines correlated with the
cell lines’ expression of CYP4F11; lines with robust expression
of CYP4F11 were sensitive to the benzothiazoles while cell
lines lacking expression of CYP4F11 were insensitive.3 This
observation raised the hypothesis that the benzothiazoles may
act as prodrugs. Indeed, LC/MS/MS analysis of the H2122
cell culture indicated that (R)-27 was metabolized by H2122
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Table 4. Activity of Prodrug and Drug Forms in Sensitive and Insensitive NSCLC Cell Lines
a
entry
cell line
H2122
H2122 + oleate
CYP4F11 status
(R)-27 IC50 (μM)
(R)-53 IC50 (μM)
(R)-54 IC50 (μM)
1
2
3
4
5
positive
positive
inhibited
negative
0.021
>50
0.18
2.1
0.008
>50
<0.010
0.009
0.020
0.012
>50
0.011
6.7
H2122 + HET0016
b
H1155-vector
b
H1155-CYP4F11
positive
0.027
1.2
a
b
CYP4F11 status of the cell line used in the experiment. Cells transduced with lentivirus containing empty vector or CYP4F11, as indicated.
Figure 6. Mouse tumor xenograft study. Tumors were formed from
H2122 cells, and treatment with (R)-27 (10 mg/kg BID) was
Figure 5. Conversion of prodrug (R)-54 into active form (R)-53 in
H2122 cells. Cells were incubated with 100 nM (R)-54 and culture
contents evaluated for (R)-54 and (R)-53 levels by LC−MS/MS.
initiated after they reached 100 mm3 size.
activity of (R)-27 is due to the inhibition of SCD and offers a
potential means to increase the effectiveness of SCD inhibitors.
No obvious differences in the behavior, appearance, or body
weight were noted among treatment groups (Figure S6).
Chemistry. Compounds were synthesized by acylating the
required amino-heterocycle with appropriate carboxylic acid in
the presence of TBTU or other coupling reagents. The
synthesis of (R)-27 is representative (Scheme 1). The addition
the CYP4F inhibitor HET0016 did not rescue toxicity from
(R)-54 (Table 4, entry 3) or block the formation of (R)-53
from (R)-54 (Figure S3), nor did the expression of CYP4F11
confer sensitivity to H1155 cells (Table 4, entry 5). Taken
together, these results indicate that both (R)-27 and (R)-54
are prodrugs that are converted to the same active drug form in
H2122 cells but not in H1155 cells. CYP4F11 is likely
responsible for the demethylation of (R)-27, whereas a
currently unidentified metabolic enzyme is capable of perform-
ing the arene oxidation to convert (R)-54 to (R)-53. By a way
of comparison, a representative noncovalent SCD inhibitor
that does not require CYP4F11 activiation (4) killed both
H2122 and H1155 cells with IC50 < 10 nM.
Scheme 1. Represenative Synthesis of N-acyl Benzothiazole
SCD Inhibitors
In Vivo Evaluation. Finally, we tested if our understanding
of the mechanism of action of (R)-27 could be exploited to
improve its in vivo activity. In a previous tumor xenograft study
with (rac)-27, we observed a reduction in the growth rate of a
tumor derived from H2122 cells, but not growth arrest.3 We
hypothesized that tumors could be scavenging unsaturated
fatty acids from dietary sources, which allowed continued
growth. To test this hypothesis, 100 mm3 tumors were formed
from H2122 cells in NOD-SCID mice, after which time mice
were treated with 20 mg/kg/d (R)-27 once daily (qd) or as a
divided dose (10 mg/kg BID).24 As shown in Figure 6, the
tumors in mice that were fed normal chow continued to grow,
albeit at a reduced rate relative to untreated controls. By
contrast, under a fat-free diet, where access to dietary
unsaturated fats is diminished, tumor growth largely arrested.
Untreated tumors grew at similar rates under both diets,
indicating that diet did not impact tumor growth. Analysis of
plasma and tumor samples taken 3 h after the final dose
indicated >5 μM concentrations of the drug form (R)-53 in
the tumors of mice from both diet groups (Figure S4).
Significantly lower levels of the drug form were observed in
plasma. This data provides further evidence that the in vivo
of phenyl Grignard to the sulfinimine 55 under conditions
developed by Ellman and co-workers provided the sulfinimide
56 as a single diastereomer, as judged by 1H NMR.25
Hydrolysis under basic conditions revealed the carboxylic
acid 57, which was then coupled with aminobenzothiaozle to
form amide 58. Final removal of the Ellman auxiliary generated
(R)-27 in optically pure form.
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50 × 4.6 mm) was used for chromatography for PK studies with the
following conditions: buffer A: dH20 + 0.1% formic acid, buffer B:
methanol +0.1% formic acid, 0−1.5 min 3% B, 1.5−2 min gradient to
100% B, 2−3.2 min 100% B, 3.2−3.5 min gradient to 3% B, 3.5−4.5
3% B. N-Benzylbenzamide (transition 212.1 to 91.1) or tolbutamide
(transition 271.2 to 91.2) both from Sigma (St. Louis, MO) were
used as internal standards (IS). Evaluation of compound stability with
cell lines employed nearly identical conditions except the gradient
utilized was 0−1.0 min 5% B, 1.0−1.5 min gradient to 100% B, 1.5−
3.5 min 100% B, 3.5−3.6 min gradient to 5% B, 3.6−4.5 5% B.
In Vitro Determination of Compound Stability with Human
Cancer Cell. H2122 and H1155 pLVX (empty vector) or H1155
CYP4F11 cells were plated at a density of 4000 cells per well in 96
well plates. After overnight adherence, media was removed and
replaced with fresh media containing 100 nM (R)-27, or 100 nM (R)-
54 +/− the CYP4A and 4F inhibitor, HET0016 (Sigma) at 1 μM.
The plates were incubated at 37 °C 5% CO2. At varying times post
compound addition, media and cells were removed and the cells were
broken open and the lysate precleared of protein by the addition of a
twofold volume of methanol containing 0.2% formic acid and 37.5
ng/mL of internal standard (N-benzylbenzamide) followed by
vigorous vortexing and centrifugation at 16,000g for 5 min. The
supernatant was analyzed by LC−MS/MS for levels of parent prodrug
(R)-27, or (R)-54 compound or the metabolite (R)-53. Compound
levels were quantitated in reference to standard curves prepared by
adding varying concentrations of (R)-27, (R)-53, or (R)-54 to
untreated blank H2122 and H1155 lysates and processing as
described above.
Pharmacokinetic Studies. Pharmacokinetic studies were per-
formed by injecting 6−7 week old CD-1 female mice (UTSW Mouse
Breeding Core Facility) with 27, at 10 mg/kg IP formulated in 10%
DMSO, 10% Kolliphor EL, 80% 50 mM lactic acid, pH 5.5; 16 at 10
mg/kg IP formulated in 10% DMSO, 10% Kolliphor EL, 5% PEG400,
75% of 0.2 M Na2CO3 buffer pH 10; and 24 formulated in 5%
DMSO, 5% Kolliphor EL, 90% of 0.2 M Na2CO3 buffer pH 10.
Animals were sacrificed in groups of three, blood was obtained by
cardiac puncture at each time point (0, 10, 30, 90, 180, 360, 960, and
1440 min post dose) using the anticoagulant ACD (acidified citrate
dextrose) and plasma isolated by centrifugation. Lung and posterior
subcutaneous fat were also collected and snap frozen in liquid
nitrogen after rinsing with PBS to remove surface adhering blood.27
Tissues were homogenized in a threefold volume (weight by volume)
of PBS to generate a homogenate. Then, 100 μL of plasma or tissue
homogenate was mixed with 200 μL of acetonitrile or methanol
containing 0.15% formic acid and 37.5 ng/mL N-benzylbenzamide IS.
The samples were vortexed 15 s, incubated at room temp for 10′, and
spun twice at 16,100g 4 °C in a refrigerated microcentrifuge. The
amount of compound present in plasma, lung, or fat was quantified by
LC/MS/MS to determine the tissue to plasma ratio of each drug.
Standard curves were generated using blank plasma (Bioreclamation,
Westbury, NY) or tissue homogenate spiked with known concen-
trations of test compound and processed as described above. The
concentrations of drug in each time-point sample were quantified
using Analyst software (Sciex). A value of threefold above the signal
obtained from blank plasma or tissue homogenate was designated the
limit of detection (LOD). The limit of quantitation (LOQ) was
defined as the lowest concentration at which back calculation yielded
a concentration within 20% of theoretical. Cmax and AUC values were
calculated using the noncompartmental analysis tool of Phoenix
WinNonLin (Certara/Pharsight, Sunnyvale, CA).
CONCLUSIONS
■
The N-acyl benzothiazole series described here shows selective
and potent cytotoxicity against cancer cell lines expressing
certain cytochrome P450 isoforms. Substantial variation on the
acyl portion of the inhibitors allowed the identification of (R)-
27, which balanced potency, solubility, and lipophilicity
sufficiently to allow proof-of-concept studies in mice. Addi-
tionally, we confirmed the structure of the active metabolite
formed from the CYP4F11-mediated demethylation of
methoxy benzothiazoles (R)-53. The same metabolite is
formed via the metabolic hydroxylation of unsubstituted
benzothiazoles, although the enzyme responsible for this
transformation is not yet known. These prodrugs are expected
to be activated inside the tumor, where they covalently inhibit
SCD and arrest tumor growth. The nature of covalent
inhibition is currently unclear, although we speculate that the
enzyme-mediated oxidation of the phenol might generate a
reactive radical. Similar chemistry underpins the APEX labeling
system developed by the Ting laboratory.26 In that system, a
simple phenol labels many proteins in a distance-dependent
manner, whereas in our SCD inhibitors, covalent adduct
formation is largely limited to SCD. Metabolic activation of the
SCD inhibitors within the liver is also likely, but SCD is not
essential in the liver, and the liver-specific SCD-knockout does
not have an obvious phenotype.19 Together, these observations
offer the promise that a tumor-activated prodrug strategy
might exploit the essentiality of SCD for tumor growth while
avoiding toxicity associated with systemic SCD inhibition.
EXPERIMENTAL SECTION
■
General. All compounds were >95% pure according to HPLC
(220, 254 nM) and 1H NMR prior to biological testing. Animal work
described in this article has been approved and conducted under the
oversight of the UT Southwestern Institutional Animal Care and Use
Committee. UT Southwestern uses the “Guide for the Care and Use
of Laboratory Animals” when establishing animal research standards.
Cell Culture and Toxicity Testing. All NSCLC cell lines were
obtained from the Minna and Gazdar laboratories at the UT
Southwestern Medical Center. They were screened for mycoplasma
by PCR and authenticated by short tandem repeat (STR) analysis
through the McDermott Core at UT Southwestern. All NSCLC cell
lines were cultured in RPMI 1640 (Sigma) supplemented with 5%
FBS (Sigma) and 2 mM L-glutamine (Sigma). For dose-response
experiments, the H2122 and H2009 cell lines were plated in 384-well
plates (Bio-one; Greiner, Inc.) at 900 cells/well and 600 cells/well in
60 μL in the above medium and were allowed to adhere overnight at
37 °C in 5% CO2. On the next day, compounds were added at twelve
half-log doses ranging from 50 μM to 50 pM (3 replicates per dose
per cell line). In all experiments, the final DMSO concentration was
maintained at 0.5%. After an incubation of 96 h under growth these
growth conditions, Cell Titer Glo reagent (Promega, Inc.) was added
to each well (10 μL of a 1:2 dilution in the above medium) and
mixed. Plates were incubated for 10 min at room temperature and
luminescence was determined for each well using an EnVision
multilabel plate reader (Perkin-Elmer, Inc.). Curve fitting for IC50’s
was performed using Analyzer module of Genedata Screener (10.1)
software suite, as previously described.
Xenograft Experiments. Female Nod-Scid mice (6−8 weeks of
age, UTSW Mouse Breeding Core Facility) were placed on one of
two specialized diets from Envigo (Madison, WI): Control containing
12.4% protein, 68.4% carbohydrate, and 4.1% fat from soybean oil
(Irradiated, Catalog# 93M) or fat-free containing 12.4% protein, 72%
carbohydrate, and 0.1% fat (Irradiated, Catalog# AIN-93M w/o
SBO). The additional carbohydrates in the latter diet, which lacked
the added soybean oil, were provided by an increase in the amount of
corn starch and maltodextrin in the formulation. Three days later, the
mice were injected with 5 × 106 H2122 cells by subcutaneous
Analytical LC−MS/MS Conditions. Compound levels for
cellular stability and in vivo pharmacokinetics (PK) were monitored
by LC/MS/MS using an AB Sciex (Framingham, MA) 4000 QTRAP
mass spectrometer coupled to a Shimadzu (Columbia, MD)
Prominence LC. Analytes were detected with the mass spectrometer
in the positive MRM (multiple reaction monitoring) mode by
following the precursor to fragment ion transitions indicated here: 27:
390.1 to 210.1; 53: 376.2 to 210.0; 54: 360.4 to 210.2; 16: 389.1 to
209.1; and 24: 392.1 to 194.1. An Agilent C18 XDB column (5 μm,
F
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injection in a single site on their left flank. Animals were weighed daily
and tumors measured with calipers twice weekly. Tumor volume was
calculated according to the formula: volume = (length × width2 ×
Pi)/6. When tumors reached approximately 100 mm3, the animals
were randomized into six groups of six mice each. Three groups
continued to receive the Control Diet and the other two groups
continued receiving the Fat-Free Diet. Therapy was initiated with
(R)-27, given BID at 10 mg/kg to one cohort of each diet type, QD at
20 mg/kg to another cohort of each diet type, or BID vehicle (10%
DMSO, 10% Kolliphor EL, 80% 50 mM lactic acid pH 3.6) to the
third cohort of each diet. Mice were treated and monitored for a total
three weeks. At the end of treatment, mice were euthanized 3 h after
the final dose and plasma and tumors collected and evaluated for
compound levels as described for pharmacokinetics. Weight loss was
17% for the mice on the Fat-Free Diet receiving (R)-27 BID, 7% for
mice receiving (R)-27 QD, 13% for the mice on the Control Diet
receiving (R)-27 BID, 5% for mice receiving (R)-27 QD, and less
than 5% for mice on both diets receiving vehicle (Figure S6).
1H), 7.21 (d, J = 8.9 Hz, 1H), 6.87 (dd, J = 8.9, 3.4 Hz, 3H), 3.86 (s,
3H), 3.82 (s, 3H). 13C NMR (400 MHz, CDCl3): δ 165.3, 163.5,
158.0, 156.9, 142.2, 133.3, 130.1, 124.3, 121.4, 115.2, 114.4, 104.2,
56.0, 55.7. ESI-MS (m/z): 315.0 [M + H]+.
3-Methoxy-N-(6-methoxybenzo[d]thiazol-2-yl)benzamide (11)
was prepared according to general procedure A, but the addition of
the reaction mixture to stirring water precipitated an oil instead. The
aqueous layer was extracted thrice with dichloromethane. The
combined organic extracts were dried over sodium sulfate,
concentrated under reduced pressure, and the residue was purified
by silica gel flash chromatography (0−5% MeOH/DCM) to afford
1
the desired product as a white solid (37 mg, 21%). H NMR (400
MHz, CDCl3): δ 11.64 (s, 1H), 7.56−7.50 (m, 2H), 7.34−7.27 (m,
2H), 7.15 (d, J = 8.9 Hz, 1H), 7.09 (ddd, J = 8.3, 2.5, 0.9 Hz, 1H),
6.87 (dd, J = 8.9, 2.5 Hz, 1H), 3.87 (s, 3H), 3.75 (s, 3H). 13C NMR
(400 MHz, CDCl3): δ 165.8, 160.1, 157.7, 157.0, 142.1, 133.5, 133.3,
130.2, 121.5, 120.0, 119.8, 115.4, 112.7, 104.1, 55.9, 55.5. ESI-MS
(m/z): 315.0 [M + H]+.
Chemical Methods. General Considerations. All reactions were
performed with commercially obtained anhydrous solvents. All
reactions and final products were monitored by liquid chromatog-
raphy mass spectrometry (LC/MS) with an Agilent Technologies
1200 series LC/MS using electrospray ionization methods. Unless
otherwise stated flash chromatography was performed with a
Teledyne CombiFlash automated chromatography system. 1H and
13C NMR spectra were recorded on a Varian Inova 400 MHz, Agilent
400 MHz, Bruker 400M Hz, or Agilent 500 MHz spectrometer.
Chemical shifts are reported relative to internal chloroform (CDCl3:
1H, δ = 7.26 ppm, 13C, δ = 77.36 ppm). Where spectra were recorded
in mixtures of deuterated solvents, the more deuterated solvent was
referenced. Coupling constants are in Hz and are reported as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and p
(pentet). Compounds 1, 31, and 43 were commercially available from
ChemDiv. Compounds 16 and 27 were previously reported.3
General Procedure A. Coupling of Heterocyclic Amines and
Carboxylic or Sulfonic Acid Chlorides. To a reaction vial equipped
with a stir bar and Teflon-lined lid were added heterocyclic amine
(0.33 mmol), pyridine (0.7 mL), and the acid chloride (0.5 mmol, 1.5
equiv). The reaction was heated to 60 °C for 16 h. After cooling to
room temperature, the mixture was diluted with water, whereupon a
precipitate formed. The precipitate was filtered, washed with water,
and dried via aspiration and then vacuum. The products thus obtained
were usually >95% by LC/MS but could be recrystallized from
absolute ethanol or purified by flash chromatography if necessary.
General Procedure B. Coupling of Acids and of Heterocyclic
Amines with HATU. To a reaction vial equipped with a stir bar and
Teflon-lined lid was added heterocyclic amine (0.5 mmol, 1 equiv),
carboxylic acid (0.55 mmol, 1.1 equiv), 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro-
phosphate (HATU, 0.55 mmol, 1.1 equiv), N,N-diisopropylethyl-
amine (1.0 mmol, 2 equiv), and N,N-dimethylformamide ([1.4 M] in
limiting reactant). The vial was flushed with nitrogen, sealed, and
stirred at room temperature for 2−16 h. The mixture was diluted with
water and the product isolated by filtration or extraction with an
organic solvent.
4-Fluoro-N-(6-methoxybenzo[d]thiazol-2-yl)benzamide (12) was
prepared according to general procedure A. The product was obtained
as a white solid (75 mg, 44%). H NMR (400 MHz, DMSO-d6): δ
8.23−8.18 (m, 2H), 7.68 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 2.6 Hz,
1H), 7.44−7.37 (m, 2H), 7.06 (dd, J = 8.8, 2.6 Hz, 1H), 3.82 (s, 3H).
13C NMR (101 MHz, DMSO-d6): δ 165.0 (JC−F = 252.5 MHz),
156.49, 131.44, 131.34, 116.03, 115.81, 115.31, 104.89, 55.86. ESI-
MS (m/z): 303.0 [M + H]+.
1
N-(6-Methoxybenzo[d]thiazol-2-yl)-4-nitrobenzamide (13) was
prepared according to general procedure A but stirred at 110 °C
overnight, then cooled and diluted with water and filtered. The orange
solid was washed with water many times, then hexanes to give 785 mg
1
of title compound in 71% yield. H NMR (400 MHz, DMSO-d6): δ
13.16 (s, 1H), 8.43−8.29 (m, 4H), 7.69 (d, J = 8.8 Hz, 1H), 7.63 (d, J
= 2.5 Hz, 1H), 7.08 (dd, J = 8.8, 2.6 Hz, 1H), 3.83 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 156.4, 149.7, 129.9, 123.7, 115.3, 104.8,
55.7. ESI-MS (m/z): 330.0 [M + H]+.
4-(Benzyloxy)-N-(6-methoxybenzo[d]thiazol-2-yl)benzamide (14)
was prepared according to general procedure A but was stirred at 110
°C overnight, then cooled and diluted with water and filtered. The
solid was purified by ISCO flash chromatography in 0−5% MeOH/
1
DCM to give desired in 23 mg of title compound in 13% yield. H
NMR (400 MHz, Chloroform-d): δ 11.56 (s, 1H), 8.01−7.92 (m,
2H), 7.47−7.34 (m, 5H), 7.32 (t, J = 1.9 Hz, 1H), 7.23 (d, J = 8.9 Hz,
1H), 7.00−6.93 (m, 2H), 6.89 (dt, J = 9.0, 1.9 Hz, 1H), 5.08 (s, 2H),
3.87 (d, J = 1.5 Hz, 3H). 13C NMR (126 MHz, DMSO): δ 165.2,
162.7, 158.1, 156.9, 142.17, 136.1, 130.1, 128.8, 128.4, 127.6, 124.5,
121.4, 115.3, 115.2, 104.2, 70.3, 55.9. ESI-MS (m/z): 391.0 [M +
H]+.
N-(6-Methoxybenzo[d]thiazol-2-yl)-4-phenylamino)benzamide
(15). 4-Iodo-N-(6-methoxybenzo[d]thiazol-2-yl)benzamide (50 mg,
0.12 mmol, prepared according to general procedure A), BrettPhos (1
mg, 2 mol %), BrettPhos precatalyst (1 mg, 1 mol %), sodium tert-
butoxide (16 mgs, 0.17 mmol), and aniline (15 μL, 0.17 mmol) were
charged into a vial, and the vial was flushed with nitrogen. To this vial
was added toluene (0.2 mL), and the vial was sealed and heated to 85
°C for 72 h. The mixture was cooled to room temperature, diluted
with water, and extracted with ethyl acetate. The combined organic
layers were concentrated under vacuum and the residue was purified
by silica gel chromatography (20%−50% ethyl acetate/hexanes) to
N-(6-Methoxybenzo[d]thiazol-2-yl) benzamide (9) was prepared
according to general procedure A. The solids were collected by
filtration, washed with water, and purified by silica gel flash
chromatography (0−5% MeOH in DCM) to afford the desired
product as a white solid (97 mg, 60%). 1H NMR (400 MHz, CDCl3):
δ 11.58 (s, 1H), 7.99 (d, J = 7.6 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1H),
7.42 (t, J = 7.8 Hz, 2H), 7.31 (d, J = 2.5 Hz, 1H), 7.26 (d, J = 0.7 Hz,
0H), 7.11 (d, J = 8.9 Hz, 1H), 6.85 (dd, J = 8.9, 2.5 Hz, 1H), 3.87 (s,
3H). 13C NMR (400 MHz, CDCl3): δ 165.9, 157.7, 157.0, 142.2,
133.3, 133.1, 132.2, 129.2, 128.0, 121.5, 115.3, 104.1, 56.0. ESI-MS
(m/z): 285.0 [M + H]+.
1
give the title compound (10 mg, 21%) as a solid: H NMR (400
MHz, Chloroform-d): δ 7.87 (d, J = 8.7 Hz, 2H), 7.38−7.30 (m, 5H),
7.13 (d, J = 7.8 Hz, 2H), 7.08 (t, J = 7.4 Hz, 1H), 6.99−6.91 (m, 4H),
6.09 (s, 1H), 3.86 (s, 3H).
4-Azido-N-(6-methoxybenzo[d]thiazol-2-yl)benzamide (17) was
prepared according to general procedure B to give 98.7 mg of desired
1
product in 54% yield. H NMR (400 MHz, DMSO-d6): δ 12.75 (s,
1H), 8.16 (d, J = 8.3 Hz, 2H), 7.73−7.47 (m, 2H), 7.25 (d, J = 8.2
Hz, 2H), 7.05 (d, J = 9.1 Hz, 1H), 3.81 (s, 3H). 13C NMR (126 MHz,
DMSO): δ 156.3, 144.0, 130.3, 119.3, 115.1, 104.7, 55.7. ESI-MS (m/
z): 391.0 [M + H]+.
4-Methoxy-N-(6-methoxybenzo[d]thiazol-2-yl)benzamide (10)
was prepared according to general procedure A. The product was
obtained as a white solid (92 mg, 52%). 1H NMR (400 MHz,
CDCl3): δ 11.47 (s, 1H), 7.95 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 2.5 Hz,
G
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Article
N-(6-Methoxybenzo[d]thiazol-2-yl)-3-methylbenzamide (18) was
prepared according to general procedure A. The orange solid isolated
was washed with water several times, then hexanes before being
recrystallized from ethanol to give 89.1 mg of title compound in 54%
chromatography (1−5% MeOH/DCM) to afford the product as a
1
white solid (97 mg, 80%). H NMR (400 MHz, DMSO-d6): δ 12.86
(br s, 1H), 8.16 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.62 (d,
J = 2.6 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.07 (dd, J = 8.8, 2.6 Hz,
1H), 3.83 (s, 3H), 3.60 (br s, 2H), 3.24 (br s, 2H), 1.67−1.52 (m,
4H), 1.46 (br s, 2H). 13C NMR (101 MHz, DMSO-d6): δ 167.9,
156.3, 140.5, 132.6, 128.5, 126.7, 121.0, 115.1, 104.7, 55.7, 47.9, 42.3,
25.9, 25.2, 24.0. ESI-MS (m/z): 396.1 [M + H]+.
1
yield. H NMR (400 MHz, DMSO-d6): δ 12.68 (s, 1H), 8.04−7.80
(m, 2H), 7.78−7.54 (m, 2H), 7.45 (d, J = 8.5 Hz, 2H), 7.06 (d, J =
8.8 Hz, 1H), 3.83 (s, 3H), 2.40 (s, 3H). 13C NMR (101 MHz,
DMSO-d6): δ 165.8, 156.2, 138.0, 133.3, 132.9, 132.0, 128.8, 128.5,
125.4, 121.0, 115.0, 104.7, 55.7, 20.9. ESI-MS (m/z): 299.0 [M + 1]+.
N-(6-Methoxybenzo[d]thiazol-2-yl)nicotinamide hydrochloride
(19·HCl). 6-Methoxybenzo[d]thiazol-2-amine (100 mg, 0.56 mmol)
and nicotinoyl chloride hydrochloride (55 mg, 0.62 mmol, 1.1 equiv)
were fused under heating with a heat gun until gas evolution ceased.
After cooling to room temperature the resulting solid was suspended
in ethanol, treated with conc. Hydrochloric acid (2 drops) and filtered
N1-Benzyl-N4-(6-methoxybenzo[d]thiazol-2-yl)terephthalamide
(23) was prepared analogously to compound 20. A mixture of 4-((6-
methoxybenzo[d]thiazol-2-yl)carbamoyl)benzoic acid (100 mg, 0.30
mmol), benzylamine (33 μL, 0.30 mmol, 1 equiv), HATU (141 mg,
0.37 mmol, 1.2 equiv), and DIPEA (80 μL, 0.46 mmol, 1.5 equiv) in
anhydrous DMF (3 mL) was stirred overnight at room temperature.
The reaction mixture was then poured into stirring water to
precipitate solids, which were collected by filtration, washed with
water. The solids were triturated with 10% NaOH and then washed
with 5% MeOH/DCM to give 73.7 mg of desired product in 58%
1
to give the title compound as a solid (143 mg, 79%): H NMR (400
MHz, DMSO-d6): δ 9.32 (dd, J = 2.2, 0.8 Hz, 1H), 8.91 (dd, J = 5.1,
1.6 Hz, 1H), 8.67 (d, J = 8.0 Hz, 1H), 7.81 (dd, J = 8.1, 5.1 Hz, 1H),
7.69 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 2.6 Hz, 1H), 7.08 (dd, J = 8.8,
2.6 Hz, 1H), 3.83 (s, 3H). LCMS (ESI) m/z 285.9 [M + 1]+.
N-(6-Methoxybenzo[d]thiazol-2-yl)-4-(4-methylpiperazine-1-
carbonyl)benzamide (20) Step 1. Methyl 4-((6-Methoxybenzo[d]-
thiazol-2-yl)carbamoyl)benzoate was prepared according to general
procedure B to provide methyl 4-((6-methoxybenzo[d]thiazol-2-
yl)carbamoyl)benzoate as a light yellow solid (168 mg, 88%), which
was used without additional purification. 1H NMR (400 MHz,
DMSO-d6): δ 13.00 (br s, 1H), 8.23 (d, J = 8.3 Hz, 2H), 8.10 (d, J =
8.2 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 2.2 Hz, 1H), 7.07
(dd, J = 8.8, 2.5 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H). 13C NMR (101
MHz, DMSO-d6): δ 166.0, 156.8, 136.6, 133.3, 129.7, 129.1, 115.6,
105.1, 56.1, 53.0. ESI-MS (m/z): 343.0 [M + H]+.
1
yield. H NMR (400 MHz, DMSO-d6): δ 12.92 (s, 1H), 9.26 (t, J =
5.9 Hz, 1H), 8.21 (d, J = 8.1 Hz, 2H), 8.05 (d, J = 8.0 Hz, 2H), 7.69
(d, J = 8.7 Hz, 1H), 7.62 (d, J = 2.5 Hz, 1H), 7.34 (d, J = 4.3 Hz, 4H),
7.25 (p, J = 4.5 Hz, 1H), 7.07 (dd, J = 8.7, 2.6 Hz, 1H), 4.51 (d, J =
6.0 Hz, 2H), 3.82 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 165.6,
156.5, 139.6, 138.0, 134.6, 133.0, 128.6, 128.6, 127.7, 127.5, 127.1,
121.2, 115.4, 104.9, 55.9, 43.0. ESI-MS (m/z): 418.0 [M + H]+.
4-(Hydroxyl(pyridin-2-yl)methyl)-N-(6-methoxybenzo[d]thiazol-
2-yl)benzamide (24). Step 1. A solution of 4-bromobenzoic acid
(1.20 g, 6.0 mmol), in anhydrous THF (12 mL) in an oven dried flask
was cooled in a dry ice/acetonitrile bath (−40 to −30 °C) before di-
n-butyl magnesium solution (1.0 M in heptane, 3.12 mL, 3.12 mmol,
0.52 equiv) was added in a slow dropwise manner, followed by the
dropwise addition of n-BuLi (1.6 M in hexanes, 4.0 mL, 6.4 mmol,
1.07 equiv). The reaction was stirred cold for an hour before the slow
addition of a solution of nicotinaldehyde (1.12 mL, 12.0 mmol, 2
equiv) in 3.7 mL of anhydrous THF. The reaction was stirred cold for
an hour and then warmed to ambient temperature. The reaction was
quenched after 2 h by the addition of 10% citric acid. The mixture was
extracted thrice with EtOAc. The combined organic layers were
washed with brine and condensed. The mixture was diluted with
DCM and washed with 1N HCl. The aqueous phase was basified with
10 N NaOH and extracted with DCM. The aqueous phase was
adjusted to pH 4−5 with 10% citric acid and HCl and extracted with
EtOAc thrice. The sticky yellow crude solid was washed with DCM to
give 4-(hydroxyl (pyridin-2-yl) methyl) benzoic acid as a white solid
(921 mg, 67% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.45 (d, J =
4.9 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.78 (t, J = 7.6 Hz, 1H), 7.54
(dd, J = 18.3, 8.0 Hz, 3H), 7.23 (dd, J = 7.5, 4.8 Hz, 1H), 6.28−6.19
(m, 1H), 5.77 (s, 1H), 5.74 (s, 1H). 13C NMR (101 MHz, DMSO-
d6): δ 167.4, 163.6, 149.4, 148.7, 137.1, 129.6, 129.3, 126.7, 122.5,
120.3, 75.5. ESI-MS (m/z): 230.0 [M + H]+.
Step 2. Compound 24 was prepared according to general
procedure B using the acid prepared in Step 1. At the end of the
reaction, water was added to the reaction to triturate the desired
product. The solid was washed with water and dried. The solid was
purified by ISCO flash chromatography in 0−10% MeOH/DCM and
then 0−100% EtOAc/hexanes to give 23.7 mg of light-yellow solid in
20% yield. 1H NMR (400 MHz, 1:1 CDCl3/CD3OD): δ 8.44 (s, 1H),
7.98 (d, J = 8.0 Hz, 2H), 7.76 (d, J = 7.9 Hz, 1H), 7.58 (d, J = 10.7
Hz, 5H), 7.24 (d, J = 6.4 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H), 5.88 (s,
1H), 3.84 (s, 3H). 13C NMR (101 MHz, 1:1 CDCl3/CD3OD): δ
167.0, 162.8, 158.1, 157.6, 149.0, 148.8, 143.1, 138.4, 133.8, 132.0,
128.8, 127.6, 123.5, 121.8, 121.7, 115.9, 104.7, 76.2, 56.2. ESI-MS
(m/z): 392.0 [M + H]+.
Step 2. To a mixture of the ester from Step 1 (855 mg, 2.5 mmol)
in THF/MeOH (1:1, 21.4 mL) was added a solution of LiOH (300
mg, 12.5 mmol, 5 equiv) in water (3.6 mL). The reaction mixture was
stirred at room temperature overnight. The reaction mixture was then
poured onto 1 N HCl to precipitate solids, which were collected by
filtration, washed with water, and dried under high vacuum to provide
4-((6-methoxybenzo[d]thiazol-2-yl)carbamoyl)benzoic acid as a
yellow solid (696 mg, 84%), which was used without further
1
purification. H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J = 8.3 Hz,
2H), 8.05 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.8 Hz, 1H), 7.61 (d, J =
2.6 Hz, 1H), 7.06 (dd, J = 8.8, 2.6 Hz, 1H), 3.83 (s, 3H). ESI-MS (m/
z): 329.0 [M + H]+, 327.0 [M − H]−.
Step 3. A mixture of 4-((6-methoxybenzo[d]thiazol-2-yl)-
carbamoyl)benzoic acid from Step 2 (100 mg, 0.30 mmol), 1-
methylpiperizine (34 μL, 0.30 mmol, 1 equiv), HATU (141 mg, 0.37
mmol, 1.2 equiv), and DIPEA (80 μL, 0.456 mmol) in anhydrous
DMF (3 mL) were stirred overnight at room temperature. The
reaction mixture was then poured into stirring water to precipitate
solids, which were collected by filtration, washed with water, and
dried under high vacuum to afford amide 20 as an off-white solid (88
mg, 70%). 1H NMR (400 MHz, DMSO-d6): δ 12.87 (br s, 1H), 8.19
(d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 2.5 Hz, 1H),
7.59 (d, J = 8.2 Hz, 2H), 7.07 (dd, J = 8.8, 2.5 Hz, 1H), 3.83 (s, 3H),
3.72 (br s, 2H), 3.41 (br s, 2H), 2.71 (br s, 4H), 2.46 (s, 3H). 13C
NMR (101 MHz, DMSO-d6): δ 168.1, 165.2, 156.8, 156.3, 142.3,
139.4, 132.9, 132.8, 128.5, 127.2, 121.0, 115.2, 109.6, 104.7, 55.7,
53.5, 44.3. ESI-MS (m/z): 411.0 [M + H]+.
N-(6-Methoxybenzo[d]thiazol-2-yl)-4-(morpholine-4-carbonyl)-
benzamide (21) was prepared analogously to compound 20. The title
compound was obtained as an off-white solid (99 mg, 82%). 1H NMR
(400 MHz, DMSO-d6): δ 12.89 (br s, 1H), 8.18 (d, J = 7.5 Hz, 2H),
7.69 (d, J = 8.6 Hz, 1H), 7.66−7.51 (m, 3H), 7.07 (d, J = 8.0 Hz,
1H), 3.83 (s, 3H), 3.78−3.45 (m, 6H), 3.34−3.25 (m, 2H). 13C NMR
(101 MHz, DMSO-d6): δ 168.1, 156.3, 139.6, 132.9, 132.8, 128.5,
127.2, 121.0, 115.2, 104.7, 66.0, 55.7. ESI-MS (m/z): 398.0 [M +
H]+.
N-(6-Methoxybenzo[d]thiazol-2-yl)-4-picolinoylbenzamide (25).
Manganese(IV) oxide (101.3 mg, 1.2 mmol, 20 equiv) was added
to a solution of alcohol 24 (23.7 mg, 0.061 mmol) in anhydrous THF
(0.65 mL). The reaction was heated at 70 °C for 3 h, when the
reaction was determined by LCMS to have gone to completion. The
reaction was cooled and filtered through a plug of Celite 545. The
plug and reaction vial were washed with 5% MeOH/DCM to give
N-(6-Methoxybenzo[d]thiazol-2-yl)-4-(piperidine-1-carbonyl)-
benzamide (22) was prepared analogously to compound 20, but the
solid collected by filtration was purified by automated flash
H
https://dx.doi.org/10.1021/acs.jmedchem.0c00899
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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1
20.4 mg of pure yellow solid in 87% yield. H NMR (400 MHz,
DMSO-d6): δ 8.77−8.71 (m, 1H), 8.27−8.19 (m, 2H), 8.15−8.03
(m, 4H), 7.77−7.64 (m, 2H), 7.61 (t, J = 3.4 Hz, 1H), 7.10−7.01 (m,
1H), 3.82 (d, J = 3.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ
193.2, 156.5, 154.1, 149.0, 139.5, 138.1, 130.8, 128.2, 127.4, 124.5,
115.3, 105.0, 55.9. ESI-MS (m/z): 390.0 [M + H]+.
((R,SS)-1,1-dimethylethylsulfinamido)(phenyl)methyl)-N-(6-
methoxybenzo[d]thiazol-2-yl)benzamide (78 mg, 88%) as a solid: 1H
NMR (400 MHz, Chloroform-d): δ 12.32 (s, 1H), 8.03 (d, J = 8.1
Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.31−7.17 (m, 6H), 6.90 (d, J =
8.9 Hz, 1H), 6.69 (dd, J = 8.9, 2.5 Hz, 1H), 5.67 (d, J = 2.6 Hz, 1H),
4.25 (d, J = 2.6 Hz, 1H), 3.83 (s, 3H), 1.24 (s, 9H). LCMS (ESI) m/z
491.8 [M − 1]−.
N-(6-Methoxybenzo[d]thiazol-2-yl)-4-nicotinoylbenzamide (26).
Step 1. 4-(hydroxy(pyridin-3-yl)methyl)benzoic acid was prepared
analogously to the acid used in the synthesis of 24. The product was
isolated as an off-white solid after washing with 5% MeOH/DCM
Step 4. The amide of Step 3 (78 mg, 0.16 mmol) was dissolved in
methanol (0.4 mL) and treated with hydrogen chloride (4.0 M in 1,4-
dioxane, 0.1 mL, 2.5 equiv) in the manner described by Ellman et al.28
3
1
1
(410 mg, 30%). H NMR (400 MHz, DMSO-d6): δ 12.94 (s, 1H),
to give (R)-27 (48 mg, 71%) as a white solid: H NMR (500 MHz,
DMSO-d6): δ 9.35 (d, J = 5.7 Hz, 3H), 8.16 (d, J = 8.3 Hz, 2H), 7.75
(d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.66−7.57 (m, 3H),
7.45 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.4 Hz, 1H), 7.08 (dd, J = 8.8, 2.6
Hz, 1H), 5.78 (d, J = 5.8 Hz, 1H), 3.83 (s, 3H). 13C NMR (101 MHz,
DMSO-d6): δ 165.2, 156.3, 142.9, 137.93, 132.82, 131.8, 128.9, 128.7,
128.5, 127.6, 127.5, 121.1, 115.2, 104.7, 56.8, 55.7. LCMS (ESI) m/z
389.9 [M + 1]+. (S)-27 was prepared analogously to (R)-27 using
(R)-methyl 4-(((tert-butylsulfinyl)imino)methyl)benzoate.
8.62 (d, J = 2.2 Hz, 1H), 8.44 (dd, J = 4.8, 1.7 Hz, 1H), 7.94−7.86
(m, 3H), 7.74 (dt, J = 7.9, 2.0 Hz, 1H), 7.57−7.50 (m, 3H), 7.34
(ddd, J = 7.9, 4.8, 0.9 Hz, 1H), 6.24 (s, 1H). 13C NMR (101 MHz,
DMSO-d6): δ 167.1, 149.7, 148.3, 147.9, 140.3, 133.9, 129.5, 129.4,
126.2, 123.4, 71.8. ESI-MS (m/z): 230.0 [M + H]+.
Step 2. 4-(hydroxyl(pyridin-3-yl)methyl)-N-(6-methoxybenzo[d]-
thiazol-2-yl) benzamide was prepared analogously to the 2-pyridyl
isomer toward compound 24. The solid was purified by ISCO flash
chromatography in 0−15% MeOH/DCM to give 33.2 mg of yellow
solid in 40% yield. 1H NMR (400 MHz, 1:1 CDCl3/CD3OD): δ 8.55
(s, 1H), 8.40 (d, J = 4.9 Hz, 1H), 8.01 (d, J = 8.0 Hz, 2H), 7.85−7.72
(m, 1H), 7.67−7.47 (m, 3H), 7.38−7.24 (m, 2H), 6.99 (dd, J = 8.9,
2.6 Hz, 1H), 5.90 (s, 1H), 3.84 (s, 3H). 13C NMR (126 MHz, 1:1
CDCl3/CD3OD): δ 167.2, 158.4, 157.9, 149.6, 148.9, 148.4, 143.3,
141.1, 136.2, 134.1, 132.4, 129.2, 127.7, 124.9, 122.0, 116.2, 104.9,
73.8, 56.4. ESI-MS (m/z): 392.0 [M + H]+.
N-(6-Methoxybenzo[d]thiazol-2-yl)-2-phenylacetamide (28) was
prepared according to general procedure A, but the residue obtained
from filtration was purified by silica gel flash chromatography and
then triturated with 1:1 DCM/Hexane to provide the product as a
1
white solid (31 mg, 18%). H NMR (400 MHz, CDCl3): δ 9.60 (s,
1H), 7.60 (d, J = 8.9 Hz, 1H), 7.40−7.31 (m, 3H), 7.31−7.23 (m,
3H), 7.02 (dd, J = 8.9, 2.6 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 2H). 13C
NMR (400 MHz, CDCl3): δ 169.3, 157.0, 156.3, 142.4, 133.5, 132.8,
129.6, 129.5, 128.2, 121.5, 115.5, 104.4, 56.0, 43.6. ESI-MS (m/z):
299.0 [M + H]+.
N-(6-Methoxybenzo[d]thiazol-2-yl)-3-phenylpropanamide (29)
was prepared according to general procedure A. The product was
obtained as a white solid (37 mg, 21%). 1H NMR (400 MHz,
CDCl3): δ 11.29 (s, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.31 (d, J = 2.5 Hz,
1H), 7.24−7.12 (m, 3H), 7.03 (d, J = 7.1 Hz, 2H), 6.98 (dd, J = 8.9,
2.5 Hz, 1H), 3.88 (s, 3H), 3.01 (t, J = 7.6 Hz, 2H), 2.73 (t, J = 7.6 Hz,
2H).13C NMR (400 MHz, CDCl3): δ 170.9, 157.7, 157.0, 141.9,
139.9, 133.2, 128.7, 128.3, 126.6, 121.1, 115.5, 104.5, 56.0, 38.2, 31.1.
ESI-MS (m/z): 313.0 [M + H]+.
Step 3. Compound 26 was prepared according to general
procedure B. After 5 h, monitoring by LC/MS revealed the presence
of desired product and aminobenzothiazole as well as complete
consumption of acid. Additional i-Pr2NEt (50 μL, 0.29 mmol, 0.6
equiv), 4-(hydroxy(pyridin-2-yl) methyl) benzoic acid (17.9 mg,
0.078 mmol, 0.15 equiv) and HATU (52.1 mg, 0.14 mmol, 0.28
equiv) was added to the reaction and stirred overnight. Water was
added to the reaction to triturate the desired product. The solid was
washed with water and dried. The solid was purified by ISCO flash
chromatography in 0−10% MeOH/DCM and then 0−100% EtOAc/
1
hexanes to give 23.7 mg of light-yellow solid in 20% yield. H NMR
(400 MHz, 1:1 CDCl3/CD3OD): δ 8.44 (s, 1H), 7.98 (d, J = 8.0 Hz,
2H), 7.76 (d, J = 7.9 Hz, 1H), 7.58 (d, J = 10.7 Hz, 5H), 7.24 (d, J =
6.4 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H), 5.88 (s, 1H), 3.84 (s, 3H). 13C
NMR (101 MHz, 1:1 CDCl3/CD3OD): δ 167.0, 162.8, 158.1, 157.6,
149.0, 148.8, 143.1, 138.4, 133.8, 132.0, 128.8, 127.6, 123.5, 121.8,
121.7, 115.9, 104.7, 76.2, 56.2. ESI-MS (m/z): 392.0 [M + H]+.
(R)-4-(Amino(phenyl)methyl)-N-(6-methoxybenzo[d]thiazol-2-
yl)benzamide hydrochloride (27) Step 1. (S)-Methyl 4-(((tert-
Butylsulfinyl)imino)methyl)benzoate (200 mg, 0.79 mmol) was
treated with phenylmagnesium bromide (1.7 M in ethyl ether, 0.51
mL, 1.1 equiv) in the manner described by Ellman et al.28 to give
methyl 4-((R,SS)-1,1-dimethylethylsulfinamido) (phenyl)methyl)-
benzoate (230 mg, 88%) as a solid after silica gel chromatography
(10% ethyl ether/methylene chloride): 1H NMR (400 MHz,
Chloroform-d): δ 8.01 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.3 Hz,
2H), 7.40−7.26 (m, 5H), 5.70 (d, J = 2.2 Hz, 1H), 3.90 (s, 3H), 1.27
(s, 9H).
Step 2. The ester from Step 1 (230 mg, 0.67 mmol) was treated
with lithium hydroxide (80 mg, 3.3 mmol, 5 equiv) in the manner
described by Ellman et al.28 to give 4-((R,SS)-1,1-
dimethylethylsulfinamido)(phenyl) methyl)benzoic acid (150 mg,
68%) as a solid: 1H NMR (500 MHz, Methanol-d4): δ 8.00−7.96 (m,
2H), 7.51−7.48 (m, 2H), 7.44−7.41 (m, 2H), 7.37−7.32 (m, 2H),
7.29−7.24 (m, 1H), 5.67 (s, 1H), 1.26 (d, J = 0.9 Hz, 9H). LCMS
(ESI) m/z 329.9 [M − 1]−.
N - ( 6 - M e t h o x y b e n z o [ d ] t h i a z o l - 2 - y l ) -
benzenesulfonamidenicotinonitrile (30) was prepared according to
general procedure A. The title compound was obtained as white solid,
1
50 mg in 28% yield. H NMR (400 MHz, DMSO-d6): δ 13.08 (s,
1H), 7.89−7.80 (m, 2H), 7.65−7.51 (m, 3H), 7.45 (d, J = 2.5 Hz,
1H), 7.21 (d, J = 8.8 Hz, 1H), 6.97 (dd, J = 8.8, 2.6 Hz, 1H), 3.75 (s,
3H). 13C NMR (400 MHz, DMSO-d6): δ 166.6, 156.1, 142.2, 132.3,
130.0, 129.1, 126.1, 125.8, 114.8, 113.6, 107.0, 55.7. ESI-MS (m/z):
321.0 [M + H]+.
N-(Benzo[d]thiazol-2-yl)-[1,1′-biphenyl]-4-carboxamide (32) was
prepared according to general procedure B on 1.7 mmol scale.
Additional biphenyl-4-carboxylic acid (165.8 mg, 0.84 mmol, 0.5
equiv) was added after 2 h when monitoring by LC/MS had
determined it was completely consumed. Two hours after the addition
of acid, another 0.4 mL of base was added, and the reaction was
stirred overnight. The reaction was diluted with water and the mixture
was filtered. The brown solid was washed with water many times and
then with hexanes. The solid was further purified by automated flash
chromatography in 100% DCM and washed with DCM to afford the
1
desired product as a white solid (197.9 mg, 39%). H NMR (400
MHz, DMSO-d6): δ 12.96 (s, 1H), 8.27−8.21 (m, 2H), 8.02 (dd, J =
8.1, 1.2 Hz, 1H), 7.90−7.84 (m, 2H), 7.82−7.74 (m, 3H), 7.55−7.40
(m, 4H), 7.34 (td, J = 7.6, 7.2, 1.1 Hz, 1H). 13C NMR (126 MHz,
DMSO): δ 144.3, 138.9, 129.2, 129.1, 128.5, 127.1, 126.9, 126.3,
123.8, 121.8. ESI-MS (m/z): 331.0 [M + H]+.
Step 3. To a reaction vial equipped with a stir bar and Teflon-lined
lid was added the carboxylic acid from Step 2 (60 mg, 0.18 mmol),
TBTU (0.6 mmol, 3.3 equiv), 4-(dimethylamino)pyridine (10 mol
%), triethylamine (0.9 mmol, 5 equiv), and N,N-dimethylformamide
(0.3 mL). The vial was flushed with nitrogen, sealed, and heated to 60
°C for 16 h. After cooling to room temperature, the mixture was
diluted with water and the product isolated by filtration to give 4-
N-(Benzo[d]thiazol-2-yl)-4-methoxybenzamide (33) was prepared
according to general procedure A. The crude solid was purified by
automated flash chromatography (0−10% MeOH/DCM) to afford
1
the desired product as a white solid 111.1 mg, 78% yield). H NMR
(400 MHz, DMSO-d6): δ 12.73 (s, 1H), 8.18−8.13 (m, 2H), 8.01
(dd, J = 7.9, 1.2 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.46 (ddd, J = 8.3,
7.2, 1.3 Hz, 1H), 7.33 (ddd, J = 8.3, 7.3, 1.2 Hz, 1H), 7.13−7.07 (m,
I
https://dx.doi.org/10.1021/acs.jmedchem.0c00899
J. Med. Chem. XXXX, XXX, XXX−XXX

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2H), 3.86 (s, 3H). 13C NMR (126 MHz, DMSO): δ 163.12, 130.7,
126.4, 123.8, 121.9, 114.2, 55.8. ESI-MS (m/z): 285.0 [M + H]+.
N-(Benzo[d]thiazol-2-yl)-3-methylbenzamide (34) was prepared
according to general procedure A in 1 mL of pyridine. The crude solid
was washed with water and air dried, then purified by automated flash
chromatography (0−5% MeOH/DCM) to afford the desired product
7.00 (d, J = 3.6 Hz, 1H). 13C NMR (400 MHz, CDCl3): δ 195.9,
165.2, 160.3, 141.3, 137.2, 136.9, 136.0, 133.3, 130.4, 130.3, 128.7,
128.2, 114.2. ESI-MS (m/z): 309.0 [M + H]+.
4-Benzoyl-N-(5-phenylthiazol-2-yl)benzamide (40) was prepared
according to general procedure B. The solids obtained from
precipitation from addition of water to the reaction was purified by
automated flash chromatography and then triturated with 1:1 DCM/
Hexane to afford the title compound as a light yellow solid (101 mg,
1
as a 120.5 mg of white solid in 90% yield. H NMR (400 MHz,
DMSO-d6): δ 12.84 (s, 1H), 8.02 (dd, J = 8.1, 1.3 Hz, 1H), 7.98 (t, J
= 1.7 Hz, 1H), 7.94 (dt, J = 6.8, 2.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H),
7.51−7.41 (m, 3H), 7.34 (ddd, J = 8.1, 7.2, 1.2 Hz, 1H), 2.40 (s, 3H).
13C NMR (126 MHz, DMSO): δ 138.3, 133.7, 129.1, 128.8, 126.4,
125.7, 123.9, 122.0, 121.1. ESI-MS (m/z): 269.0 [M + H]+.
1
60% yield). H NMR (400 MHz, DMSO-d6): δ 12.98 (s, 1H), 8.26
(d, J = 8.4 Hz, 2H), 8.00 (s, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.82−7.76
(m, 2H), 7.76−7.64 (m, 3H), 7.60 (t, J = 7.6 Hz, 2H), 7.44 (t, J = 7.7
Hz, 2H), 7.33 (t, J = 7.4 Hz, 1H). 13C NMR (400 MHz, DMSO-d6):
δ 195.8, 140.6, 136.9, 135.9, 133.6, 131.9, 130.2, 130.0, 129.7, 129.2,
128.9, 128.1, 126.2. ESI-MS (m/z): 385.0 [M + H]+.
N-(Benzo[d]thiazol-2-yl)nicotinamide (35) was prepared accord-
ing to general procedure B. No precipitation was observed when water
was added to the reaction mixture. The mixture was then diluted with
EtOAc and washed several times with water and then brine. The off-
white precipitate from the organic layer was collected by filtration and
found to be 42.9 mg of pure desired product. The organic layer was
dried over Na2SO4, filtered, condensed and purified by automated
flash chromatography in 0−10% MeOH/DCM to give an additional
4-Benzoyl-N-(4-phenylthiazol-2-yl)benzamide (41) was prepared
according to general procedure B. The crude reaction product was
triturated with water and the mixture was filtered. The isolated solid
was washed with water several times with water and then with
hexanes. The solid was recrystallized from ethanol to afford the title
1
compound as yellow needles (138.4 mg, 63% yield). H NMR (400
1
80.9 mg desired product. Total isolated yield = 123.8 mg, 73%. H
MHz, DMSO-d6): δ 12.99 (s, 1H), 8.28 (d, J = 8.0 Hz, 2H), 7.96 (d, J
= 7.7 Hz, 2H), 7.87 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 7.6 Hz, 2H), 7.71
(d, J = 8.5 Hz, 2H), 7.60 (t, J = 7.5 Hz, 2H), 7.45 (t, J = 7.6 Hz, 2H),
7.34 (t, J = 7.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6): δ 195.3,
164.6, 158.4, 149.3, 140.3, 136.5, 135.3, 134.3, 133.20, 129.7, 129.5,
128.8, 128.7, 128.4, 127.9, 125.8, 108.8. ESI-MS (m/z): 385.0 [M +
H]+.
NMR (400 MHz, DMSO-d6): δ 13.16 (s, 1H), 9.26 (dd, J = 2.3, 0.9
Hz, 1H), 8.82 (dd, J = 4.8, 1.7 Hz, 1H), 8.47 (dt, J = 8.0, 2.0 Hz, 1H),
8.04 (dd, J = 8.0, 1.3 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.61 (ddd, J =
8.0, 4.8, 0.9 Hz, 1H), 7.49 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 7.36 (ddd, J
= 8.2, 7.3, 1.2 Hz, 1H). 13C NMR (101 MHz, DMSO-d6): δ 153.3,
149.5, 136.3, 126.5, 124.1, 123.9, 122.1. ESI-MS (m/z): 256.0 [M +
H]+.
N-(5-Aminobenzo[d]thiazol-2-yl)-4-methoxybenzamide (42). A
vial was charged with 4-methoxy-N-(5-nitrobenzo[d]thiazol-2-yl)-
benzamide (50 mg, 0.15 mmol) and palladium on activated carbon
(10%) and the mixture was suspended in ethanol (1 mL) and conc.
hydrochloric acid (20 μL). The vial was sealed and equipped with a
hydrogen balloon. After evacuation and back filling of the vial, the
mixture was stirred at room temperature 4 h, at which TLC indicated
completion. The mixture was filtered through a pad of Celite 545 and
the pad was washed copiously with ethyl acetate to give the title
N-(Benzo[d]thiazol-2-yl)-4-benzoylbenzamide (36) was prepared
according to general procedure B on a 0.1 mmol scale. Thus,
benzo[d]thiazol-2-amine (15 mg, 0.1 mmol), 4-benzoylbenzoic acid
(24 mg, 0.11 mmol, 1.1 equiv), HATU (41 mg, 0.11 mmol, 1.1
equiv), and N,N-diisopropylethylamine (30 μL, 0.2 mmol, 2 equiv)
were dissolved in anhydrous N,N-dimethylformamide (1 mL) and the
mixture was stirred for 16 h at room temperature. The crude mixture
was diluted with water, and the resulting solid was filtered and dried
1
by aspiration to give the title compound (10 mg, 27%). H NMR
1
compound: H NMR (500 MHz, DMSO-d6): δ 8.12 (d, J = 8.7 Hz,
(400 MHz, Chloroform-d): δ 8.16 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5
Hz, 2H), 7.90 (d, J = 7.0 Hz, 1H), 7.80 (d, J = 7.0 Hz, 2H), 7.72 (d, J
= 7.8 Hz, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 2H), 7.47
(d, J = 8.2 Hz, 1H), 7.39 (t, J = 8.2 Hz, 1H).LCMS (ESI) m/z 359
[M + 1]+.
2H), 7.51 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 8.6 Hz, 2H), 6.89 (d, J =
2.1 Hz, 1H), 6.60 (dd, J = 8.3, 2.0 Hz, 1H), 3.84 (s, 3H); LCMS
(ESI) m/z 300.0 [M + 1]+.
4-Benzoyl-N-(6-propoxybenzo[d]thiazol-2-yl)benzamide (44). 4-
Benzoyl-N-(6-(prop-2-yn-1-yloxy)benzo[d]thiazol-2-yl)benzamide
(49 (see below), 30 mg, 0.07 mmol) and palladium on carbon (20%,
Degussa type, 10 mg) were charged into a round-bottom flask,
followed by ethyl acetate (1 mL). The flask was purged with nitrogen,
then equipped with a hydrogen balloon attached to a three-way inlet,
and subsequently evacuated and backfilled with hydrogen 4 times.
The mixture was stirred at room temperature for 1 h, and was then
filtered through Celite 545, and the Celite pad was washed copiously
with ethyl acetate. The filtrate was concentrated under vacuum and
the residue triturated with ethyl ether/methanol to give the title
N-(1H-Benzo[d]imidazole-2-yl)-4-benzoylbenzamide (37) was
prepared according to general procedure B on a 0.1 mmol scale.
Thus, 1H-benzo[d]imidazole-2-amine (13 mg, 0.1 mmol), 4-
benzoylbenzoic acid (24 mg, 0.11 mmol, 1.1 equiv), HATU (41
mg, 0.11 mmol, 1.1 equiv), and N,N-diisopropylethylamine (34 μL,
0.2 mmol, 2 equiv) were dissolved in anhydrous N,N-dimethylforma-
mide (1 mL) and stirred overnight. The crude mixture was diluted
with water, and the resulting solid was filtered and dried by aspiration
to give the title compound (10 mg, 27%). 1H NMR (400 MHz,
DMSO-d6): δ 12.47 (s, 2H), 8.30 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 8.3
Hz, 2H), 7.79 (d, J = 7.0 Hz, 2H), 7.72 (t, J = 7.4 Hz, 1H), 7.60 (t, J =
7.6 Hz, 2H), 7.46 (dd, J = 5.9, 3.2 Hz, 2H), 7.17 (dd, J = 5.9, 3.2 Hz,
2H). LCMS (ESI) m/z 342 [M + 1]+.
N-(Benzo[d]oxazol-2-yl)-4-benzoylbenzamide (38) was prepared
according to general procedure B. The crude mixture was diluted with
water, and the resulting solid was filtered and dried by aspiration to
give the title compound (112 mg, 33%): 1H NMR (400 MHz,
DMSO-d6): δ 8.23 (d, J = 8.1 Hz, 2H), 7.89−7.84 (m, 2H), 7.81−
7.76 (m, 2H), 7.72 (t, J = 7.4 Hz, 1H), 7.66−7.55 (m, 4H), 7.38−
7.27 (m, 2H). ESI-MS (m/z): 343 [M + 1]+.
4-Benzoyl-N-(thiazol-2-yl)benzamide (39) was prepared according
to general procedure B. The reaction mixture was added to stirring
water, and the precipitated solids were collected by filtration, washed
with water, and purified by automated flash chromatography (0−3%
MeOH/DCM) to give the title compound as a white solid (132 mg,
43% yield). 1H NMR (400 MHz, CDCl3): δ 12.83 (s, 1H), 8.14 (d, J
= 7.9 Hz, 2H), 7.93 (d, J = 7.9 Hz, 2H), 7.84 (d, J = 8.2 Hz, 2H), 7.64
(t, J = 6.9 Hz, 1H), 7.53 (t, J = 7.7 Hz, 2H), 7.05 (d, J = 3.6 Hz, 1H),
1
compound (15 mg, 52%) as a golden solid: H NMR (400 MHz,
Chloroform-d): δ 10.99 (s, 1H), 8.08 (d, J = 8.3 Hz, 2H), 7.86 (d, J =
8.2 Hz, 2H), 7.79−7.73 (m, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.50 (t, J =
7.7 Hz, 2H), 7.38−7.31 (m, 2H), 6.96 (dd, J = 8.9, 2.5 Hz, 1H), 3.97
(t, J = 6.5 Hz, 2H), 1.84 (h, J = 7.1 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H);
13C NMR (101 MHz, Chloroform-d): δ 195.9, 165.5, 158.1, 156.9,
141.9, 141.6, 137.0, 135.6, 133.5, 133.4, 130.7, 130.5, 128.8, 128.3,
121.5, 116.0, 105.4, 70.50, 23.0, 10.9. LCMS (ESI) m/z 414.9 [M −
1]−.
4-Benzoyl-N-(6-(2-bromoethoxy)benzo[d]thiazol-2-yl)benzamide
(45). Step 1. A solution of 2-aminobenzo[d]thiazol-6-ol (50 mg, 0.3
mmol) in acetone (3 ml) was treated with potassium carbonate (208
mg, 1.5 mmol, 5 equiv) and 1,3-dibromopropane (153 μL, 1.5 mmol,
5 equiv). The mixture was stirred at room temperature for 16 h, then
diluted with water and extracted with ethyl acetate. The organic layer
was dried (sodium sulfate), filtered, and concentrated under vacuum
to give the crude 6-(3-bromopropoxy)benzo[d]thiazol-2-amine (91
mg), which was not purified but used as is in the next step.
J
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Step 2. This material was treated with 4-benzoylbenzoic acid (69
mg, 0.3 mmol), TBTU (193 mg, 0.6 mmol), triethylamine (125 μL),
DMAP (10 mol %), and DMF (0.3 mL) in the manner described in
General Procedure B to give the title compound (14 mg, 9%) as a
solid containing a unidentified impurity (m, 3.5 ppm): 1H NMR (400
MHz, Chloroform-d): δ 11.04 (s, 1H), 8.09 (d, J = 8.1 Hz, 2H), 7.87
(d, J = 8.1 Hz, 2H), 7.81−7.73 (m, 2H), 7.63 (t, J = 7.4 Hz, 1H), 7.50
(t, J = 7.7 Hz, 2H), 7.36 (dd, J = 5.7, 3.2 Hz, 2H), 6.96 (dd, J = 8.8,
2.4 Hz, 1H), 4.17 (t, J = 5.8 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.35
(p, J = 6.0 Hz, 2H); 13C NMR (101 MHz, Chloroform-d): δ 195.9,
156.5, 141.8, 137.1, 135.4, 133.5, 130.8, 130.5, 128.9, 128.1, 121.8,
105.6, 66.4, 32.7, 30.3. LCMS (ESI) m/z 495.0 [M]+.
Step 2. 4-benzoyl-N-(6-(prop-2-yn-1-yloxy)benzo[d]thiazol-2-yl)-
benzamide. The amine from step 1 (50.1 mg, 0.25 mmol), 4-
benzoylbenzoic acid (55.9 mg, 0.25 mmol, 1 equiv), HATU (116.8
mg, 0.31 mmol, 1.2 equiv), triethylamine (56 μL, 0.32 mmol, 1.2
equiv), and DMF (2.4 mL) were treated in the manner similar as
described in general procedure B to give 49 as cream colored solid
1
(90.3 mg, 89%). H NMR (400 MHz, DMSO-d6): δ 13.01 (s, 1H),
8.33−8.24 (m, 2H), 7.88 (d, J = 8.1 Hz, 2H), 7.83−7.76 (m, 2H),
7.71 (ddd, J = 14.1, 9.0, 2.7 Hz, 3H), 7.60 (t, J = 7.6 Hz, 2H), 7.14
(dd, J = 8.8, 2.6 Hz, 1H), 4.87 (d, J = 2.4 Hz, 2H), 3.60 (q, J = 2.6 Hz,
1H). 13C NMR (101 MHz, DMSO-d6): δ 195.3, 154.2, 140.4, 136.5,
133.1, 129.7, 129.5, 128.7, 128.5, 115.6, 106.4, 79.2, 78.23, 56.1.
LCMS (ESI) m/z 413.0 [M + H]+.
4-Benzoyl-N-(5-bromo-6-methoxybenzo[d]thiazol-2-yl)benzamide
(46). 4-benzoyl-N-(6-methoxybenzo[d]thiazol-2-yl)benzamide (16,
50 mg, 0.13 mmol) was suspended in methylene chloride/methanol
(10:1, 1.7 mL) and the resulting mixture was treated with bromine
(10 μl, 0.19 mmol, 1.5 equiv). After stirring for 16 h at room
temperature, the solvent was removed under vacuum and the resulting
residue was purified by silica gel chromatography (1% MeOH/
Prop-2-yn-1-yl (4-(Benzo[d]thiazol-2-ylcarbamoyl)phenyl)-
carbamate (50). Step 1. A mixture of alkyne 31 (326 mg, 1.09
mmol), iron powder (304 mg, 5.45 mmol, 5 equiv), ammonium
chloride (88 mg, 1.64 mmol, 1.5 equiv) in EtOH/water (3:1, 6 mL)
was stirred at 90 °C in a sealed vial for 8 h. The reaction mixture was
allowed to cool and the iron powder pelleted with a magnet. The
supernatant was partitioned between dichloromethane and saturated
sodium bicarbonate solution and the aqueous phase was extracted
with dichloromethane (3×). The combined extracts were dried over
sodium sulfate and condensed to afford the crude 4-amino-N-
(benzo[d]thiazol-2-yl)benzamide as a white solid (245 mg, 83%)
which was carried forward without further purification. 1H NMR (400
MHz, Methanol-d4): δ 7.84 (dd, J = 8.3, 6.3 Hz, 3H), 7.73 (d, J = 8.1
Hz, 1H), 7.45−7.37 (m, 1H), 7.29 (t, J = 7.6 Hz, 1H), 6.72 (d, J = 8.6
Hz, 2H). 13C NMR (101 MHz, Methanol-d4): δ 167.9, 161.0, 154.9,
150.0, 133.4, 131.2, 127.1, 124.7, 122.2, 121.5, 120.3, 114.6, 49.9. ESI-
MS (m/z): 270. 0 [M + H]+.
1
CH2Cl2) to give the title compound as a tan solid: H NMR (400
MHz, Chloroform-d): δ 10.28 (s, 1H), 8.12−8.07 (m, 2H), 7.96−
7.92 (m, 2H), 7.83−7.78 (m, 3H), 7.64 (t, J = 7.4 Hz, 1H), 7.55−
7.48 (m, 3H), 7.35 (s, 1H), 3.98 (s, 3H); LCMS (ESI) m/z 467.0
[M]+.
Methyl 2-(4-Benzoylbenzamido)benzo[d]thiazole-6-carboxylate
(48) was prepared according to general procedure B. The reaction
was stirred overnight, and the title compound was precipitated by the
addition of water to the reaction. The mixture was filtered and the
solid was washed with water copiously and then air dried to give 148.1
mg of desired compound in 74% yield. 1H NMR (400 MHz, DMSO-
d6): δ 13.32 (s, 1H), 8.67 (s, 1H), 8.28 (d, J = 7.9 Hz, 2H), 8.03 (d, J
= 8.5 Hz, 1H), 7.92−7.68 (m, 6H), 7.59 (t, J = 7.6 Hz, 2H), 3.88 (s,
3H). 13C NMR (101 MHz, DMSO-d6): δ 195.5, 166.2, 140.8, 136.6,
133.4, 130.0, 129.8, 129.0, 128.9, 127.4, 125.0, 124.2, 52.4. ESI-MS
(m/z): 417.0 [M + H]+.
Step 2. Triethylamine (24 μL, 0.17 mmol, 1.4 equiv) was added
dropwise to a cold solution of the amine from Step 1 (33.3 mg, 0.12
mmol) in 2 ml of THF at −78 °C (dry ice/acetone bath). The
mixture was stirred cold for 10 min before the dropwise addition of
propargyl chloroformate (22 μL, 0.22 mmol, 1.8 equiv). The reaction
was stirred overnight in the melting cold bath. The reaction mixture
was condensed to dryness, taken up in ethyl acetate and washed with
water, brine and dried over Na2SO4, filtered and condensed. The
crude mixture was purified by automated flash chromatography in 0−
5% MeOH/DCM to give 20.6 mg of the title compound in 47%
N-(4-Benzoylphenyl)benzo[d]thiazole-2-carboxamide (48) was
prepared according to general procedure B. After stirring overnight,
additional carboxylic acid (0.2 equiv, 0.076 mmol) and HATU (0.2
equiv, 0.072 mmol) were added to the reaction. The reaction mixture
was poured onto stirring water but no solids precipitated. The water
was extracted with EtOAC thrice. The combined extracts were
washed with 5% LiCl and brine, dried over anhydrous sodium sulfate,
and condensed. The residue was purified by silica gel flash
chromatography: first using (1−3% MeOH in DCM) and then (2−
30% EtOAc in hexanes). The residue obtained from the second
column was purified further by silica gel preparative TLC in 100%
1
isolated yield. H NMR (400 MHz, Methanol-d4): δ 8.05−7.98 (m,
2H), 7.89−7.84 (m, 1H), 7.78−7.73 (m, 1H), 7.67−7.61 (m, 2H),
7.43 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 7.34−7.27 (m, 1H), 4.79 (d, J =
2.3 Hz, 2H), 2.96 (t, J = 2.4 Hz, 1H). 13C NMR (101 MHz,
Methanol-d4): δ 154.8, 145.1, 133.6, 130.7, 127.9, 127.5, 125.2, 122.6,
121.9, 119.3, 79.4, 76.6, 53.8. ESI-MS (m/z): 352. 0 [M + H]+.
N-(6-Methoxybenzo[d]thiazol-2-yl)-4-(4-(prop-2-yn-1-yloxy)-
benzoyl)benzamide (51) was prepared according to general
procedure B. After 12 h, the reaction mixture was poured onto
stirring water which precipitated solids that were collected by
filtration, washed with water, and purified twice by silica gel flash
chromatography: first using (0−4% MeOH in DCM) then (0−1%
MeOH in DCM). The title compound was obtained as a light yellow
solid (119 mg, 64%). 1H NMR (400 MHz, DMSO-d6): δ 12.99 (br s,
1H), 8.27 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.80 (d, J =
8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 2.6 Hz, 1H), 7.17
(d, J = 8.9 Hz, 2H), 7.07 (dd, J = 8.8, 2.6 Hz, 1H), 4.95 (d, J = 2.3 Hz,
2H), 3.83 (s, 3H), 3.67 (t, J = 2.3 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6): δ 193.9, 161.2, 156.3, 141.1, 132.2, 129.6, 129.3, 128.4,
115.2, 114.9, 104.7, 78.9, 78.7, 55.8, 55.7. ESI-MS (m/z): 443.0 [M +
H]+.
1
DCM to afford the product as a white solid (33 mg, 24%). H NMR
(400 MHz, DMSO-d6): δ 11.51 (br s, 1H), 8.31−8.22 (m, 2H), 8.13
(d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.77−7.71 (m, 2H),
7.71−7.54 (m, 5H). 13C NMR (101 MHz,, DMSO-d6): δ 194.7,
164.4, 158.7, 152.7, 142.3, 137.4, 136.5, 132.5, 132.4, 130.9, 129.5,
128.5, 127.3, 127.3, 124.3, 123.2, 120.2. ESI-MS (m/z): 359.0 [M +
H]+.
4-Benzoyl-N-(6-(prop-2-yn-1-yloxy)benzo[d]thiazol-2-yl)-
benzamide (49). Step 1. Propargyl bromide (245 mL, 2.2 mmol, 1.2
equiv, 80%w/w in tol) was added to a suspension of 2-aminobenzo-
[d]thiazol-6-ol (300.7 mg, 1.8 mmol) and potassium carbonate (302.9
mg, 2.2 mmol, 1.2 equiv) in DMF (3.6 mL). The reaction was stirred
overnight at 60 °C. The cooled reaction was diluted with water and
extracted three times with EtOAc. The combined organic extracts
were washed with water a few times and then with brine. The organic
layer was dried over Na2SO4, filtered and condensed. The crude
mixture was purified by automated flash chromatography in 0−5%
MeOH/DCM to give 97.9 mg of 6-(prop-2-yn-1-yloxy)benzo[d]-
N1-(Benzo[d]thiazol-2-yl)-N4-(prop-2-yn-1-yl)terephthalamide
(52) Step 1. Methyl 4-(benzo[d]thiazol-2-ylcarbamoyl)benzoate was
prepared analogously to methyl 4-((6-methoxybenzo[d]thiazol-2-
yl)carbamoyl)benzoate described above for the synthesis of amides
1
i
thiazol-2-amine in 26% yield. H NMR (400 MHz, Methanol-d4) δ
20−23 but with 1.5 equiv of Pr2NEt. The crude product was
7.29 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 2.5 Hz, 1H), 6.92 (dd, J = 8.8,
2.6 Hz, 1H), 4.71 (d, J = 2.4 Hz, 2H), 2.92 (t, J = 2.4 Hz, 1H). 13C
NMR (101 MHz, Methanol-d4): δ 168.8, 155.1, 148.1, 133.1, 119.5,
115.9, 108.5, 80.3, 77.0, 57.9. LCMS (ESI) m/z: 205.0 [M + H]+.
obtained as an off-white solid (413 mg, 99%), which was carried
forward directly. 1H NMR (400 MHz, DMSO-d6): δ 13.14 (br s, 1H),
8.25 (d, J = 8.5 Hz, 2H), 8.10 (d, J = 8.5 Hz, 2H), 8.03 (d, J = 7.7 Hz,
1H), 7.79 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.35 (t, J = 7.1
K
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Hz, 1H), 3.90 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 167.0,
165.6, 136.2, 133.0, 131.7, 131.6, 131.3, 129.3, 128.8, 128.7, 126.3,
123.8, 121.9, 120.2, 109.6, 67.4, 52.5, 38.1, 29.8, 28.4, 23.3, 22.4, 13.9,
10.8. ESI-MS (m/z): 313.0 [M + H]+.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00899.
Step 2. 4-(Benzo[d]thiazol-2-ylcarbamoyl) benzoic acid was
prepared analogously to 4-((6-methoxybenzo[d]thiazol-2-yl) carba-
moyl) benzoic acid described above for the synthesis of amides 20−
23). The crude product was obtained as a white solid (397 mg, 100%)
Supplementary figures, chemical methods, NMR spectra,
representative HPLC traces, spectra, selected chromato-
grams, and molecular Formula Strings with structures of
tested compounds and IC50 values (PDF)
1
1
in 90% purity (from HNMR) which was used directly. H NMR
(400 MHz, DMSO-d6): δ 13.24 (s, 1H), 8.23 (d, J = 8.5 Hz, 2H),
8.09 (d, J = 8.5 Hz, 2H), 8.03 (d, J = 7.4 Hz, 1H), 7.79 (d, J = 8.0 Hz,
1H), 7.48 (t, J = 7.1 Hz, 1H), 7.35 (t, J = 7.1 Hz, 1H). 13C NMR (101
MHz, DMSO-d6): δ 166.6, 135.8, 134.4, 131.6, 131.4, 129.4, 128.7,
126.3, 123.9, 121.9, 120.2. ESI-MS (m/z): 299.0 [M + H]+, 296.9 [M
− H]−.
(CSV)
AUTHOR INFORMATION
■
Corresponding Authors
Step 3. Amide 52 was prepared analogously to compound 20. The
crude product was obtained as a white solid and washed with 5%
MeOH/DCM to give 69.7 mg of product in 81% yield. 1H NMR (400
MHz, DMSO-d6): δ 13.17−12.91 (m, 1H), 9.16 (t, J = 5.5 Hz, 1H),
8.22 (d, J = 8.1 Hz, 2H), 8.02 (t, J = 7.7 Hz, 3H), 7.79 (d, J = 8.1 Hz,
1H), 7.48 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 4.09 (dd, J =
5.5, 2.5 Hz, 2H), 3.17 (d, J = 2.5 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6): δ 165.2, 137.4, 128.5, 127.6, 126.3, 123.8, 121.9, 81.1,
73.1, 28.7. ESI-MS (m/z): 336.0 [M + H]+.
(R)-4-(amino(phenyl)methyl)-N-(6-hydroxybenzo[d]thiazol-2-yl)-
benzamide (53). Fresh boron tribromide (1.0 M in DCM, 0.212 mL,
0.21 mmol, 3 equiv) added to a solution of 4-((R,SS)-tert-
butylsulfinyl)amino)(phenyl)methyl)-N-(6-methoxybenzo[d]thiazol-
2-yl)benzamide (intermediate in the synthesis of (R)-27, 34.8 mg,
0.070 mmol) in 0.7 mL DCM. The reaction was stirred overnight,
then diluted with DCM and washed with saturated Na2CO3 solution.
The aqueous layer containing colored solids, was filtered and washed
to give crude product which was purified via automated flash
chromatography in 0−10% MeOH/DCM (+0.05% Et3N) to give 10.9
mg of the title compound in 41% yield. In our experience using a
previously used bottle of BBr3 gave a mixture of 4-(R,SS)-tert-
butylsulfinyl)amino)(phenyl)methyl)-N-(6-hydroxybenzo[d]thiazol-
2-yl)benzamide and title compound. 1H NMR (400 MHz, Methanol-
d4): δ 8.03−7.97 (m, 2H), 7.58 (t, J = 9.1 Hz, 3H), 7.35 (dd, J = 16.6,
9.2 Hz, 4H), 7.28−7.20 (m, 2H), 6.94 (dd, J = 8.7, 2.5 Hz, 1H), 5.24
(s, 1H). 13C NMR (101 MHz, Methanol-d4): δ 168.2, 158.5, 156.2,
152.0, 146.1, 143.4, 134.9, 132.7, 129.9, 129.6, 128.7, 128.6, 128.4,
122.6, 116.8, 107.5, 60.9. MS (m/z): 480.0 [M + H]+.
N-(R)-4-(Amino(phenyl)methyl)-N-(benzo[d]thiazol-2-yl)-
benzamide hydrochloride (54). Step 1. N-(Benzo[d]thiazol-2-yl)-4-
((1R,SS)-((tert-butylsulfinyl)amino) (phenyl) methyl)benzamide was
prepared according to general procedure B. The desired product was
isolated by triturating the crude reaction mixture with water. The
mixture was filtered and the solid obtained was washed with water and
then hexanes. The solid was purified by preparative TLC in 2%
MeOH/DCM to provide 34.4 mg of the title compound in 61% yield.
1H NMR (400 MHz, CD3OD): δ 8.03−7.97 (m, 2H), 7.84 (dd, J =
7.8, 1.2 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 8.3 Hz, 2H),
7.46−7.36 (m, 3H), 7.35−7.20 (m, 4H), 5.68 (d, J = 4.6 Hz, 1H),
1.25 (s, 9H). 13C NMR (101 MHz, CD3OD): δ 167.8, 160.6, 149.6,
148.7, 143.4, 133.3, 132.8, 129.8, 129.5, 128.8, 128.6, 127.3, 125.0,
122.4, 121.6, 64.3, 57.5, 23.2. ESI-MS (m/z): 464.0 [M + H]+.
Step 2. Hydrogen chloride (4 M in dioxane, 52 mL, 0.21 mmol, 3
equiv) was added to a suspension of the sulfinamide from Step 1
(34.4 mg, 0.074 mmol) in methanol (0.51 mL) and stirred. The
reaction had gone to completion within 3 h and was condensed to
give 30.2 mg of light yellow solid in quantitative yield. 1H NMR (400
MHz, DMSO-d6): δ 12.94 (s, 1H), 9.21 (dd, J = 18.1, 5.7 Hz, 2H),
8.19 (d, J = 8.3 Hz, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 8.1 Hz,
1H), 7.71 (d, J = 8.3 Hz, 2H), 7.59−7.52 (m, 2H), 7.52−7.42 (m,
3H), 7.42−7.30 (m, 2H), 5.79 (q, J = 5.3 Hz, 1H). 13C NMR (101
MHz, DMSO-d6): δ 165.7, 159.1, 143.1, 143.0, 138.0, 137.9, 132.0,
131.6, 129.1, 128.94, 128.71, 127.70, 127.65, 126.4, 124.0, 122.0,
120.5, 56.9, 56.8. ESI-MS (m/z): 360.0 [M + H]+.
Noelle S. Williams − Department of Biochemistry, UT
Southwestern Medical Center, Dallas, Texas 75390-9038,
United States; Email: Noelle.williams@utsouthwestern.edu
Deepak Nijhawan − Department of Biochemistry and
Department of Internal Medicine, Division of Hematology and
Oncology, UT Southwestern Medical Center, Dallas, Texas
75390-9038, United States; orcid.org/0000-0001-6253-
4268; Email: deepak.nijhawan@utsouthwestern.edu
Joseph M. Ready − Department of Biochemistry, UT
Southwestern Medical Center, Dallas, Texas 75390-9038,
United States; orcid.org/0000-0003-1305-9581;
Email: joseph.ready@utsouthwestern.edu
Authors
Stephen Gonzales − Department of Biochemistry, UT
Southwestern Medical Center, Dallas, Texas 75390-9038,
United States
Jacinth Naidoo − Department of Biochemistry, UT
Southwestern Medical Center, Dallas, Texas 75390-9038,
United States
Giomar Rivera-Cancel − Department of Biochemistry and
Department of Internal Medicine, Division of Hematology and
Oncology, UT Southwestern Medical Center, Dallas, Texas
75390-9038, United States
Sukesh Voruganti − Department of Biochemistry, UT
Southwestern Medical Center, Dallas, Texas 75390-9038,
United States
Prema Mallipeddi − Department of Biochemistry, UT
Southwestern Medical Center, Dallas, Texas 75390-9038,
United States
Panayotis C. Theodoropoulos − Department of Biochemistry
and Department of Internal Medicine, Division of Hematology
and Oncology, UT Southwestern Medical Center, Dallas, Texas
75390-9038, United States
Sophie Geboers − Department of Biochemistry, UT
Southwestern Medical Center, Dallas, Texas 75390-9038,
United States
Hong Chen − Department of Biochemistry, UT Southwestern
Medical Center, Dallas, Texas 75390-9038, United States
Francisco Ortiz − Department of Biochemistry, UT
Southwestern Medical Center, Dallas, Texas 75390-9038,
United States
Bruce Posner − Department of Biochemistry, UT Southwestern
Medical Center, Dallas, Texas 75390-9038, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00899
Notes
The authors declare no competing financial interest.
L
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Kapur, P.; Pedrosa, I.; Brugarolas, J.; Kittler, R.; Ye, J. Unsaturated
fatty acids stimulate tumor growth through stabilization of β-catenin.
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ACKNOWLEDGMENTS
■
Financial support from the CPRIT (RP180457) and the Welch
Foundation (I-1612 to J.M.R., I-1879 to D.N.) is acknowl-
edged. We thank Emauela Copota for technical assistance.
ABBREVIATIONS
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A. I.; Zilbershtein, T. M.; Ivanov, A. V.; Cole, A. G.; Henderson, I.;
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AUC, area under the curve; Cmax, maximal plasma concen-
tration; CTD2, cancer target discovery development; CYP,
cytochrome; DCM, dichloromethane; HPLC, high pressure
liquid chromatography; IP, intraperitoneal; NSCLC, nonsmall
cell lung cancer; PK, pharmacokinetic; SCD, stearoyl-CoA
desaturase; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetrame-
thylaminium tetrafluoroborate
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
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