Thioimidazoline based compounds reverse glucocorticoid resistance in human acute lymphoblastic leukemia xenografts

Article abstract of DOI:10.1039/c5ob00779h

Glucocorticoids form a critical component of chemotherapy regimens for pediatric acute lymphoblastic leukemia (ALL) and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. A high-throughput screen identified four thioimidazoline-containing compounds that reversed dexamethasone resistance in an ALL xenograft derived from a chemoresistant pediatric ALL. The lead compound (1) was synergistic when used in combination with the glucocorticoids, dexamethasone or prednisolone. Synergy was observed in a range of dexamethasone-resistant xenografts representative of B-cell precursor ALL (BCP-ALL) and T-cell ALL. We describe here the synthesis of twenty compounds and biological evaluation of thirty two molecules that explore the structure-activity relationships (SAR) of this novel class of glucocorticoid sensitizing compounds. SAR analysis has identified that the most effective dexamethasone sensitizers contain a thioimidazoline acetamide substructure with a large hydrophobic moiety on the acetamide. This journal is

Full text of DOI:10.1039/c5ob00779h

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Thioimidazoline based compounds reverse
glucocorticoid resistance in human acute
Cite this: DOI: 10.1039/c5ob00779h
lymphoblastic leukemia xenografts†
Cara E. Toscan,a,b,c Marwa Rahimi,b,c Mohan Bhadbhade,b Russell Pickford,d
Shelli R. McAlpine*b and Richard B. Lock*a,c
Glucocorticoids form a critical component of chemotherapy regimens for pediatric acute lymphoblastic
leukemia (ALL) and the initial response to glucocorticoid therapy is a major prognostic factor, where
resistance is predictive of poor outcome. A high-throughput screen identified four thioimidazoline-
containing compounds that reversed dexamethasone resistance in an ALL xenograft derived from a
chemoresistant pediatric ALL. The lead compound (1) was synergistic when used in combination with the
glucocorticoids, dexamethasone or prednisolone. Synergy was observed in a range of dexamethasone-
resistant xenografts representative of B-cell precursor ALL (BCP-ALL) and T-cell ALL. We describe here
the synthesis of twenty compounds and biological evaluation of thirty two molecules that explore the
structure–activity relationships (SAR) of this novel class of glucocorticoid sensitizing compounds. SAR
analysis has identified that the most effective dexamethasone sensitizers contain a thioimidazoline acet-
amide substructure with a large hydrophobic moiety on the acetamide.
Received 18th April 2015,
Accepted 23rd April 2015
DOI: 10.1039/c5ob00779h
www.rsc.org/obc
Various strategies have been adopted to identify drugs that
reverse glucocorticoid resistance. Using a gene expression pro-
Introduction
Acute lymphoblastic leukemia (ALL) is the most common pedi- filing approach, the mTOR inhibitor, rapamycin, was identi-
atric cancer.1 Although five year survival rates are approaching fied as a glucocorticoid sensitizer.7 The BCL-2 antagonist,
90%, pediatric ALL remains one of the most common causes obatoclax, was shown to overcome glucocorticoid resistance as
of death from disease in children due to its high incidence.2 the BCL-2 family plays an essential role in regulating gluco-
Treatment of ALL consists of three phases of chemotherapy: corticoid induced cell death.8,9 AKT inhibition was also shown
remission induction, intensification, and continuation to restore glucocorticoid receptor translocation to the nucleus
therapy, administered over 2 to 2.5 years.3 Glucocorticoids, in resistant T-cell ALL,10 and the AKT inhibitor, MK2206, was
such as dexamethasone and prednisolone, are frequently used identified as a glucocorticoid sensitizer. We have previously
in all phases and are critical components of remission induc- established primary biopsies from pediatric ALL patients as
tion therapy protocols.4 Glucocorticoids are also used as a xenografts in immune deficient mice, and reported that their
prognostic indicator, where resistance to initial glucocorticoid in vivo and ex vivo dexamethasone sensitivity correlated with
therapy is predictive of poor outcome.5 As resistance to gluco- patient outcome.11,12 Using this clinically relevant pediatric
corticoids is common at relapse, its pharmacologic reversal ALL xenograft model, we have shown that dexamethasone
may lead to improved outcomes for children with intrinsic/ resistance can be partially reversed both ex vivo and in vivo,
acquired glucocorticoid resistant ALL.6
with the histone deacetylase inhibitor vorinostat, or the
sunitinib analog and receptor tyrosine kinase inhibitor
SU11657.13,14 However, none of these candidates were specifi-
cally developed as glucocorticoid sensitizers.
aSchool of Women’s and Children’s Health, UNSW Australia, Sydney 2052, Australia.
E-mail: rlock@ccia.unsw.edu.au
bSchool of Chemistry, UNSW Australia, Sydney 2052, Australia.
To identify a glucocorticoid sensitizer specifically designed
to reverse dexamethasone resistance,
a high-throughput
E-mail: s.mcalpine@unsw.edu.au
screening (HTS) assay was performed using the pediatric ALL
xenograft, ALL-19.15 The xenograft, ALL-19, was derived from
an aggressive and chemoresistant pediatric ALL that induced
early fatality in the patient.11,12 ALL-19 exhibits dexametha-
sone resistance both ex vivo and in vivo and is representative of
cChildren’s Cancer Institute, Lowy Cancer Research Center, UNSW Australia, Sydney
2052, Australia
dBioanalytical Mass Spectrometery Facility, UNSW Australia, Sydney 2052, Australia
†Electronic supplementary information (ESI) available: Additional biological
data and characterization data are available. See DOI: 10.1039/c5ob00779h
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Fig. 1 Novel dexamethasone sensitizers identified from HTS of human
leukemia xenografts. Thioimidazoline substructure shown in red.
the most common pediatric ALL subtype, B-cell precursor ALL
(BCP-ALL).11,12 A 40 000 compound HTS assay identified four
thioimidazoline containing compounds (compounds 1–4) that
overcame dexamethasone resistance in ALL-19 cells (Fig. 1).15
However, not all thioimidazoline containing compounds are
dexamethasone sensitizers, since an additional twenty four
compounds with this substructure were included in the 40 000
compounds screened, but did not show any dexamethasone
sensitizing effect (ESI Fig. 1†). Described here is the synthesis
of compounds 1 and 3 and twenty novel thioimidazoline con-
taining compounds that were based on 1–4. Biological evalu-
ation of these twenty synthesized compounds as well as twelve
purchased compounds produced a structure–activity relation-
ship (SAR) of this novel class of glucocorticoid sensitizing com-
pounds. Within these thirty two compounds, we identified
three that are more potent dexamethasone sensitizers than
compounds 1–4.
Fig. 2 Synergistic antileukemic effects of 1 and glucocorticoids ex vivo
against ALL xenograft cells. (a) ALL-19 cells were exposed to 1, Dex, or
both in combination at a fixed-ratio of concentrations for 48 h. Cell via-
bility was assessed by Alamar Blue assay. (b) ALL-19 cells were exposed
to 1, Pred, or both in combination at a fixed-ratio of concentrations for
48 h. Cell viability was assessed by Alamar Blue assay. (c) ALL-19 cells
were treated with 10 µM 1, 10 µM Dex, or both in combination. Cell via-
bility was determined by flow cytometry at various time points up to
72 h. Each data point represents the mean SEM of three independent
experiments.
Modifying the timing of compound addition confirmed
that dexamethasone sensitization was maximal when ALL-19
cells were treated simultaneously with 1 and dexamethasone
(ESI Fig. 4†). A time course experiment was performed by flow
cytometry with a fixed concentration of 10 µM 1 and dexa-
methasone, alone and in combination. The combination
caused a marked decrease in cell viability compared to the
single agents, with <15% viable cells remaining after 72 h
(Fig. 2c).
Results and discussion
To determine if 1 only sensitized ALL-19 cells to dexametha-
sone or whether it was broadly active, fixed-ratio combination
cytotoxicity assays were performed against an additional five
xenografts. The panel consisted of dexamethasone-sensitive
and -resistant xenografts, representative of BCP-ALL, T-cell
ALL, and Mixed Lineage Leukemia-rearranged ALL. Interestingly
1 was synergistic with dexamethasone in all dexamethasone-
resistant xenografts, but did not further potentiate the effects
of dexamethasone in dexamethasone-sensitive xenografts (ESI
Fig. 5 and Table 3†). Therefore, 1 is a glucocorticoid sensitizer
that is specific to dexamethasone-resistant ALL.
Compounds 5–16, (Fig. 3) were designed based on 1. Com-
pounds 5, 6, and 7 were chosen for testing as they represented
the pharmacophores of 1. Thus, testing these molecules will
determine whether these were the active components for sensi-
tizing the cells to dexamethasone. Compound 8 was a dimer-
ized variation of 5. Compounds 9–16 are analogs of 1, with
modifications to the acetamide side chain in order to evaluate
ALL-19 xenograft cells are highly resistant to dexamethasone
ex vivo, displaying minimal cell death when treated with 300
µM dexamethasone. To determine whether compounds were
synergistic with dexamethasone the Bliss-Additivity model was
used.16 Compound 1 was selected as the lead candidate as it
exerted the highest single level of synergism with dexametha-
sone of 1–4 (ESI Fig. 2 and Table 1†). Ex vivo fixed-ratio combi-
nation cytotoxicity assays showed that the 1/dexamethasone
combination was synergistic against ALL-19 (Fig. 2a and ESI
Table 1†). As single agents, 1 has modest activity (IC50 = 19.8
µM) and dexamethasone has minimal affect (IC50 > 40 µM) on
ALL-19 cells ex vivo, but when used in combination only 6.1
µM of each is required to inhibit 50% of cell survival relative to
the control (Fig. 2a). Almost identical results were observed
when the same assay was repeated using flow cytometry, a
direct measure of cell viability (ESI Fig. 3†). Synergy was also
observed with prednisolone (Fig. 2b and ESI Table 2†), hence 1
is a broad glucocorticoid sensitizer.
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Reacting 5 and the chloroacetyl 6 generated 1 in an overall
reasonable yield (31%). The crystal structure of 1 shows the
cycloheptane ring is distorted and adopts two conformations
in the final compound (Scheme 1b). The conformation of the
cycloheptane ring in the intermediate 6, is similar to one of
the conformations displayed in the final compound 1 (ESI
Fig. 6†). Synthesis of compounds 6–7, and 10–15 was accom-
plished using the same approach as described for
1
(Scheme 1a), with overall final yields ranging from 18–36%.
Compounds 2–5, 8, 9, and 16 were commercially available.
The ex vivo anti-leukemic activity of all sixteen compounds
was tested alone and in combination with dexamethasone
against ALL-19 (ESI Fig. 7†). The IC50 values were calculated
from the fixed-ratio combination cytotoxicity assays, where
compounds were tested at a 1 : 1 ratio with dexamethasone on
ALL-19 (Table 1). To determine whether compounds were
synergistic, additive or antagonistic with dexamethasone, the
Bliss-Additivity model was used.16 Deviation from Bliss-Addi-
tivity (BA) was calculated at each tested dose, where synergy is
defined as a positive deviation, additive effect as no deviation,
and antagonism as a negative deviation (Table 1 and ESI
Table 4†).16 While a synergistic combination effect is required,
the combination also needs to be potent to warrant further
development. A synergistic and potent compound was defined
as a compound that resulted in a combination IC50 less than
compound 1 (combination IC50 < 6.1 µM) and was synergistic
with dexamethasone.
The Compounds 5 and 6 had little effect as single agents
and were not synergistic in combination with dexamethasone,
which indicates that both the right and left hand sides of the
molecule are required for activity. Furthermore, 1–4 all contain
a thioimidazoline substructure, which alone has no dexa-
methasone sensitizing effect, thus, molecules containing just
this element are not necessarily going to be dexamethasone
sensitizers. Removing the ring system at the R position (7) or
addition of a second thioimidazoline group (8) results in the
Fig. 3 Compounds designed to investigate the SAR of 1.
the impact of changing that moiety. Compound 9 had the
methylene between the thiazole and the amide bond removed,
while 10 had two methylene units. Compounds 11–16 all had
variation to the side chains on the acetamide moiety.
Compound 1 was synthesized in order to produce reason-
able quantities of material for extensive testing. Chloroacetyl 6
was formed via amidation between 17 and 18 (Scheme 1a).
Table 1 Antileukemic activity of compounds alone and in combination
with dexamethasone ex vivo against ALL-19 xenograft cells
Cmpd
IC50 (μM)
Combo
IC50 (μM)
Median
BA
Combo
effect
Compound
1
2
3
4
5
6
7
8
19.8 1.2
15.4 3.5
23.7 1.4
12.9 0.3
>40
34.5 1.0
>40
>40
>40
>40
27.5 4.4
32.1 4.2
25.1 2.2
15.1 1.3
15.9 1.1
14.6 0.8
6.1 1.0
5.0 1.6
10.2 1.1
7.6 0.3
>40
>40
>40
>40
>40
>40
12.4 1.6
14.5 1.7
11.7 0.6
7.6 0.7
7.5 0.4
7.5 0.7
0.08
0.08
0.11
0.07
−0.03
0.00
0.05
0.04
−0.07
−0.15
0.11
0.07
0.15
0.08
0.11
0.05
Synergy
Synergy
Synergy
Synergy
Antag.
Additive
Synergy
Synergy
Antag.
9
10
11
12
13
14
Antag.
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Scheme 1 (a) General synthesis of dexamethasone sensitizers with sec-
ondary acetamide. (b) Crystal structure of compounds 1 and 6. *R = 15
amine from Table 1 for compounds 1, 3, 4, 6, 10–16.
16
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loss of single and combination activity, which indicates that
the R group is important, although this differs considerably
between 1–4.
Eliminating the carbon between the sulfur and carbonyl
groups (9) or the addition of an extra carbon (10) destroys
single agent activity and dexamethasone sensitizing effects.
Thus, one carbon between the sulfur and the carbonyl is
required to sensitize ALL cells to dexamethasone. While the
ring structures at the R position in structures 11–16 differ con-
siderably, all the structures were synergistic, although none
were as potent as compound 1.
Comparing 12 and 13, the addition of a second cyclohexane
improves both the single agent and combination activity. Com-
pounds 14 and 15 are enantiomers of each other and yet they
have the same activity, which suggests that the biological
target responsible for dexamethasone sensitization can accom-
modate a relatively large group at several orientations. These
data are supported by the fact that 4 and 13 are both relatively
potent while containing two large hydrophobic groups orien-
tated at different angles. It appears that the pocket accommo-
dating the hydrophobic side chain is quite large. Indeed, a
large hydrophobic pocket can accommodate an adamantly
moiety, as shown by compound 16’s impressive single agent
and combination activity, which is similar to that seen with
1–4, 14 and 15. Thus, these promising results suggest that
increasing the hydrophobic nature at the R position has posi-
tive effects on the combination potency and combination
effect.
The ex vivo half-lives of 1–4, dexamethasone and the
standard chemotherapy agent vincristine, were determined
using mouse liver microsomes (ESI Fig. 8 and Table 5†). While
dexamethasone showed the greatest stability with a half-life
>90 min, the half-lives of 3 and vincristine were comparable
(29.2 min and 25.4 min, respectively). The half-lives of 1, 2,
and 4 were considerably poorer than 3 (2.0 min, 9.4 min, and
8.4 min, respectively), hence 3 shows the greatest stability of
the analogues tested.
Fig. 4 Compounds designed to investigate the SAR of 3.
As compound 3 exerted the highest median synergy with
dexamethasone of compounds 1–4 in ALL-19 and also dis-
played the longest half-life, a second generation of glucocorti-
coid sensitizers were developed based on compound 3 and the
SAR developed from 1–16. Compound 3 was synthesized in
order to produce reasonable quantities of material for testing,
and to verify its activity was repeatable. We designed and syn-
thesized twelve molecules, where we varied the R1 and R2 posi-
tions on the acetamide (Fig. 4). Variation of R1 with R2 = H
generated 9 derivatives, compounds 19–27. Incorporation of
the isopropyl group based on 3 produced compounds 28–30
(Fig. 4). Finally, inclusion of a large steric methylcyclohexyl
moiety generated derivatives 31–34 (Fig. 4).
The synthesis of 3 and derivatives 28–34 involved the
addition of 18 refluxed with the appropriate alkyl bromide
35 (isopropyl or methylcyclohexyl) (Scheme 2). Chloroacetyl
chloride 17, is reacted with product 36, generating acetamide
37. Reaction between the acetamide and thioimidazoline
5 generates analogs 28–34 (38). The synthesis of compounds
Scheme 2 General synthesis of dexamethasone sensitizers with tertiary
acetamide. *R1 and R2 are from the amine shown in Table 2 for com-
pounds 3, 19–34.
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19–22 and 27, which contain a secondary acetamide, is
shown in Scheme 1. Compounds 23–26 were commercially
available.
Table 2 Antileukemic activity of compounds alone and in combination
with dexamethasone ex vivo against ALL-19 xenograft cells
Cmpd
IC50 (μM)
Combo
IC50 (μM)
Median
BA
Combo
effect
The ex vivo anti-leukemic activity of all sixteen compounds
and synthesized 3 were tested alone and in combination with
dexamethasone against the xenograft, ALL-19, using fixed-ratio
combination cytotoxicity assays (ESI Fig. 9†). The IC50 values
were calculated from the fixed-ratio combination cytotoxicity
assays, where compounds were tested at a 1 : 1 ratio with dexa-
methasone (Table 2). It is important to remember that in
dexamethasone-resistant xenografts, including ALL-19, dexa-
methasone is completely inactive by itself. Thus, only the com-
pounds or the combination will be active.
To determine whether compounds were synergistic, additive
or antagonistic with dexamethasone, the deviation from Bliss-
Additivity (BA) was calculated at each tested dose, where
synergy is defined as a positive deviation, additive effect as no
deviation, and antagonism as a negative deviation (Table 2
and ESI Table 6†).16
Comparing analogs 19–27, where R2 = H, showed that mole-
cules 19–24 and 27 were synergistic, while compounds 25 and
26 were antagonistic. However, none of the compounds met
the potency criteria of a combination IC50 below that of com-
pound 1 (IC50 < 6.1 µM). Compounds where R2 = an isopropyl
moiety (28–30) were more potent sensitizers than where R2 = H
(20–22). Specifically, 30 was synergistic and potent, with a com-
bination IC50 = 5.0 µM. The exception to this is when the aro-
matic ring is placed 4 carbons from the acetamide moiety (21
and 29), where the addition of an isopropyl group reduces
both potency and synergy with dexamethasone.
Compound
18.1 2.0
9.2 0.5
0.09
Synergy
>40
31.8 4.8
18.8 2.2
0.04
0.10
Synergy
Synergy
37.9 2.6
29.0 3.9
20.1 1.4
14.8 1.0
13.4 0.8
0.07
0.04
Synergy
Synergy
>40
21.5 1.9
9.7 2.2
>40
0.03
0.09
Synergy
Synergy
Antag.
23.2 2.4
>40
−0.02
>40
>40
−0.01
0.03
Antag.
>40
22.8 4.2
9.2 0.9
Synergy
Synergy
18.1 1.2
0.05
Evaluating compounds 31–34, where R2 = a methyl cyclo-
hexyl moiety, showed that all compounds were synergistic and
more potent sensitizers than where R2 = H (19–22) or R2 = an
isopropyl moiety (3, 28–30). Specifically, 32 and 34 where
synergistic and potent, with combination IC50 values of 4.7 µM
and 6.0 µM respectively. As observed in the isopropyl series,
compound 33, where R2 = a methyl cyclohexyl moiety and the
aromatic ring is placed 4 carbons from the acetamide moiety,
was the least potent sensitizer out of 31–34.
Analysis of the structure–activity relationships of these
molecules showed that the optimal distance between the aro-
matic ring (R1) and the acetamide moiety is 2 or 3 carbons,
where 32 is 2 carbons, but 30 and 34 are both 3 carbons. Mole-
cules possessing 0 or 4 carbons between the aromatic group
and the amide nitrogen (3, 19, 21, 23–27, 29, 31 and 33) all
had higher combination IC50 values than their 2–3 carbon
counterparts (20, 22, 28, 30, 32, 34). These data suggest that
the aromatic group is optimally interacting with the biological
target when placed 2–3 carbons away from the acetamide.
Furthermore, having a tertiary acetamide, compounds 28–34,
generated more potent compounds than molecules that were
secondary acetamides. Interestingly, the secondary acetamides
showed that the electronegative substituent F or the electron
donating substituent methyl, do not enhance the potency of
the molecule. However, a Cl substituent appears to play an
important role in binding to the target.
22.4 1.3
11.3 1.4
15.4 0.1
5.0 0.6
0.00
0.08
Additive
Synergy
15.0 0.7
9.5 1.3
6.6 0.5
4.7 0.3
7.5 0.5
6.0 0.1
0.12
0.10
0.07
0.03
Synergy
Synergy
Synergy
Synergy
14.2 0.4
10.5 1.0
While various strategies have been used to identify com-
pounds that reverse glucocorticoid resistance, including bioin-
formatics and testing candidates based on mechanism of
action, we designed an unbiased HTS assay which identified
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sone sensitizing effect and the thioimidazoline acetamide
substructure cannot be altered without losing dexamethasone
sensitizing effect. Furthermore, we have identified three mole-
cules from the series, 30, 32 and 34, that are synergistic and
more potent than 1. Our data suggest that increasing the
hydrophobic bulk at positions R1 and R2 decreases the IC50
value when used in combination with dexamethasone. Future
development of this novel class of dexamethasone sensitizers
will focus on defining the mechanism by which these mole-
cules function as glucocorticoid sensitizers.
Experimental section
General biological procedures
Cell culture tested, water-insoluble dexamethasone and predni-
solone were used in all experiments (Sigma-Aldrich, Castle
Hill, Australia). Cell viability was determined using ViaCount
(Merck Millipore, Billerica, MA) or Resazurin reagent (aka
Alamar Blue). Flow cytometry was performed using a Guava
easyCyte flow cytometer (Merck Millipore, Billerica, MA). Com-
pounds 1 to 4 were identified from a high-throughput screen
of a random selection of 40 000 compounds from the Austra-
lian Cancer Research Foundation Drug Discovery Centre for
Childhood Cancer diversity library.15
Fig. 5 (a) Novel dexamethasone sensitizer identified from HTS of the
T-cell ALL cell line, CUTLL1.17 (b) ALL-19 xenograft cells were exposed
to J9, Dex or both in combination at a fixed-ratio of concentrations for
48 h. Cell viability was assessed by Alamar Blue assay. (c) ALL-31 xeno-
graft cells were exposed to J9, Dex, or both in combination at a fixed-
ratio of concentrations for 48 h. Cell viability was assessed by Alamar
Blue assay. Each data point represents the mean SEM of three inde-
pendent experiments.
Ex vivo cell culture
The development and characterization of a series of pediatric
ALL xenografts derived from patient biopsies have been pre-
viously described.11 All assays were performed using myco-
plasma-free and validated stocks of xenograft cells. For
all experiments described in this manuscript, xenograft
cells were retrieved from cryostorage and resuspended in
RPMI-1640 medium (Invitrogen Life Technologies, Gaithers-
burg, MD) supplemented with 10% fetal bovine serum (FBS;
Invitrogen Life Technologies), penicillin (100 U mL−1), strepto-
mycin (100 µg mL−1), and L-glutamine (2 mM) (complete
RPMI). Cells were centrifuged at 490g for 5 minutes, aspirated,
and washed with complete RPMI. The cells were resuspended
in QBSF-60 medium (Quality Biological, Gaithersburg, MD)
supplemented with Flt-3 ligand (20 ng mL−1; Amgen, Thou-
sand Oaks, CA), penicillin (100 U mL−1), streptomycin (100 µg
mL−1), and L-glutamine (2 mM) (complete QBSF) at a cell con-
centration previously optimized for each xenograft (1–5 × 106
cells mL−1). Viability was determined by the exclusion of 0.2%
trypan blue (Sigma-Aldrich). For all experiments, cells were
seeded and equilibrated at 37 °C, 5% CO2, for 12–16 h prior to
drug treatment.
this novel class of thioimidazoline-based glucocorticoid sensi-
tizers. Cantley et al., performed an HTS assay in the T-cell ALL
cell line, CUTLL1, to identify the dexamethasone sensitizer, J9
(Fig. 5a).17 J9 was able to reverse dexamethasone resistance in
CUTLL1 cells by upregulating the glucocorticoid receptor.17
We tested J9 in a panel of xenografts representative of dexa-
methasone–resistant T-cell ALL (ALL-31, ETP-2), dexametha-
sone-resistant BCP-ALL (ALL-19, ALL-50), and dexamethasone-
sensitive T-cell and BCP-ALL (ALL-16 and ALL-54, respectively),
however no sensitizing effect was observed (Fig. 5b and c,
Supple. Figure 10, Supple. Table 7). These conflicting results
are likely to be due to the experimental models employed.
ALL cell lines undergo multiple passages and transformations
in vitro, this process appears to select for mutations that are
infrequently observed in primary patient cells.18,19 ALL xeno-
graft cells are never passaged in vitro and harvested cells
remain highly representative of the immunophenotypes and
gene expression profiles observed in the original patient
sample.11,12,20
Combination cytotoxicity assays
Conclusion
Xenograft cells were retrieved from cryostorage, washed with
In summary, we have identified a novel class of dexametha- complete RPMI and resuspended in complete QBSF at a cell
sone sensitizing compounds that contain a thioimidazoline concentration previously optimized for each xenograft (1–5 ×
group. Compound 1 is a glucocorticoid sensitizer that acts 106 cells mL−1), and 100 µL was seeded in 96-well clear,
specifically on glucocorticoid-resistant ALL. SAR analysis indi- U-bottom tissue culture treated plates (In Vitro Technologies,
cates that thioimidazoline alone does not have any dexametha- VIC, Australia). Plates were equilibrated at 37 °C, 5% CO2, for
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12–16 h prior to compound treatment. Compounds were seri- some solution was immediately added to acetonitrile (1 : 1
ally diluted in complete QBSF medium and added in triplicate dilution) and incubated at 0 °C (0 min time point).
wells. Cells were treated with compound and dexamethasone/
The remaining compound/microsome solution was incu-
prednisolone simultaneously at a fixed-ratio of concentrations bated at 37 °C and samples were taken, diluted with aceto-
1
4
1
2
corresponding to , , 1, 2 and 4 times the IC50 values for each nitrile (1 : 1) and incubated at 0 °C at the following time
drug independently and in combination. Where the IC50 points; 5 min, 15 min, 30 min, 60 min and 90 min. Each time
values for each compound and dexamethasone were >10 µM, point sample was centrifuged at 10 000g for 3 minutes at 0 °C
cells were treated with the following concentrations; 2.5 µM, and the supernatant was analyzed by mass spectroscopy. Com-
5 µM, 10 µM, 20 µM and 40 µM, independently and in combi- pound stability in liver microsomes over time was calculated
nation. Following 48 h incubation at 37 °C, 5% CO2, cell viabi- by plotting the area under the curve for each time point as a
lity was assessed by mitochondrial activity assay (Resazurin percentage of the 0 min time point. Results presented are the
cell viability assay, 6 h incubation) or by flow cytometry (Via- mean standard error of the mean (SEM) of a minimum of 3
Count reagent). Cell viability was calculated as a percentage of independent experiments. Half-life values were calculated
untreated controls. Results presented are the mean standard from one phase exponential decay curves.
error of the mean (SEM) of a minimum of 3 independent
experiments. IC50 values were calculated from cumulative sur-
General chemical procedures
vival curves.
All chemical reagents and purchased compounds were used
without further purification (Sigma-Aldrich, Enamine Ltd,
Princeton BioMolecular Research Inc.). All reactions were per-
Calculation of combination effect
To determine whether compounds were synergistic, additive or formed with anhydrous solvents under N2 atmosphere. Reac-
antagonistic with dexamethasone/prednisolone, the Bliss- tions were monitored by thin-layer chromatography (TLC)
Additivity model was used.16 The Bliss-Additivity model pre- using silica coated aluminium plates (250 µM Whatman®,
dicts the additive effect for two single compounds (A and B) at 4861-820) and visualized with UV light (λ = 254 nm) and the
a single concentration as;
developing agents; potassium permanganate and ninhydrin.
Solvent was removed in vacuo using a Buchi RE121 rotatory
evaporator. Flash chromatography was performed using
Davisil® silica gel (60 Å, particle size 40–63 μM).
Reversed-phase HPLC purification was performed on a Shi-
madzu Prominence High-performance LCMS 2010EV system
(Phenomenex® Jupiter C18 column, 4 μm, 250 × 10 mm). The
Mobile phase was composed of deionised water (solvent A),
and HPLC grade methanol (solvent B). The flow rate was 2 mL
min−1 and the following elution gradient was employed;
0–10 min, 100% solvent A; 10–25 min, gradient increase from
0% solvent B to 100% solvent B; 25–35 min, 100% solvent B;
Bliss-Additive effect ¼ ðFaA þ FaBÞ ꢀ ðFaA ꢁ FaBÞ
where, FaA is the fraction of cells affected by compound A
alone at a single concentration, and FaB is the fraction of cells
affected by compound B alone at a single concentration.
To determine the effect of the combination (C) at a single
concentration, the deviation from the calculated Bliss-Additive
effect is calculated as;
Deviation from Bliss-Additivity ðBAÞ
¼ ðexperimental FaCÞ ꢀ ðcalculated Bliss-Additive FaCÞ
where, ‘experimental FaC’ is the fraction of cells affected by 35–55 min, gradient decrease from 100% solvent B to 0%
the combination of A and B at a single concentration, and
‘calculated Bliss-Additive FaC’ is the calculated additive effect
of A and B at a single concentration.
solvent B.
LC/MS analysis was performed on a Shimadzu Prominence
High-performance LCMS 2010EV system (Waters Symmetry®
Deviation from Bliss-Additivity (BA) was calculated at each
C18 column, 3.5 μm, 4.6 × 75 mm) connected to a Shimadzu
tested dose, where synergy is defined as a positive deviation, LCMS 2010EV mass spectrometer running in positive electro-
additive effect as no deviation, and antagonism as a negative spray ionization (ESI+) mode. The Mobile phase was composed
deviation.
of deionised water with 0.1% (v/v) formic acid (solvent A), and
HPLC grade acetonitrile with 0.1% (v/v) formic acid (solvent
B). The flow rate was 0.5 mL min−1 and the following elution
Half-life assays
Half-life assays were performed to determine compound stabi- gradient was employed; 0–4 min, gradient increase from 30%
lity. Compounds were tested individually ex vivo in liver micro- solvent B to 100% solvent B; 4–12 min, 100% solvent B;
somes. Compound (final concentration 1 µM) was equilibrated 12–16 min, gradient decrease from 100% solvent B to 30%
at 37 °C for 5 min in purified water (Gibco Ultrapure distilled solvent B.
water) supplemented with 20% potassium phosphate (0.5 M,
HRMS analysis was performed using a Thermo LTQ Orbi-
pH 7.4), 5% NADPH Regenerating System Solution A (BD Bio- trap XL ESI/APCI with UPLC system at the Bioanalytical Mass
sciences, catalogue number 451220), and 1% NADPH Regener- Spectrometry Facility within the Mark Wainwright Analytical
ating System Solution B (BD Biosciences, catalogue number Centre at the University of New South Wales. NMR spectra
451200). Liver microsomes (final concentration 0.5 mg mL−1
,
were performed at ∼298 K on a Bruker Avance III 300 MHz
BD Biosciences, catalogue number 452701) were added to the spectrometer. Chemical shifts (δ) were reported in ppm and
compound solution and a portion of the compound/micro- were calibrated with the residual solvent resonance.
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To perform X-ray crystallography, suitable single crystals of (s, 2H), 3.99 (s, 4H), 4.87 (m, 1H, J = 6.69 Hz), 7.14–7.24 (m,
1 and 6 were selected under a polarizing microscope (Leica 2H), 7.46–7.55 (m, 3H), 11.07 (br, 2H). 13C NMR (CDCl3,
M165Z), mounted on a MicroMount (MiTeGen, USA) consist- 300 MHz) δ (ppm) 20.64, 33.64, 45.85, 48.01, 129.65, 129.69,
ing of a thin polymer tip with a wicking aperture. The X-ray 130.22, 136.46, 167.45, 170.85. LCMS(ESI): calcd for
diffraction measurements were performed on a Bruker kappa- C14H20N3OS+ [M+] = 278.1322, found 278.30.
II CCD diffractometer at 150 K, employing a IµS Incoatec
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-benzhydrylacetamide
Microfocus Source with Mo-Kα radiation (λ = 0.710723 Å). A (4). Purchased from Enamine Ltd, Ukraine, catalogue number
single crystal, mounted on the goniometer using cryo loops for T6256376. LCMS(ESI): calcd for C18H20N3OS+ [M+] = 326.1322,
intensity measurements, was coated with paraffin oil and then found 326.30.
quickly transferred to the cold stream using an Oxford Cryo
Imidazolidine-2-thione (5). Purchased from Sigma-Aldrich,
stream attachment. Symmetry related absorption corrections Castle Hill, Australia catalogue number 03940. 1H NMR
using the program SADABS (Bruker, AXS Inc., Wisconsin, USA, ((CD3)2SO, 300 MHz) δ (ppm) 3.48 (s, 4H), 7.93 (br, 2H). LCMS
2001) were applied and the data were corrected for Lorentz (ESI): calcd for C3H6N2S [M] = 102.0252, found 102.90.
and polarisation effects using Bruker APEX2 software (Bruker,
1,4-Bis((4,5-dihydro-1H-imidazol-2-yl)thio)butane (8). Pur-
AXS Inc., Wisconsin, USA, 2007). The structure was solved by chased from Sigma-Aldrich, Castle Hill, Australia, catalogue
direct methods and the full-matrix least-square refinement number CCA001895. LCMS(ESI): calcd for C10H20N4S2+ [M+] =
was carried out using XL in Olex2.21,22 The non-hydrogen 260.1118, found [1/2 M+] 130.30.
atoms were refined anisotropically. The molecular graphic was
generated using program Olex2.22
S-(4,5-Dihydro-1H-imidazol-2-yl)cyclohexylthiocarbamate (9).
Purchased from Sigma-Aldrich, Castle Hill, Australia, catalogue
MS analysis of half-life assay samples was performed using number CCA002504. LCMS(ESI): calcd for C10H17N3OS [M] =
a ThermoFisher Scientific Quantum Access mass spectrometer 227.1085, found 227.90.
and Accela UHPLC pump with a CTC Analytics PAL auto-
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N((3s,5s,7s)adamanta-
sampler at the Bioanalytical Mass Spectrometry Facility within nyl)lacetamide (16). Purchased from Enamine Ltd, Ukraine,
the Mark Wainwright Analytical Centre at the University of catalogue number Z48834237. LCMS(ESI): calcd for
New South Wales. Compounds were quantified using ultra- C11H20N3OS+ [M+] = 294.1635, found 294.00.
high performance liquid chromatography coupled to mass
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(4-fluorophenyl)-
spectrometry. Separation was performed on a Phenomenex acetamide (23). Purchased from Enamine Ltd, Ukraine, cata-
Kinetix Column (2.1 × 50 mm) held at 30 degrees C using a logue number Z48834313. LCMS(ESI): calculated for
5 minute gradient of 0.1% formic acid in water vs. acetonitrile. C11H13FN3OS+ [M+] = 254.0758, found 254.30.
Compounds were detected in the Selected Reaction Monitoring N-(4-Chlorophenyl)-2-((4,5-dihydro-1H-imidazol-2-yl)thio)-acet-
mode with transitions, polarity and instrument source con- amide (24). Purchased from Vitas-M Laboratory Ltd, the Neth-
ditions being optimized for each compound before analysis.
erlands, catalogue number STL112120). LCMS(ESI): calculated
for C11H13ClN3OS+ [M+] = 270.0462, found 270.25.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(p-tolyl)acetamide
Purchased compounds 1–5, 8, 9, 16, 23–26 and J9
All purchased compounds were used without further (25). Purchased from Chembridge, San Diego, CA, and catalo-
purification. gue number 9038095. LCMS(ESI): calculated for C12H16N3OS+
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-cycloheptylacetamide [M+] = 250.1009, found 250.35.
(1). Purchased from Enamine Ltd, Ukraine, catalogue number
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(o-tolyl)acetamide
T0511-1822. 1H NMR (CDCl3, 300 MHz) δ (ppm) 1.34–1.77 (26). Purchased from Sigma-Aldrich, Castle Hill, Australia,
(m, 10H), 1.83–1.99 (m, 2H), 3.73–3.88 (m, 1H), 3.97 (s, 4H), catalogue number L120413. LCMS(ESI): calculated for
4.10 (s, 2H), 9.25 (d, 1H, J = 7.68 Hz) 11.13 (s, 2H). 13C NMR C12H16N3OS+ [M+] = 250.1009, found 249.95.
(CDCl3, 300 MHz) δ (ppm) 24.17, 28.00, 33.45, 34.15, 45.03,
52.06, 167.82, 172.16. LCMS(ESI): calcd for C12H22N3OS+ catalogue number RGNCY_0013. 1H NMR ((CD3)2SO,
[M+] = 256.1478, found 256.30. 300 MHz) δ (ppm) 0.88–0.96 (m, 2H), 1.04–1.10 (m, 2H),
J9. Purchased from Reagency Pty Ltd, Victoria, Australia,
2-(((4,5-Dihydro-1H-imidazol-2-yl)thio)methyl)benzo[d]thiazole 1.87–1.98 (m, 1H), 6.67 (s, 1H), 7.43–7.47 (m, 2H), 8.08 (s, 1H),
(2). Purchased from Enamine Ltd, Ukraine, catalogue number 8.59–8.64 (m, 2H). 13C NMR ((CD3)2SO, 300 MHz) δ (ppm)
1
T0512-8364. H NMR (CD3OD, 300 MHz) δ (ppm) 4.00 (s, 4H), 11.05, 14.33, 39.99, 120.73, 124.14, 145.11, 150.18, 157.67,
4.93 (s, 2H), 7.21 (d, 2H, J = 8.02 Hz), 7.70 d, 2H, J = 7.67 Hz) 163.55, 168.45. LCMS(ESI): calcd for C12H12N4 [M + H] =
13C NMR (CD3OD, 300 MHz) δ (ppm) 13.02, 19.89, 22.29, 213.1142, found 213.30. HRMS(ESI): calcd for C12H12N4 [M +
31.34, 31.74, 45.46, 121.76, 122.59, 125.54, 125.88, 126.53, H] = 213.1142, found 213.1134.
128.39, 140.24, 142.18, 152.41. LCMS(ESI): calcd for
General procedure for synthesis of intermediate 6 (Scheme 1)
C11H12N3S2+ [M+] = 250.0467, found 250.20.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenyl- The appropriate primary amine (18, 1 equiv., 7 mmol) was
acetamide (3). Purchased from Princeton BioMolecular added drop wise to a mixture of triethylamine (1.2 equiv.,
Research Inc., NJ, USA, catalogue number OSSK_003145. 1H 8.4 mmol) in dichloromethane (4.5 mL) under nitrogen atmos-
NMR (CDCl3, 300 MHz) δ (ppm) 1.11 (d, 6H, J = 6.62 Hz), 3.60 phere at 0 °C. Chloroacetyl chloride (17, 1.2 equiv., 8.4 mmol)
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was added drop wise at 0 °C, then the reaction mixture was 7.77 Hz) 11.15 (s, 2H). 13C NMR (CDCl3, 300 MHz) δ (ppm)
stirred at room temperature for 20 h. The reaction mixture was 24.19, 28.02, 33.45, 34.15, 45.04, 52.09, 167.85, 172.21. HRMS
concentrated in vacuo and the residue washed with ice water (ESI): calcd for C12H22N3OS+ [M + H] = 256.1478, found
(3 × 20 mL) and filtered. The solid residue was purified by 256.1481.
flash chromatography on silica gel.
Synthesis of compound 3
General procedure for synthesis of intermediate 36 (Scheme 2)
N-Isopropylaniline (intermediate 3–36). Aniline (18, 3 mL,
The appropriate primary amine (18, 1 equiv., 30 mmol) was
30 mmol), isopropyl bromide (35, 3.4 mL, 40 mmol), aceto-
added drop wise to a mixture of the appropriate alkyl bromide
(35, 1.1 equiv., 33 mmol) in acetonitrile (30 mL) under nitro-
gen atmosphere. The reaction mixture was refluxed for 1–5 h,
nitrile (30 mL). Crude yield 3.48 g, 85%. LCMS: calcd for
C9H13N+ [M + H] = 135.10, found 134.90.
2-Chloro-N-isopropyl-N-phenylacetamide
(intermediate
then cooled to room temperature. The reaction mixture was
washed with sodium hydrogen carbonate and extracted with
dichloromethane (3 × 20 mL). The organic layers were com-
bined and dried over Na2SO4, filtered, and the solvent evapor-
ated under reduced pressure. The crude product was used for
the synthesis of intermediate 37 without further purification.
3–37). N-Isopropylaniline (3–36, 2 g, 15 mmol), triethylamine
(1.8 g, 18 mmol), dichloromethane (9.9 mL), chloroacetyl
chloride (17, 2 g, 18 mmol). Purified by flash chromatography
on silica gel (EtOAc : Hexane, 1 : 4) to yield pure 3–37 (pale
yellow solid, 2.71 g, 85% yield). LCMS: calcd for C11H14ClNO+
[M + H] = 211.08, found: 211.10.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenyl-
acetamide (3). 2-Chloro-N-isopropyl-N-phenylacetamide (3–37,
1 g, 5.0 mmol), N,N′-ethylenethiourea (5, 0.58 g, 5.6 mmol),
acetonitrile (50 mL). Purified by recrystallization (CH2Cl2 and
hexane) to yield compound 3 (white solid, 73% purified yield).
1H NMR (CDCl3, 300 MHz) δ (ppm) 1.08 (s, 3H), 1.11 (s, 3H),
3.78 (s, 2H), 3.93 (s, 4H), 4.89 (5ry, 1H), 7.31–7.527 (m, 5HAr).
13C NMR (CDCl3, 300 MHz) δ (ppm) 20.66, 34.41, 45.77, 47.91,
129.53, 129.80, 130.10, 136.52, 166.88, 170.56. LCMS calcd for
C14H19N3OS+ [M + H] = 278.12, found: 278.35.
General procedure for synthesis of intermediate 37 (Scheme 2)
The appropriate secondary amine (36, 1 equiv., 15 mmol) was
added drop wise to a mixture of triethylamine (1.2 equiv.,
18 mmol) in dichloromethane (15 mL) under nitrogen atmos-
phere at 0 °C. Chloroacetyl chloride (17, 1.2 equiv., 18 mmol)
was added drop wise at 0 °C, then the reaction mixture was
stirred at room temperature for 20 h. The reaction mixture was
concentrated in vacuo and the residue washed with ice water (3
× 20 mL) and filtered. The solid residue was purified by flash
chromatography on silica gel.
Synthesis of compound 7
General procedure for synthesis of final compounds (Schemes
1 and 2)
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)acetic acid (7). 2-((4,5-
Dihydro-1H-imidazol-2-yl)thio)acetic acid was synthesized based
on the method of Kushakova et al.23 Chloroacetic acid
(720.1 mg, 7.6 mmol) and N,N′-ethylenethiourea (5, 516.2 mg,
5.1 mmol) were stirred at room temperature for 8 h in acetone
(10 mL). The precipitate was concentrated in vacuo and puri-
fied by recrystallization (CH2Cl2 and hexane) to yield com-
The appropriate chloro acetamide (6 or 37, 1 equiv., 0.1 M) was
stirred with N,N′-ethylenethiourea (5, 1.5 equiv.) in acetonitrile
under nitrogen atmosphere for 72 h. The precipitate was
concentrated in vacuo and purified by either HPLC or
recrystallization.
1
Synthesis of compound 1
pound 7 (white solid, 228.4 mg, 28% purified yield). H NMR
((CD3)2SO, 300 MHz) δ (ppm) 3.82 (s, 4H), 4.32 (s, 2H), 10.64
(br, 2H). 13C NMR ((CD3)2SO, 300 MHz) δ (ppm) 33.79, 45.13,
168.31, 168.50. HRMS(ESI): calcd for C5H9N2O2S+ [M + H] =
161.0379, found 161.0377.
2-Chloro-N-cycloheptylacetamide (intermediate 1–6, com-
pound 6). Cycloheptylamine (18, 0.89 mL, 7 mmol), triethyl-
amine (1.17 mL, 8.4 mmol), dichloromethane (4.5 mL),
chloroacetyl chloride (17, 0.66 mL, 8.4 mmol). Purified by
flash chromatography on silica gel (EtOAc : Hexane, 1 : 4) to
yield pure 1–6 (pale yellow solid, 1163.4 mg, 88% yield). 1H
NMR (CDCl3, 300 MHz) δ (ppm) 1.40–1.72 (m, 10H), 1.86–2.02
Synthesis of compound 10
3-Chloro-N-cycloheptylpropanamide (intermediate 10-6).
(m, 2H), 3.90–4.04 (m, 1H), 4.02 (s, 2H), 6.50 (br, 1H). 13C Cycloheptylamine (18, 0.89 mL,
7
mmol), triethylamine
NMR (CDCl3, 300 MHz) δ (ppm) 23.97, 27.93, 34.82, 42.77, (1.17 mL, 8.4 mmol), dichloromethane (4.1 mL), 3-chloro-
50.88, 164.59. propionyl chloride (17, 0.80 mL, 8.4 mmol). Purified by flash
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-cycloheptylaceta- chromatography on silica gel (EtOAc : Hexane, 1 : 4) to yield
mide (1). 2-Chloro-N-cycloheptylacetamide (1–6, 409.8 mg, pure 10-6 (clear oil, 1012.5 mg, 71%). 1H NMR (CDCl3,
2.2 mmol), N,N′-ethylenethiourea (5, 326.4 mg, 3.2 mmol), 300 MHz) δ (ppm) 1.34–1.71 (m, 10H), 1.84–2.01 (m, 2H), 2.58
acetonitrile (21 mL). Purified by recrystallization (CH2Cl2 and (t, 2H, J = 6.50 Hz), 3.78 (t, 2H, J = 6.50 Hz), 3.88–4.08 (m, 1H),
hexane) to yield compound 1 (white solid, 322.2 mg, 58% iso- 5.81 (br, 1H). 13C NMR (CD3OD, 300 MHz) δ (ppm) 23.90,
lated yield, 204.1 mg, 37% purified yield). 1H NMR (CDCl3, 27.88, 34.69, 38.6, 39.9, 50.49, 50.60, 124.92, 130.96, 165.33,
300 MHz) δ (ppm) 1.34–1.80 (m, 10H), 1.84–1.99 (m, 2H), 169.57. HRMS(ESI): calcd for C10H18ClNONa [M + Na] =
3.74–3.88 (m, 1H), 3.97 (s, 4H), 4.11 (s, 2H), 9.26 (d, 1H, J = 226.0975, found 226.0967.
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3-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-cycloheptylpropan- 1.54–1.65 (m, 1H), 1.72–1.98 (m, 5H), 3.59–3.74 (m, 1H), 4.00
amide (10). 3-Chloro-N-cycloheptylpropanamide
(10-6, (s, 4H), 4.13, (s, 2H), 9.24 (d, 1H, J = 7.95 Hz), 11.16 (s, 2H). 13
C
300.0 mg, 1.5 mmol), N,N′-ethylenethiourea (5, 225.7 mg, NMR (CDCl3, 300 MHz) δ (ppm) 24.75, 25.30, 32.03, 33.46,
2.2 mmol), acetonitrile (15 mL). Purified by HPLC to yield 45.83, 49.79, 168.21, 172.19. HRMS(ESI): calcd for
compound 10 (white solid, 143.6 mg, 36% isolated yield, 12% C11H20N3OS+ [M + H] = 242.1322, found 242.1319.
1
purified yield). H NMR (CDCl3, 300 MHz) δ (ppm) 1.38–1.75
(m, 10H), 1.83–1.97 (m, 2H), 2.91 (t, 2H, J = 6.74 Hz), 3.47
(t, 2H, J = 6.47 Hz), 3.81–3.93 (m, 1H), 3.99 (s, 4H), 8.10
(d, 1H, J = 7.84 Hz). 13C NMR (CDCl3, 300 MHz) δ (ppm) 24.19,
27.56, 28.02, 34.66, 36.96, 45.85, 51.48, 169.95, 170.74. HRMS
(ESI): calcd for C13H24N3OS+ [M + H] = 270.1635, found
270.1632.
Synthesis of compound 13
2-Chloro-N,N-dicyclohexylacetamide (intermediate 13-6).
Dicyclohexylamine (18, 1.39 mL, 7 mmol), triethylamine
(1.17 mL, 8.4 mmol), dichloromethane (4.5 mL), chloroacetyl
chloride (17, 0.66 mL, 8.4 mmol). Purified by flash chromato-
graphy on silica gel (EtOAc : Hexane, 1 : 4) to yield pure 13-6
(brown solid, 912.0 mg, 51% yield). 1H NMR (CD3OD,
300 MHz) δ (ppm) 1.09–1.96 (m, 18H), 2.41 (q, 2H, J = 12.12
Hz), 3.00–3.19 (m, 1H), 3.51–3.66 (m, 1H), 4.19 (s, 2H).
13C NMR (CD3OD, 300 MHz) δ (ppm) 26.20, 26.50, 26.76,
27.33, 30.55, 32.05, 44.09, 57.61, 60.35, 168.06. HRMS(ESI):
Synthesis of compound 11
2-Chloro-N-cyclopentylacetamide (intermediate 11-6). Cyclo-
pentylamine (18, 0.69 mL, 7 mmol), triethylamine (1.17 mL,
8.4 mmol), dichloromethane (4.5 mL), chloroacetyl chloride
(17, 0.66 mL, 8.4 mmol). Purified by flash chromatography on
silica gel (EtOAc : Hexane, 1 : 4) to yield pure 11-6 (pale orange
solid, 562.3 mg, 50% yield). 1H NMR (CDCl3, 300 MHz)
δ (ppm) 1.35–1.49 (m, 2H), 1.54–1.77 (m, 4H), 1.93–2.08 (m,
2H), 4.01 (s, 2H), 4.14–4.27 (m, 1H), 6.48 (br, 1H). 13C NMR
(CDCl3, 300 MHz) δ (ppm) 23.67, 32.91, 42.68, 51.54, 165.30.
HRMS(ESI): calcd for C7H12ClNONa [M + Na] = 184.0505,
found 184.0500.
calcd for
280.1439.
C14H24ClNONa [M + Na] = 280.1444, found
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N,N-dicyclohexyl-
acetamide (13). 2-Chloro-N,N-dicyclohexylacetamide (13-6,
300.0 mg, 1.2 mmol), N,N′-ethylenethiourea (5, 178.3 mg,
1.8 mmol), acetonitrile (18 mL). Purified by HPLC to yield
compound 13 (white solid, 288.1 mg, 77% isolated yield, 60%
1
purified yield). H NMR (CD3OD, 300 MHz) δ (ppm) 1.07–1.92
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-cyclopentylacetamide
(11). 2-Chloro-N-cyclopentylacetamide (11-6, 200.0 mg,
1.2 mmol), N,N′-ethylenethiourea (5, 326.4 mg, 189.6 mg,
1.9 mmol), acetonitrile (12 mL). Purified by recrystallization
(CH2Cl2 and hexane) to yield compound 11 (yellow crystals,
202.3 mg, 74% isolated yield, 180.4 mg, 66% purified yield).
1H NMR (CDCl3, 300 MHz) δ (ppm) 1.50–1.73 (m, 4H),
1.73–1.86 (m, 2H), 1.86–2.07 (m, 2H), 4.00 (s, 4H), 4.06–4.18
(m, 1H), 4.12 (s, 2H), 9.34 (d, 1H, J = 7.26 Hz), 11.16 (s, 2H).
(m, 18H), 3.36 (q, 2H, J = 11.61 Hz), 3.02–3.21 (m, 1H),
3.50–3.66 (m, 1H). 3.96 (s, 4H), 4.46 (s, 2H). 13C NMR (CD3OD,
300 MHz) δ (ppm) 26.14, 26.48, 26.61, 27.31, 30.77, 31.90,
39.16, 46.66, 57.75, 60.26, 166.21, 171.96. HRMS(ESI): calcd for
C17H30N3OS+ [M + H] = 324.2104, found 324.2103.
Synthesis of compound 14
(R)-2-Chloro-N-(1-cyclohexylethyl)acetamide (intermediate
13C NMR (CDCl3, 300 MHz) δ (ppm) 23.89, 32.39, 33.32, 45.84, 14-6). (S)-(+)-1-Cyclohexylethylamine (18, 1.04 mL, 7 mmol),
52.28, 168.58, 172.18. HRMS(ESI): calcd for C10H18N3OS+ [M + dichloromethane (4.5 mL), chloroacetyl chloride (17, 0.66 mL,
H] = 228.1165, found 228.1163.
8.4 mmol). Purified by flash chromatography on silica gel
(EtOAc : Hexane, 1 : 4) to yield pure 14-6 (pale orange solid,
828.2 mg, 58%). H NMR (CDCl3, 300 MHz) δ (ppm) 0.91–1.45
Synthesis of compound 12
1
2-Chloro-N-cyclohexylacetamide (intermediate 12-6). Cyclo- (m, 6H), 1.15 (d, 3H, J = 6.56 Hz), 1.63–1.84 (m, 5H), 3.80–3.96
hexylamine (18, 0.80 mL, 7 mmol), triethylamine (1.17 mL, (m, 1H), 4.07 (s, 2H), 6.42 (br, 1H). 13C NMR (CDCl3, 300 MHz)
8.4 mmol), dichloromethane (4.5 mL), chloroacetyl chloride δ (ppm) 17.73, 26.10, 26.32, 28.91, 28.99, 42.89, 50.00, 164.96.
(17, 0.66 mL, 8.4 mmol). Purified by flash chromatography on HRMS(ESI): calcd for C10H18ClNONa [M + Na] = 226.0975,
silica gel (EtOAc : Hexane, 1 : 4) to yield pure 12-6 (pale orange found 226.0971.
solid, 614.1 mg, 50% yield). 1H NMR (CDCl3, 300 MHz)
δ (ppm) 1.13–1.30 (m, 3H), 1.32–1.49 (m, 2H), 1.59–1.81 (m, ethyl)acetamide
(R)-2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(1-cyclohexyl-
(14). (R)-2-Chloro-N-(1-cyclohexylethyl)acet-
3H), 1.89–2.00 (m, 2H), 3.74–3.88 (m, 1H), 4.05 (s, 2H) 6.45 (br, amide (14-6, 400.0 mg, 2.0 mmol), N,N′-ethylenethiourea (5,
1H). 13C NMR (CDCl3, 300 MHz) δ (ppm) 24.71, 25.41, 32.80, 300.9 mg, 3.0 mmol), acetonitrile (20 mL). Purified by HPLC to
42.74, 48.65, 164.81. HRMS(ESI): calcd for C8H14ClNONa [M + yield compound 14 (white oil, 216.3 mg, 41% isolated yield,
Na] = 198.0662, found 198.0655.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-cyclohexylacetamide 0.90–1.32 (m, 4H), 1.18 (d, 3H, J = 7.00 Hz), 1.37–1.53 (m, 1H),
(12). 2-Chloro-N-cyclohexylacetamide (12-6, 300.0 mg, 1.60–1.90 (6H), 3.66–3.81 (m, 1H), 4.00 (s, 4H), 4.16 (s, 2H),
32% purified yield). 1H NMR (CDCl3, 300 MHz) δ (ppm)
1.7 mmol), N,N′-ethylenethiourea (5, 261.7 mg, 2.6 mmol), 9.13 (d, 1H, J = 8.95 Hz), 11.16 (br, 2H). 13C NMR (CDCl3,
acetonitrile (17 mL). Purified by HPLC to yield compound 12 300 MHz) δ (ppm) 17.20, 26.09, 26.13, 26.34, 29.10, 29.27,
(white oil, 247.0 mg, 60% isolated yield, 52% purified yield). 33.49, 42.66, 45.84, 51.17, 168.45, 172.17. HRMS(ESI): calcd for
1H NMR (CDCl3, 300 MHz) δ (ppm) 1.13–1.51 (m, 4H), C13H24N3OS+ [M + H] = 270.1635, found 270.1632.
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1
Synthesis of compound 15
81% yield). H NMR (CD3OD, 300 MHz) δ (ppm) 2.81 (t, 2H,
J = 7.43 Hz), 3.45 (t, 2H, J = 8.21 Hz), 3.99 (s, 2H), 7.15–7.32 (m,
5H). 13C NMR (CD3OD, 300 MHz) δ (ppm) 36.26, 42.34, 43.10,
127.40, 129.48, 129.78, 140.18, 169.14. HRMS(ESI): calcd for
C10H12ClNONa+ [M + Na] = 220.0505, found 220.0503.
(S)-2-Chloro-N-(1-cyclohexylethyl)acetamide
(intermediate
15-6). (R)-(−)-1-Cyclohexylethylamine (18, 1.04 mL, 7 mmol),
dichloromethane (4.5 mL), chloroacetyl chloride (17, 0.66 mL,
8.4 mmol). Purified by flash chromatography on silica gel
(EtOAc : Hexane, 1 : 4) to yield pure 15-6 (pale orange solid,
1036.3 mg, 73%). 1H NMR (CDCl3, 300 MHz) δ (ppm)
0.91–1.45 (m, 6H), 1.15 (d, 3H, J = 6.77 Hz), 1.64–1.84 (m, 5H),
3.81–3.96 (m, 1H), 4.07 (s, 2H), 6.42 (br, 1H). 13C NMR (CDCl3,
300 MHz) δ (ppm) 17.73, 26.10, 26.32, 28.91, 28.99, 42.89,
50.00, 164.96. HRMS(ESI): calcd for C10H18ClNONa [M + Na] =
226.0975, found 226.0971.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-phenethylacetamide
(20). 2-Chloro-N-phenethylacetamide
(20-6,
550.0
mg,
2.8 mmol), N,N′-ethylenethiourea (5, 430.0 mg, 4.2 mmol),
acetonitrile (28 mL). Purified HPLC to yield compound 20
(white solid, 500.5 mg, 76%). 1H NMR (300 MHz, CDCl3)
δ (ppm) 2.87 (t, J = 7.22 Hz, 2H), 3.52 ppm (t, J = 7.22 Hz, 2H),
3.91 (s, 2H), 4.00 (s, 4H), 7.23–7.35(m, 5HAr). 13C NMR
(300 MHz, CDCl3) δ (ppm) 32.81, 35.50, 43.81, 51.62, 126.52,
128.71, 137.93, 168.61, 171.14. LCMS: calcd for C13H17N3OS+
[M + H] = 264.11, found 264.00.
(S)-2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(1-cyclohexyl-
ethyl)acetamide
(15). (S)-2-Chloro-N-(1-cyclohexylethyl)acet-
amide (15-6, 400.0 mg, 2.0 mmol), N,N′-ethylenethiourea (5,
300.9 mg, 3.0 mmol), acetonitrile (20 mL). Purified by HPLC to
yield compound 15 (white oil, 253.9 mg, 48% isolated yield,
36% purified yield). 1H NMR (CDCl3, 300 MHz) δ (ppm)
0.91–1.33 (m, 4H), 1.17 (d, 3H, J = 6.80 Hz), 1.38–1.53 (m, 1H),
1.60–1.98 (6H), 3.65–3.80 (m, 1H), 3.99 (s, 4H), 4.16 (s, 2H),
9.12 (d, 1H, J = 8.70 Hz), 11.15 (br, 2H). 13C NMR (CDCl3,
300 MHz) δ (ppm) 17.20, 26.09, 26.12, 26.34, 29.10, 29.26,
33.49, 42.66, 45.84, 51.15, 168.42, 172.13. HRMS(ESI): calcd for
C13H24N3OS+ [M + H] = 270.1635, found 270.1631.
Synthesis of compound 21
2-Chloro-N-phenylbutylacetamide
(intermediate
21-6).
4-Phenylbutan-1-amine (18, 1 mL, 6 mmol), triethylamine
(1 mL, 7.6 mmol), dichloromethane (3.4 mL), chloroacetyl
chloride (17, 0.6 mL, 7.6 mmol). Purified by flash chromato-
graphy on silica gel (EtOAc : Hexane, 3 : 7) to yield pure 21-6
(990.0 mg, 87% yield). LCMS: calcd for C12H16ClNO+ [M + H] =
225.09, found 226.00.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(4-phenylbutyl)acet-
amide (21). 2-Chloro-N-phenylbutylacetamide (21-6, 910.0 mg,
4.0 mmol), N,N′-ethylenethiourea (5, 610.0 mg, 6.0 mmol),
acetonitrile (39 mL). Purified by recrystallization (CH2Cl2 and
hexane) to yield compound 21 (white solid, 790.0 mg, 76%).
1H NMR (300 MHz, CDCl3) δ (ppm) 1.65 (d, J = 6.99, 4H),
2.60–2.64 (t, J = 7.42, 2H), 3.26–3.28 (d, J = 6.24, 2H), 3.94 (s,
4H), 4.08 (s, 2H), 7.15–7.28 (m, 5HAr). 13C NMR (300 MHz,
CDCl3) δ (ppm) 28.50, 28.70, 33.45, 35.42, 40.10, 45.81, 125.74,
128.30, 128.40, 142.19, 168.98, 171.80. LCMS: calcd for
C15H21N3OS+ [M + H] = 292.14, found 292.35.
Synthesis of compound 19
2-Chloro-N-phenylacetamide (intermediate 19-6). Aniline
(18, 0.64 mL, 7 mmol), triethylamine (1.17 mL, 8.4 mmol),
dichloromethane (4.5 mL), chloroacetyl chloride (17, 0.66 mL,
8.4 mmol). Purified by flash chromatography on silica gel
(EtOAc : Hexane, 1 : 4) to yield pure 19-6 (dark green solid,
1
996.5 mg, 84% yield). H NMR (CDCl3, 300 MHz) δ (ppm) 4.18
(s, 2H), 7.17 (t, 1H, J = 7.51 Hz), 7.36 (t, 2H, J = 7.68 Hz), 7.55
(dd, 2H, J = 8.02 Hz, J = 1.02 Hz), 8.26 (br, 1H). 13C NMR
(CDCl3, 300 MHz) δ (ppm) 42.99, 120.25, 125.35, 129.23,
136.77, 163.94. HRMS(ESI): calcd for C8H8ClNO [M + H] =
170.0374, found 170.0368.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-phenylacetamide Synthesis of compound 22
(19). 2-Chloro-N-phenylacetamide (19-6, 300.0 mg, 1.8 mmol),
2-Chloro-N-(3,3-diphenylpropyl)acetamide (intermediate 22-
N,N′-ethylenethiourea (5, 271.0 mg, 2.7 mmol), acetonitrile
(18 mL). Purified by recrystallization (CH2Cl2 and hexane) to
yield compound 19 (white solid, 312.2 mg, 75% isolated yield,
83.5 mg, 20% purified yield). 1H NMR ((CD3)2SO, 300 MHz)
δ (ppm) 3.86 (s, 4H), 4.37 (s, 2H), 7.08 (t, 1H, J = 7.33 Hz), 7.33
(t, 2H, J = 7.33 Hz), 7.62 (d, 2H, J = 7.89 Hz), 10.46 (br, 2H),
10.82 (br, 1H). 13C NMR ((CD3)2SO, 300 MHz) δ (ppm) 35.10,
45.16, 119.19, 123.79, 128.82, 138.61, 164.36, 168.92. HRMS(ESI):
calcd for C11H14N3OS+ [M + H] = 236.0852, found 236.0851.
6). Diphenylpropylamine (18, 3 g, 14 mmol), triethylamine
(1.7 mL, 20 mmol), dichloromethane (7.5 mL), chloroacetyl
chloride (17, 1.7 mL, 20 mmol). Purified by flash chromato-
graphy on silica gel (EtOAc : Hexane, 1 : 1) to yield pure 22-6
(2.94 g, 84% yield). LCMS: calcd for C17H18ClNO+ [M + H] =
287.11, found 288.10.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(3,3-diphenylpropyl)-
acetamide
(22). 2-Chloro-N-(3,3-diphenylpropyl)acetamide
(22-6, 2.0 g, 7.0 mmol), N,N′-ethylenethiourea (5, 850.0 mg,
8.0 mmol), acetonitrile (70 mL). Purified by HPLC to yield
compound 22 (75% yield). 1H NMR (300 MHz, CDCl3) δ (ppm)
Synthesis of compound 20
2-Chloro-N-phenethylacetamide (intermediate 20-6). 2-Phenyl- 2.34 (q, J = 7.87 Hz, 2H), 3.12 (t, J = 7.14 Hz, 2H), 3.87 (s, 2H),
ethan-1-amine (18, 1 mL, 8 mmol), triethylamine (1.33 mL, 3.95 (s, 4H), 4.03 (t, J = 7.82 Hz, 1H), 7.13–7.35 (m, 10HAr). 13C
10 mmol), dichloromethane (4.8 mL), chloroacetyl chloride NMR (300 MHz, CDCl3) δ (ppm) 33.41, 34.40, 39.02, 45.79,
(17, 0.8 mL, 10 mmol). Purified by flash chromatography on 48.56, 126.32, 127.88, 128.53, 144.06, 169.02, 171.85. LCMS:
silica gel (EtOAc : Hexane, 1 : 4) to yield pure 20-6 (850.0 mg, calcd for C20H23N3OS+ [M + H] = 354.16, found 354.45.
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Synthesis of compound 27
(35, 1.3 mL, 14 mmol), acetonitrile (13 mL). Crude yield 3.76 g,
62%.
2-Chloro-N-(5,6,7,8-tetrahydronaphthalen-2-yl)acetamide
(intermediate 27-6). 5,6,7,8-Tetrahydro-2-naphthylamine (18,
1000 mg, 7.0 mmol), triethylamine (1.17 mL, 8.4 mmol),
dichloromethane (4.5 mL), chloroacetyl chloride (17, 0.66 mL,
8.4 mmol). Purified by flash chromatography on silica gel
(EtOAc : Hexane, 1 : 4) to yield pure 27-6 (black solid,
1406.3 mg, 90% yield). 1H NMR (CDCl3, 300 MHz) δ (ppm)
1.81 (m, 4H), 2.77 (d, 4H, J = 6.28 Hz), 4.19 (s, 2H), 7.06 (d, 1H,
J = 7.91 Hz), 7.22–7.30 (m, 2H), 8.17 (br, 1H). 13C NMR (CDCl3,
300 MHz) δ (ppm) 23.02, 23.15, 28.93, 29.49, 42.19, 117.74,
120.75, 129.64, 133.98, 134.36, 138.02, 163.69. HRMS(ESI):
calculated for C12H14ClNONa+ [M + Na] = 246.0662, found
246.0655.
2-Chloro-N-isopropyl-N-(4-phenylbutyl)acetamide
(inter-
mediate 29–37). N-Isopropyl-4-phenylbutan-1-amine (29–36,
2.0 g, 10 mmol), triethylamine (1.0 mL, 12 mmol), dichloro-
methane (5.4 mL), chloroacetyl chloride (17, 1 mL, 12 mmol).
Purified by flash chromatography on silica gel (EtOAc : Hexane,
1 : 1) to yield pure 29–37 (1.72 g, 85% yield). LCMS: calcd for
C15H22ClNO+ [M + H] = 267.14, found 268.50.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-(4-
phenylbutyl)acetamide (29). 2-Chloro-N-isopropyl-N-(4-phenyl-
butyl)acetamide (29–37, 800 mg, 2.3 mmol), N,N′-ethyle-
nethiourea (5, 370 mg, 3.6 mmol), acetonitrile (20 mL).
Purified by HPLC to yield compound 29 (white solid, 70% pur-
1
ified yield). H NMR (300 MHz, CDCl3) δ (ppm) 1.10–1.21 (m,
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(5,6,7,8-tetrahydro-
naphthalen-2-yl)acetamide (27). 2-Chloro-N-(5,6,7,8-tetrahydro-
2-naphthyl)acetamide (22-6, 400.0 mg, 1.8 mmol), N,N′-ethylene-
thiourea (5, 274.0 mg, 2.7 mmol), acetonitrile (18 mL). Purified
by HPLC to yield compound 27 (white solid, 420.3 mg, 81%
isolated yield, 153.9 mg, 30% purified yield). 1H NMR
((CD3)2SO, 300 MHz) δ (ppm) 1.71 (m, 4H), 2.66 (d, 4H, J =
5.37 Hz), 3.86 (s, 4H), 4.30 (s, 2H), 6.99 (d, 1H, J = 8.80 Hz),
7.22–7.39 (m, 2H), 10.38 (br, 2H), 10.59 (s, 1H). 13C NMR
((CD3)2SO, 300 MHz) δ (ppm) 23.14, 23.25, 28.71, 29.43, 35.51,
45.69, 117.32, 119.89, 129.59, 132.62, 136.44, 137.32, 164.56,
169.38. HRMS(ESI): calculated for C15H20N3OS+ [M + H] =
290.1322, found 290.1322.
6H), 1.60 (s, 4H), 2.61 (d, J = 7.22, 2H), 3.17 (s, 2H), 3.90 (s,
4H), 4.21 (s, 2H), 7.13–7.30 (m, 5HAr). 13C NMR (300 MHz,
CDCl3) δ (ppm) 20.12, 20.37, 21.26, 21.39, 29.16, 29.27, 35.61,
45.89, 125.72, 125.84, 125.99, 128.34, 166.67, 170.18. LCMS
calcd for C18H27N3OS+ [M + H] = 334.19, found 334.35.
Synthesis of compound 30
N-Isopropyl-3,3-diphenylpropan-1-amine (intermediate 30–36).
Diphenylpropylamine (18, 2.0 g,
9
mmol), isopropyl
bromide (35, 1.0 mL, 10 mmol), acetonitrile (18 mL). Crude
yield 2.2 g, 79%.
2-Chloro-N-(3,3-diphenylpropyl)-N-isopropylacetamide (inter-
mediate
30–37). N-Isopropyl-3,3-diphenylpropan-1-amine
Synthesis of compound 28
(30–36, 1.8 g, 7.1 mmol), triethylamine (1.1 mL, 8.5 mmol),
dichloromethane (3.5 mL), chloroacetyl chloride (17, 0.7 mL,
8.5 mmol). Purified by flash chromatography on silica gel
(EtOAc : Hexane, 3 : 2) to yield pure 30–37 (1.94 g, 83% yield).
LCMS: calcd for C20H24ClNO+ [M + H] = 392.15, found 393.20.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-(3,3-diphenylpropyl)-
N-Phenethylpropan-2-amine (intermediate 28–36). Phenyl-
ethanamine (18, 3.0 mL, 24 mmol), isopropyl bromide (35,
2.5 mL, 26 mmol), acetonitrile (30 mL). Crude yield 2.69 g, 84%.
2-Chloro-N-isopropyl-N-phenethylacetamide (intermediate
28–37). N-Phenethylpropan-2-amine
(28–36,
800.0
mg,
N-isopropylacetamide
(30). 2-Chloro-N-(3,3-diphenylpropyl)-
4.9 mmol), triethylamine (0.78 mL, 5.9 mmol), dichloro-
methane (3.0 mL), chloroacetyl chloride (17, 2 g, 18 mmol).
Purified by flash chromatography on silica gel (EtOAc : Hexane,
2 : 3) to yield pure 28–37 (910 mg, 78% yield). LCMS: calcd for
C13H16ClNO+ [M + H] = 239.11, found 240.14.
N-isopropylacetamide (30–37, 1000 mg, 3.0 mmol), N,N′-ethyle-
nethiourea (5, 370 mg, 3.6 mmol), acetonitrile (20 mL). Puri-
fied by HPLC to yield compound 30 (white solid, 71% purified
yield). 1H NMR (300 MHz, CDCl3) δ (ppm) 1.09–1.16 (dd, J =
6.4, J = 6.4, 6H), 2.35 (s, 2H), 3.09–3.28 (dt, J = 7.9, J = 8.1, 2H),
3.94 (s, 4H), 3.99 (s, 1H), 4.11 (t, J = 8.5, 1H), 4.46 (s, 2H),
7.17–7.35 (m, 10HAr). 13C NMR (300 MHz, CDCl3) δ (ppm)
20.18, 21.20, 34.25, 41.02, 45.65, 45.74, 49.66, 50.02, 126.50,
127.65, 128.61, 143.63, 144.00, 165.93, 170.40. LCMS calcd for
C23H29N3OS+ [M + H] = 396.20, found 396.45.
2-((4,5-Dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phen-
ethylacetamide
(28). 2-Chloro-N-isopropyl-N-phenethyl-
acetamide (28–37, 600 mg, 2.5 mmol), N,N′-ethylenethiourea
(5, 300 mg, 3.0 mmol), acetonitrile (25 mL). Purified by HPLC
to yield compound 28 (white solid, 79% purified yield). 1H
NMR (300 MHz, CDCl3) δ (ppm) 1.24–1.28 (m, 6H), 2.81–2.90
(dt, J = 7.98, J = 8.12, 2H), 3.39 (t, J = 7.15, 1H), 3.54 (t, J = 7.14,
1H), 3.86 (s, 1H), 3.97 (s, 4H), 4.28–4.36 (5ry, J = 6.17, 1H), 4.41 Synthesis of compound 31
(s, 2H), 7.18–7.33 (m, 5HAr). 13C NMR (300 MHz, CDCl3) δ.
N-(Cyclohexylmethyl)aniline (intermediate 31–36). Aniline
(18, 3.1 g, 30 mmol), methylcyclohexane bromide (35, 5.0 mL,
36 mmol), acetonitrile (30 mL).
(ppm) 20.27, 21.24, 35.20, 37.67, 43.56, 45.75, 49.91, 126.53,
128.60, 128.65, 137.79, 139.02, 165.80, 170.22. LCMS calcd for
C16H23N3OS+ [M + H] = 306.16, found: 306.35.
2-Chloro-N-(cyclohexylmethyl)-N-phenylacetamide
(inter-
mediate 31–37). N-(Cyclohexylmethyl)aniline (31–36, 2.0 g,
7.5 mmol), triethylamine (1.2 mL, 9.0 mmol), dichloro-
Synthesis of compound 29
N-Isopropyl-4-phenylbutan-1-amine (intermediate 29–36). methane (3.6 mL), chloroacetyl chloride (17, 0.7 mL,
Phenylbutanamine (18, 2.0 mL, 13 mmol), isopropyl bromide 9.0 mmol). Purified by flash chromatography on silica gel
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(EtOAc : Hexane, 3 : 2) to yield pure 31–37 (2.51 g, 98% yield). nitrile (18 mL). Purified by HPLC to yield compound 33 (white
1
LCMS: calcd for C15H20ClNO+ [M + H] = 265.12, found 266.10.
solid, 77% purified yield). H NMR (300 MHz, CDCl3) δ (ppm)
N-(Cyclohexylmethyl)-2-((4,5-dihydro-1H-imidazol-2-yl)thio)- 0.89–1.19 (m, 6H), 2.63 (dt, J = 7.42, 2H), 3.14 3.26 (dd, J = 6.8,
N-phenylacetamide (31). 2-Chloro-N-(cyclohexylmethyl)-N-phenyl- 2H), 3.26 3.44 (dt, J = 7.3, 2H), 3.92 (s, 2H), 3.97 (s, 2H), 4.25
acetamide (31–37, 1000 mg, 3.8 mmol), N,N′-ethylenethiourea (s, 1H), 4.28 (s, 1H), 7.14–7.30 (m, 5HAr). 13C NMR (300 MHz,
(5, 460 mg, 4.5 mmol), acetonitrile (36 mL). Purified by HPLC CDCl3) δ (ppm) 25.84, 28.73, 30.83, 35.53, 45.84, 125.90,
to yield compound 31 (white solid, 71% purified yield). 1H 128.40, 128.43, 141.92, 167.17, 167.28, 170.34. LCMS calcd for
NMR (300 MHz, CDCl3) δ (ppm) 0.97–1.10 (m, 6H), 1.70 (d, J = C22H33N3OS+ [M + H] = 388.23, found 388.15.
10.9, 10H), 3.58 (s, 2H), 3.62 (d, J = 7.45, 2H), 4.02 (s, 4H),
7.23–7.52 (m, 5HAr). 13C NMR (300 MHz, CDCl3) δ (ppm)
25.65, 26.25, 30.67, 33.09, 35.89, 45.85, 56.09, 127.66, 129.30,
130.63, 140.68, 168.34, 170.81. LCMS calcd for C18H25N3OS+
[M + H] = 332.17, found 332.50.
Synthesis of compound 34
N-(Cyclohexylmethyl)-3,3-diphenylpropan-1-amine
(inter-
mediate 34–36). 3,3-Diphenylpropan-1-amine (18, 2.0 mL,
10 mmol), methylcyclohexane bromide (35, 1.9 mL, 14 mmol),
acetonitrile (6 mL).
Synthesis of compound 32
2-Chloro-N-(cyclohexylmethyl)-N-(3,3-diphenylpropyl)acet-
amide (intermediate 34–37). N-(Cyclohexylmethyl)-4-phenyl-
butan-1-amine (34–36, 2.0 g, 6.5 mmol), triethylamine (1.0 mL,
7.8 mmol), dichloromethane (21.3 mL), chloroacetyl chloride
(17, 0.6 mL, 7.8 mmol). Purified by flash chromatography on
silica gel (EtOAc : Hexane, 1 : 3) to yield pure 34–37 (1.35 g,
80% yield). LCMS: calcd for C24H30ClNO+ [M + H] = 384.96,
found 385.22.
N-(Cyclohexylmethyl)-2-phenylethan-1-amine (intermediate
32–36). Phenylethanamine (18, 2.0 mL, 16 mmol), methyl-
cyclohexane bromide (35, 2.4 mL, 17 mmol), acetonitrile
(36 mL).
2-Chloro-N-(cyclohexylmethyl)-N-phenethylacetamide (inter-
mediate 32–37). N-(Cyclohexylmethyl)-2-phenylethan-1-amine
(32–36, 0.5 g, 2.3 mmol), triethylamine (0.4 mL, 2.8 mmol),
dichloromethane (1.2 mL), chloroacetyl chloride (17, 0.2 mL,
2.8 mmol). Purified by flash chromatography on silica gel
(EtOAc : Hexane, 1 : 4) to yield pure 32–37 (330 mg, 61% yield).
LCMS: calcd for C17H24ClNO+ [M + H] = 293.15, found 294.30.
N-(Cyclohexylmethyl)-2-((4,5-dihydro-1H-imidazol-2-yl)thio)-
N-(Cyclohexylmethyl)-2-((4,5-dihydro-1H-imidazol-2-yl)thio)-
N-(3,3-diphenylpropyl)acetamide (34). 2-Chloro-N-(cyclohexyl-
methyl)-N-(3,3-diphenylpropyl)acetamide (34–37, 1000 mg,
2.6 mmol), N,N′-ethylenethiourea (5, 220 mg, 2.2 mmol), aceto-
nitrile (11 mL). Purified by HPLC to yield compound 34 (white
N-phenethylacetamide
(32). 2-Chloro-N-(cyclohexylmethyl)-
1
solid, 77% purified yield). H NMR (300 MHz, CDCl3) δ (ppm)
N-phenethylacetamide (32–37, 200 mg, 6.8 mmol), N,N′-ethylene-
thiourea (5, 83 mg, 0.8 mmol), acetonitrile (4.5 mL). Purified
by HPLC to yield compound 32 (white solid, 77% purified
0.67–1.20 (m, 6H), 1.32–1.72 (m, 6H), 2.19–2.37 (m, 3H), 3.10
(d, J = 7.14, 1H), 3.24 (t, J = 7.14, 2H), 3.38 (dt, J = 7.33, J = 7.91,
1H), 3.85 (dt, J = 7.75, J = 7.75, 1H), 3.90 (s, 4H), 4.08 (t, J =
7.75, 1H), 4.20 (s, 1H), 4.39 (s, 1H), 7.16–7.29 (m, 10HAr).
13C NMR (300 MHz, CDCl3) δ (ppm) 25.6, 25.8, 26.1, 30.4, 30.7,
32.5, 35.7, 35.9, 36.6, 37.1, 45.7, 48.1, 49.1, 51.7, 54.9, 126.5,
126.6, 127.6, 127.7, 128.6, 128.8, 143.6, 143.9, 166.8, 167.1,
170.3. LCMS calcd for C27H35N3OS+ [M + H] = 449.66, found
450.66.
1
yield). H NMR (300 MHz, CDCl3) δ (ppm) 0.91–1.66 (m, 6H),
2.86–2.89 (m, 2H), 3.15 3.20 (dd, J = 6.88, J = 6.35, 2H), 3.75 (d,
J = 7.32, 2H), 3.97 (d, J = 6.44, 4H), 7.15–7.30 (m, 5HAr). 13C
NMR (300 MHz, CDCl3) δ (ppm) 25.76, 30.49, 37.43, 45.74,
126.55, 127.01, 128.84, 129.22, 137.66, 138.41, 167.27, 170.46,
170.57 LCMS calcd for C20H29N3OS+ [M + H] = 360.20, found
360.40.
Synthesis of compound 33
N-(Cyclohexylmethyl)-4-phenylbutan-1-amine (intermediate
33–36). Phenylbutylanamine (18, 2.0 mL, 10 mmol), methyl-
Abbreviations
cyclohexane bromide (35, 1.9 mL, 14 mmol), acetonitrile ALL
(6 mL).
2-Chloro-N-(cyclohexylmethyl)-N-(4-phenylbutyl)acetamide Dex
Acute lymphoblastic leukemia
BCP-ALL B-cell precursor ALL
Dexamethasone
(intermediate 33–37). N-(Cyclohexylmethyl)-4-phenylbutan-1- eq
amine (33–36, 2.6 g, 16 mmol), triethylamine (1.7 mL, HTS
13 mmol), dichloromethane (5.3 mL), chloroacetyl chloride Pred
(17, 1.4 mL, 13 mmol). Purified by flash chromatography on TEA
silica gel (EtOAc : Hexane, 1 : 3) to yield pure 33–37 (1.35 g, TLC
80% yield). LCMS: calcd for C19H28ClNO+ [M + H] = 321.19,
found 320.55.
equivalent
High throughput screening
Prednisolone
Triethyl amine
Thin layer chromatography
Author contributions
N-(Cyclohexylmethyl)-2-((4,5-dihydro-1H-imidazol-2-yl)thio)-
N-(4-phenylbutyl)acetamide
methyl)-N-(4-phenylbutyl)acetamide
(33). 2-Chloro-N-(cyclohexyl- The manuscript was written through contributions of all
(33–37, 1000 mg, authors. All authors have given approval to the final version of
3.1 mmol), N,N′-ethylenethiourea (5, 380 mg, 3.7 mmol), aceto- the manuscript.
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china, E. Paietta, J. Racevskis, J. M. Rowe, M. S. Tallman,
G. Basso, J. P. Meijerink, C. Cordon-Cardo, A. Califano and
A. A. Ferrando, Cancer Cell, 2013, 24, 766–776.
Funding sources
This research was funded by an NHMRC grant. R.B.L. is
funded by an NHMRC fellowship. C.E.T. is funded by a 11 N. L. M. Liem, R. A. Papa, C. G. Milross, M. A. Schmid,
Research Excellence Award from UNSW.
M. Tajbakhsh, S. Choi, C. D. Ramirez, A. M. Rice,
M. Haber, M. D. Norris, K. L. MacKenzie and R. B. Lock,
Blood, 2004, 103, 3905–3914.
Conflict of interest
12 R. B. Lock, N. Liem, M. L. Farnsworth, C. G. Milross,
C. Xue, M. Tajbakhsh, M. Haber, M. D. Norris,
G. M. Marshall and A. M. Rice, Blood, 2002, 99, 4100–4108.
13 P. S. Bachmann, R. Gorman, R. A. Papa, J. E. Bardell,
J. Ford, U. R. Kees, G. M. Marshall and R. B. Lock, Cancer
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The authors declare no competing financial interests.
Acknowledgements
14 P. S. Bachmann, R. G. Piazza, M. E. Janes, N. C. Wong,
C. Davies, A. Mogavero, V. A. Bhadri, B. Szymanska,
G. Geninson, V. Magistroni, G. Cazzaniga, A. Biondi,
D. Miranda-Saavedra, B. Göttgens, R. Saffery, J. M. Craig,
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Children’s Cancer Institute is affiliated with UNSW Australia
and the Sydney Children’s Hospitals Network.
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