Inhibitors of apoptosis in lymphocytes: Synthesis and biological evaluation of compounds related to pifithrin-α

Article abstract of DOI:10.1021/jm0502034

The chemoprotection of cells from apoptosis induced by toxins or ionizing radiation could be important for biodefense and in the treatment of acute injuries. We describe a series of small heterocycles, including fused benzothiazoles, benzimidazoles, and related compounds, that abrogate thymocyte apoptosis induced by dexamethasone and γ-irradiation. To optimize the protective activity of the previously reported pifithrin-α (PFT-α, 1), various derivatives and analogues of this and the corresponding ring-closed imidazobenzothiazole (IBT, 39) were synthesized. The aromatic analogues of 39 were more protective than 39, while the aromatic analogues of 1 were not active. Compound 19 containing a pyrrolidinyl substituent on the phenyl ring provided potent antiapoptotic activity (EC₅₀ of 1.31 μM compared to 4.16 μM for 1). Modification of aromatic 39 with a pyrrolidinyl para substituent (compound 60) enhanced the activity, lowering the EC₅₀ to 0.35 μM. Also, 60 provided significant protection against γ-irradiation-induced apoptosis, as expected. Compounds 19 and 60 may be promising for potential clinical development.

Full text of DOI:10.1021/jm0502034

J. Med. Chem. 2005, 48, 6409-6422
6409
Inhibitors of Apoptosis in Lymphocytes: Synthesis and Biological Evaluation of
Compounds Related to Pifithrin-r
Sylvie D. Barche´chath,‡ Rommel I. Tawatao,† Maripat Corr,† Dennis A. Carson,† and Howard B. Cottam*,†
Department of Chemistry and Biochemistry and Department of Medicine, University of California, San Diego,
La Jolla, California 92093-0663
Received March 4, 2005
The chemoprotection of cells from apoptosis induced by toxins or ionizing radiation could be
important for biodefense and in the treatment of acute injuries. We describe a series of small
heterocycles, including fused benzothiazoles, benzimidazoles, and related compounds, that
abrogate thymocyte apoptosis induced by dexamethasone and γ-irradiation. To optimize the
protective activity of the previously reported pifithrin-R (PFT-R, 1), various derivatives and
analogues of this and the corresponding ring-closed imidazobenzothiazole (IBT, 39) were
synthesized. The aromatic analogues of 39 were more protective than 39, while the aromatic
analogues of 1 were not active. Compound 19 containing a pyrrolidinyl substituent on the phenyl
ring provided potent antiapoptotic activity (EC50 of 1.31 µM compared to 4.16 µM for 1).
Modification of aromatic 39 with a pyrrolidinyl para substituent (compound 60) enhanced the
activity, lowering the EC50 to 0.35 µM. Also, 60 provided significant protection against
γ-irradiation-induced apoptosis, as expected. Compounds 19 and 60 may be promising for
potential clinical development.
Introduction
into the cytoplasm where it can associate with ATP,
apoptosis-activating factor-1, and procaspase-9. This
apoptosome complex cleaves procaspase-9 to caspase-
9, which in turn cleaves procaspase-3, initiating the
cascade of protease activation in the execution phase
of apoptosis.8,9
The prevention of untoward cell death has become an
important goal for pharmacologic intervention in a
variety of clinical settings. Profound disability can result
from apoptotic cell death and tissue injury due to
ischemia, chemotherapeutic agents, ionizing radiation,
or hyperthermia. Recently, a small molecule, 2-(2-imino-
4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone hy-
drobromide (PFT-R, 1), was originally identified from a
broad screen of 10 000 compounds to inhibit cell death
from γ-radiation.1 In addition, this compound was
shown to protect mice from lethal genotoxic stress,1 focal
cortical ischemic injury, and neuronal excitotoxic dam-
age.2,3 This protection by 1 has been attributed to
inhibition of p53 transactivation. Supporting evidence
has included diminished nuclear accumulation with
decreased p53 DNA binding activity and decreased
caspase activity and suppression of mitochondrial dys-
function.2,3 Furthermore, the transcription of apoptosis-
associated gene products p53, Bax, and p21 was inhib-
ited.4
However, the activity of 1 is not limited to inhibiting
the transactivation of p53. This small molecule has been
found to also suppress the heat shock and glucocorticoid
signaling pathways.5 Glucocorticoid-induced cell death
is independent of p53.6 This pathway, however, also
induces the transcription and activity of the proapop-
totic BH3 (Bcl-2 homology)-only protein p53-upregulated
modulator of apoptosis (PUMA).7 When this death-
inducing signal reaches the mitochondria, cell-death-
related events ensue. The inner mitochondrial mem-
brane loses its potential, and cytochrome c is released
Hence, 1 has been reported to diminish p53-depend-
ent and -independent mitochondria-mediated cell death
in vitro and in vivo.1,5 The multiplicity of molecular
pathways that are reportedly influenced by 1 suggested
that the cytoprotective effect could be further optimized.
Furthermore, 1 is not stable under physiological condi-
tions and spontaneously undergoes ring closure to form
the imidazo[2,1-b]benzothiazole (IBT) 39.10,11 Despite
recent reports by Zhu12 and Pietrancosta11 describing
a few of the compounds prepared here, the biologically
active form of 1 (open versus closed ring) has not
previously been formally determined. Elucidation of the
active ring structure would allow investigation of fur-
ther chemical modifications that may enhance the
potency of the compound. In addition, alternative ring
structures, such as the quaternary salts recently re-
ported from our laboratories, may also provide greater
potency.13 Hence, we report the synthesis and structure-
activity relationship of novel derivatives and analogues
of 1 and corresponding closed ring counterparts with
enhanced potency and stability. To determine activity,
we used a p53-independent assay of cell death wherein
mouse thymocytes were treated with dexamethasone in
the presence or absence of test compounds. In addition,
we confirmed the activity of the most potent compounds
in a p53-dependent apoptosis assay.
Chemistry
* To whom correspondence should be addressed. Telephone: (858)
534 5424. Fax: (858) 534 5399. E-mail: hcottam@ucsd.edu.
‡ Department of Chemistry and Biochemistry.
† Department of Medicine.
With PFT-R (1) as the lead compound with known
biological activity, lead optimization strategies were
applied to establish a structure-activity relationship.
10.1021/jm0502034 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/07/2005

6410 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Barche´chath et al.
Scheme 1a
Scheme 4
products with concomitant dehydration. Therefore, we
elected to prepare the corresponding closed ring deriva-
tives 20-41. The corresponding IBT derivatives 20-
41 and 48-60 were then prepared by ring closure in
refluxing ethanol or in methoxyethanol when higher
temperatures were needed (Scheme 1). Compounds 3,
17, 20, and 21 have been reported by Pietrancosta11 and
3, 8, 13, 14, 16, 17, and 66 by Zhu12 as p53 inactivators
and neuroprotective agents.
To study the effects of ring closure on activity, two
strategies were employed. First, to prepare derivatives
that would undergo limited spontaneous ring closure
under normal physiological conditions, we alkylated
2-aminobenzothiazole with benzyl chloride to obtain 61,
a product incapable of ring closure in refluxing ethanol
because it lacks the carbonyl group. In addition, the
para methyl substituted product 62 was synthesized in
the same manner, giving a molecule closer in shape to
that of 1 (Scheme 2). In a second approach to hinder
ring closure, we protected the exocyclic nitrogen of the
2-aminobenzothiazole using p-toluenesulfonyl choride to
yield 63 before alkylating with 2-bromo-4′-methyl-
acetophenone to give 64 (Scheme 3). In addition, a non-
ring-closable PFT-R analogue 65 was prepared by reac-
tion of 2-bromo-4′-methylacetophenone with 2-mercap-
tobenzothiazole, resulting exclusively in the S-alkylation
product (Scheme 4). Compound 66, which was synthe-
sized by alkylation of 2-amino-4,5,6,7-tetrahydroben-
zothiazole with ethyl bromoacetate, appeared to be
resistant to ring closure in refluxing ethanol even
though it possesses a carbonyl group â to the ring
nitrogen and should in theory be able to form a ring
(Scheme 5).
a (a) Toluene, room temp, 24-48 h; (b) ethanol or methoxyetha-
nol, reflux, 90 min; (c) toluene, room temp, 24-48 h; (d) ethanol
or methoxyethanol, reflux, 1.5 h.
The starting 2-aminotetrahydrobenzothiazoles used to
prepare PFT-R and derivatives 2-19 were obtained
following the procedure described by Singh et al.10 in
which the appropriate cyclohexanone was reacted with
thiourea in the presence of iodine (Scheme 1a). For the
synthesis of the aromatic analogues of PFT-R, 42-47,
the starting 2-aminobenzothiazoles were commercially
available (Scheme 1b). Alkylation of the 2-aminoben-
zothiazoles with various R-haloacetophenones provided
the corresponding N-alkylated compounds 1-19 and
42-47, with alkylation occurring exclusively at the ring
nitrogen (Scheme 1). In protic solvents the ring alky-
lated fused thiazoles often formed aromatic ring-closed
Scheme 2
Scheme 3

Inhibitors of Apoptosis in Lymphocytes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6411
Scheme 5
by reaction with 2-chlorocyclohexanone to form the
imidazole 70 (Scheme 7). An isomer of 70, compound
72, was prepared by alkylation of a 2-mercaptobenz-
imidazole with the appropriate acetophenone to form
exclusively the S-alkylated compound 71 that was
further ring-closed using PPA, formed in situ as de-
scribed by Mahfouz et al.17 for a related compound
(Scheme 8). Finally, the PFT-R and IBT analogues
lacking the benzo ring, 73 and 74, respectively, were
prepared following the basic alkylation and ring-closure
procedure described earlier.
Related ring systems were designed to maintain the
overall shape relative to IBT. Heterocycles containing
various combinations or arrangements of nitrogen atoms
were prepared. Anthranilonitrile refluxed with meth-
oxyethanol in the presence of formamidine acetate
generated 4-aminoquinazoline,18 which was subse-
quently used for the synthesis of the 1,2,4-triazolo[1,5-
c]quinazoline 79 and imidazo[1,2-c]quinazoline 81
(Scheme 9). First, 4-aminoquinazoline was refluxed with
hydrazine hydrate for 6 h to form quinazolin-4-yl-
hydrazine. A Schiff base 77 was obtained by reacting
this substituted hydrazine derivative with tolualdehyde.
Ring closure to 78 occurred by addition of acetic
anhydride, and finally, deacetylation gave the desired
quinazoline 79. Second, 4-aminoquinazoline was alky-
lated with 2-bromo-4′-methylacetophenone to produce
a mixture of the open ring 80 and closed ring 81
products, which were then separated by flash chroma-
tography on silica gel.
The effects of substitution at the para position of the
phenyl ring of 1 and corresponding derivatives were
investigated. A variety of substituents was selected on
the basis of their electronic and lipophilic properties.
The electronic (Hammett σ) and the lipophilic (Hansch
π) values calculated for each substituent were plotted
in a two-dimensional graph, the so-called Craig plot,14
and substituents were selected from each quadrant for
preparation. These para-substituted derivatives in both
the open ring (Table 1) and closed ring series (Table 2)
are indicated by the R1 variable substitution.
Analogues in the PFT and IBT series still containing
the thiazole moiety were synthesized in order to evalu-
ate how modifications on parts of the molecule other
than the para position of the phenyl group would
influence the biological activity. The phenyl ring was
replaced by a methyl group in the synthesis of 68. Thus,
the open ring structure 67 was prepared by reacting
2-aminobenzothiazole with propargyl bromide followed
by addition of sodium to refluxing ethanol to effect ring
closure (Scheme 6).15 An isomeric version of IBT with
the phenyl substituent attached to C-3 rather than C-2
was made by first forming the substituted 2-mercap-
toimidazole 69 as described by Maeda et al.,16 followed
Another fused quinazoline, the imidazo[1,2-a]quinazo-
line 85, was prepared starting with the alkylation of
isatoic anhydride using 2-bromo-4′-methylacetophenone
Table 1. Survival of Cells Treated with PFT-R and Analogues When Challenged with DEX
average cell
survival (%)a
standard
deviation (%)
P
compd
class
R1
R2
R3
(Mann-Whitney U test)
1
I
Me
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
72
17
21
31
36
40
40
48
54
56
57
58
59
59
65
65b
66
70
77
20
21
33
62
64
66
9
17
0
0.0001
0.0364
0.0553
0.0910
0.7037
0.7182
0.2255
0.1070
0.0098
0.0027
0.0009
0.0003
0.0020
0.0017
0.0009
0.0001
0.0019
0.0044
0.0009
0.0514
0.0364
0.4273
0.0001
0.0009
0.0009
2
I
OCOPh
3
I
NO2
4
I
Br
1
5
I
CF3
12
1
18
17
17
10
3
6
I
CN
7
I
F
8
I
F
H
F
9
I
H
10
11
12
13
14
15
16
17
18
19
42
43
44
45
46
47
I
N3
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
Cl
I
2-naphthyl
4
9
I
H
I
Cl
10
1
I
H
I
Ph
5
I
OMe
2
I
Et2N
9
I
pyrrolidinyl
H
3
II
II
II
II
II
II
14
11
3
Me
cyclopentyl
2-naphthyl
pyrrolidinyl
Ph
5
3
3
a Control average % cell survival: 72 ( 4. Dexamethasone average % cell survival: 36 ( 7. b Toxic at 10 µM. Tested at 5 µM.

6412 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Barche´chath et al.
Table 2. Survival of Cells Treated with IBT and Analogues When Challenged with DEX
average cell
standard
deviation (%)
P
compd
class
R1
R2
R3
R4
R5
survival (%)a
(Mann-Whitney U test)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
48
49
50
51
52
53
54
55
56
57
58
59
60
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
NO2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
Cl
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
11
18
19
20
21
21
23
27
27
28
33
34
35
35
36
37
44
50
50
54
57
75
16
19b
22
24b
27
36
39
59
68b
73
73b
75b
78b
2
14
7
0.0358
0.0829
0.0173
0.0364
0.0553
0.0514
0.0829
0.1265
0.4114
0.3167
0.2494
0.6481
0.7122
0.4292
0.9273
0.7149
0.3154
0.3863
0.0057
0.0051
0.0003
0.0019
0.0553
0.0009
0.0829
0.0829
0.4114
0.9517
0.8286
0.0009
0.0009
0.0019
0.0003
0.0009
0.0003
H
H
COOH
H
8
OH
H
1
CN
H
11
14
14
16
19
5
OCOPh
H
CF3
H
Me
H
OMe
H
F
H
H
H
7
Cl
H
22
7
Br
H
Cl
H
19
8
H
H
F
H
11
5
N3
H
pyrrolidinyl
H
6
Me
H
10
1
2-naphthyl
H
Ph
H
5
furanyl
H
13
10
10
10
22
2
Me
OH
H
Br
Me
H
Me
NH2
H
cyclopentyl
Me
Br
H
3
2
Ph
Me
OMe
H
14
1
H
Me
H
5
2-naphthyl
pyrrolidinyl
H
2
H
1
a Control average % cell survival: 72 ( 4. Dexamethasone average % cell survival: 36 ( 7. b Toxic at 10 µM. Tested at 5 µM.
Scheme 6
Scheme 7
(Scheme 10). The product 82 was reacted with S-
methylthiopseudourea, followed by refluxing in diglyme
under basic conditions to give the ring-closed derivative
84 whose keto group was further transformed to a chloro
by treatment with phosphorus oxychloride to give the
imidazo[1,2-a]quinazoline 85 (Scheme 10). The 2-(5-
chloro-2-iminobenzoxazol-3-yl)-1-p-tolylethanone 86, an
open-ring-related derivative, was prepared following the
same procedure as that used for the alkylation of the
thiazoles.
Biology Section
To examine the relative protective effect of the
compounds, a reliable and relatively rapid screening
assay was required. We and others have found that the
structural similarity of PFT-R and IBT with luciferin
rendered luciferase-based reporter gene assays for apop-
tosis unreliable.19 Instead, we assayed fluorescent dye
retention described below in thymocytes after dexa-
methasone-induced apoptosis. Dexamethasone (DEX)
treatment of lymphocytes from the thymus initiates a

Inhibitors of Apoptosis in Lymphocytes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6413
Scheme 8
Scheme 9
Scheme 10
signaling pathway that converges on the mitochondria-
mediated molecular executioner cascade, leading to
apoptosis within 6 h.20 The 6 h time point was chosen
because PFT-R was reported to inhibit DEX-induced
apoptosis within this incubation period, and in our
hands it was found to be reliable and reproducible.5
All of the compounds were screened in the thymocyte
assay for cytotoxicity and their ability to prevent dexa-
methasone-induced cell death. Murine thymocytes were
exposed to 10 or 5 µM of each compound in the presence
or absence of DEX for 6 h and then assayed for their in
vitro viability by 3,3′-dihexyloxacarbocyanine iodide
(DiOC6) and propidium iodide (PI) staining and by flow
cytometry. When cell death signals converge on the
mitochondria, there is a loss of the inner mitochondrial
membrane potential and cells can no longer retain the
dye DiOC6 as an indicator of apoptosis. Once cells lose
the integrity of the outer cell membrane, then the dye
propidium iodide is no longer excluded from these cells.
The percentage of viable cells that retained DiOC6 and
excluded PI was compared to cells treated with DEX
alone. Compounds that significantly enhanced survival

6414 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Barche´chath et al.
ring. Class II structures also had the open PFT platform
but contained the aromatic benzo ring. Class III and
class IV compounds had the closed ring structure of IBT
bearing the saturated and aromatic rings, respectively.
These series of compounds were used to compare the
influence of an open versus closed ring structure, a
saturated benzo ring versus an aromatic ring, and the
characteristics of the para substituent on cytoprotective
activity. Additional compounds were synthesized to
evaluate other isoteric ring systems for activity (Tables
3 and 4). Several of these compounds were generated
with obligately open ring structures to determine if this
was the optimal scaffold to suppress mitochondrial
death in DEX treated thymocytes.
Figure 1. Kinetics of ring closure from PFT-R 1 to IBT 39. A
200 µM solution of PFT-R in water with 0.1% DMSO was kept
at room temperature and, at the indicated time points,
quantitated by HPLC. IBT precipitated and was not reliably
quantitated by this method.
As a primary screen for biological activity, each
compound was tested for its ability to prevent DEX-
induced cell death (Tables 1-4). Notably, all of the
compounds that prevented DEX-induced cell death were
derivatives of 1 and 39. That is, none of the other
isosteric ring analogues were cytoprotective (see Tables
3 and 4). The open PFT structure with a saturated benzo
ring proved to be the best scaffold. While PFT-R showed
impressive cytoprotective activity, with an average of
72% cells remaining alive at the end of the assay,
compound 19, bearing a pyrrolidinyl substituent at the
para position of the phenyl, was even more protective
with 77% cell survival. Another compound (18) bearing
a tertiary amine substituent, the diethylamino group,
also enhanced survival significantly compared to control
cells treated with DEX. Moreover, compounds 15 (o-Cl),
16 (p-Ph), and 17 (p-OMe) were also fairly active.
Compounds with similar substituents proved to be
active in the class II series (Table 1). While none of the
compounds in this class were as protective as PFT-R,
three of them showed a cell survival average higher
than 60%. Compounds 45 (p-naphthyl), 46 (p-pyrroli-
dinyl), and 47 (p-phenyl) were also titrated to obtain
their EC50.
Some compounds in the ring closed IBT series also
enhanced survival of thymocytes treated with DEX
(Table 2). Here again, a phenyl substituent (41) im-
proved cytoprotective activity in the class III series.
However, there were compounds in the aromatic series
with greater activity (class IV, Table 2). Again, the
pyrrolidinyl (60), naphthyl (59), and methyl (58) sub-
stituents were among the most active. Compound 57
without a susbstituent on the phenyl, as well as
compound 56 with a methoxyl group on the benzo ring,
also showed enhanced cytoprotective activity.
Figure 2. Inhibition of dexamethasone-induced thymocyte
cell death. Murine thymocytes were pretreated with graded
quantities of the indicated compounds. Apoptosis was then
induced with 5 µM dexamethasone. After 6 h the cells were
stained with DiOC6 and PI and assayed for survival by flow
cytometry. Shown are the percentages of living cells at the
end of 6 h for three independent experiments.
in repeated determinations (P e 0.01) were then se-
lected for a dose response study to determine the EC50
(Tables 5 and 6 and Figure 2).
Results and Discussion
PFT-R (1) has been described as a small-molecule
inhibitor of programmed cell death.1 However, as men-
tioned above, 1 does not remain in the open ring form
in protic solution. After 16 h in solution under physi-
ological conditions, 1 is largely ring-closed and converted
to IBT (39) (Figure 1). Hence, a biological assay that
could be performed during a relatively short period of
time was necessary to investigate whether the closed
ring or the open ring form of the compound was
responsible for cytoprotective activity. It has been well
established that murine thymocytes rapidly undergo
apoptotic cell death after exposure to glucocorticoids
such as DEX,21 and recently glucocorticoid-induced
thymocyte death was shown to be inhibited by 1.5 To
evaluate the features associated with the cytoprotective
effect of 1, compounds were synthesized on the basis of
the PFT-R or IBT structural platforms (summarized
above and in Tables 1-4).
Titration of those compounds that provided cytopro-
tection of at least 60%, a value arbitrarily chosen,
allowed for a more precise comparison using EC50 values
(Tables 5 and 6). Overall, the same substituents seemed
to improve activity in several classes. Compounds with
lipophilic substituents such as tertiary amines (18, 19,
and 60) or large aromatic groups (12, 16, 41, and 59)
showed similar and even greater cytoprotection than
that of PFT-R or IBT. To explore the contribution of the
para pyrrolidinyl group to the activity of the most potent
compounds, we selected an isosteric mimic of this group,
the cyclopentyl group, and prepared derivatives in
classes II and IV (44 and 53, respectively) for evaluation
in the thymocyte protection assay. Neither compound
The PFT and IBT analogues were divided into four
classes (Tables 1 and 2). Class I compounds included
the open ring structure of PFT with a saturated benzo

Inhibitors of Apoptosis in Lymphocytes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6415
Table 3. Survival of Cells Treated with PFT-R and IBT
Analogues When Challenged with DEX
Table 4. Survival of Cells Treated with Various Compounds
with Ring Structures Related to PFT-R and IBT When
Challenged with DEX
a Control average % cell survival: 72 ( 4. Dexamethasone
average % cell survival: 36 ( 7.
a Control average % cell survival: 72 ( 4. Dexamethasone
average % cell survival: 36 ( 7.

6416 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Barche´chath et al.
Table 5. EC50 (µM) of Compounds in Thymocyte Apoptosis
Assay
Scheme 11
compd
EC50 (µM)
95% confidence interval (µM)
9
10
11
13
14
15
17
18
56
57
9.69
5.68
7.28
6.06
5.37
5.72
7.54
1.84
4.44
10.23
NDa
4.61-7.00
3.86-13.70
4.64-7.91
3.26-8.82
4.08-8.01
6.71-8.48
1.51-2.24
2.86.6.89
8.49-12.32
a ND: 95% confidence interval not determined.
Table 6. EC50 (µM) of Compounds in Thymocyte Apoptosis
Assay
vival, as can be seen in the following examples. In one
case, the imine at C-2 of an open ring compound (63)
was protected by a tosyl group in order to prevent
formation of the imidazole ring. With or without alky-
lation by the usual acetophenone, compounds 63 or 64,
respectively, did not show significant ability to protect
cells against DEX-induced apoptosis (Table 3). Another
example is illustrated by the alkylation of 2-amino-
4,5,6,7-tetrahydrobenzothiazole with a benzyl (61) or
tolyl group (62) (Table 3). With just the carbonyl group
missing, these structures were much closer in shape to
that of 1 but were less active. Also, replacement of the
4-methylacetophenone by a propargyl group (67) led to
a compound showing diminished cytoprotective activity
(68, Table 3).
EC50
95% confidence
R1 substituent
class
compd
(µM)
interval (µM)
methyl
I
1
43
39
58
16
47
41
55
12
45
40
59
19
46
38
60
4.16
>10
2.01
1.22
2.29
7.26
3.79
8.12
4.02
>10
4.77
4.02
1.31
9.84
>10
0.35
2.95-5.88
methyl
II
III
IV
I
methyl
1.45-2.80
0.95-1.57
1.76-2.98
6.02-8.76
2.78-5.16
4.08-13.75
3.07-5.28
methyl
phenyl
phenyl
II
III
IV
I
II
III
IV
I
phenyl
phenyl
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
pyrrolidinyl
pyrrolidinyl
pyrrolidinyl
pyrrolidinyl
3.95-5.75
3.45-4.69
0.94-1.82
2.78-34.83
II
III
IV
0.11-1.08
In general aromatic closed ring analogues demon-
strated improved cytoprotective activity, with 60 and
58 being the most potent compounds in this assay. In
contrast, the aromatic series of PFT-R analogues exhib-
ited weaker protective ability. However, in both 1 and
39 the saturated fused benzo ring was a feature that
conferred protective ability, since both 73 and 74,
lacking this ring, were devoid of protective activity
(Table 3). An additional critical feature for bioactivity
was the position of the phenyl substituent. Compound
70, similar to 39 except that the phenyl ring is attached
to C-3 instead of the usual C-2, was not active (Table
3). The fully aromatic 72, an isomer of 58 but having
the sulfur and nitrogen ring atoms interchanged, showed
a much weaker ability to protect the cells relative to 58
(Tables 3 and 4).
Finally, to examine the influence of the imine nitrogen
in the open ring compounds (classes I and II), an isostere
of the imino group, the oxo function was considered.
Thus, the 2-oxo derivatives in both classes (compounds
76 and 75, respectively) were prepared (Scheme 11).
Both compounds showed very weak activity, suggesting
that the imine function plays a vital role in the bioac-
tivity of the open ring compounds, particularly for those
of class I, such as 1.
was found to have significant activity. Thus, the pres-
ence of the amine nitrogen appears to be essential for
enhanced bioactivity.
Following leads for the most biologically active com-
pounds in the primary screen, families of compounds
were evaluated for structural features that optimized
activity (Table 6). Thus, aromatic and nonaromatic
derivatives of 1 and 39 that were para-substituted on
the phenyl moiety with a methyl, phenyl, naphthyl, or
pyrrolidinyl group were synthesized. The aromatic
analogues of 39 (class IV) were highly active, whereas
this modification in the aromatic PFT-R series (class II)
diminished the activity. The addition of large aryl para
substituents modestly enhanced activity except for the
para phenyl group in the class IV series, which para-
doxically diminished cytoprotective ability. Adding the
para pyrrolidinyl or diethylamino group largely im-
proved activity as opposed to the primary amine 21
(Table 2). Interestingly, the para pyrrolidinyl on the IBT
template (class III) abrogated all cytoprotective activity
(compound 38). Hence, modifying IBT with an aromatic
ring (class IV) and a para pyrrolidinyl group revealed
two features that in combination improved thymocyte
survival. Compound 39 was modestly more cytoprotec-
tive than 1 as judged by the EC50 values, but over half
of the 10 most potent compounds were derivatives of 1
(class I) (Tables 5 and 6). However, it is unlikely that
the biological activity of 1 is limited to its conversion to
the ring closed 39 counterpart during the 6 h assay
because only about 25% conversion takes place within
this time period (Figure 1). In addition, other com-
pounds specifically designed to maintain an open ring
structure did not necessarily promote thymocyte sur-
Several compounds had greater cytoprotective activity
compared with 1, particularly a few members of classes
I and IV. The three most potent new compounds were
19, 58, and 60, which had antiapoptotic activity in the
thymocyte protection assay at low micromolar or sub-
micromolar concentrations while displaying direct cy-
totoxic activity at 20-fold higher concentrations. More-
over, 60 protected thymocytes from apoptosis induced
by γ-irradiation (Figure 3), as expected for a p53-

Inhibitors of Apoptosis in Lymphocytes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6417
Figure 4. Effect of compounds 1 and 60 on PUMA protein
expression in mouse thymocytes. Thymocytes were pretreated
with 10 µM of the indicated compounds and then exposed to 5
µM of dexamethasone for 6 h. The cytosolic proteins were
fractionated and then separated on a 4-12% gradient gel by
SDS-PAGE and transferred to a PDVF membrane, which was
probed with antibodies to PUMA and actin.
Figure 3. Inhibition of γ-irradiation-induced apoptosis. Ten
million thymocytes per well were pretreated with 5 µM of the
indicated compounds for 30 min and then irradiated with 6
Gy. After 6 h the cells were stained with DiOC6 and PI and
assayed for survival by flow cytometry. Shown are the average
percent survival data pooled from two experiments and the
SEM. Compounds that significantly improved viability after
irradiation are indicated by an asterisk (P < 0.01 by Students
t test).
Experimental Section
Chemistry. Melting points were obtained on a Mel-temp
II capillary melting point apparatus and are uncorrected.
Proton nuclear magnetic resonance spectra were obtained on
a Varian Unity 500 at 499.8 MHz or on a Varian Mercury at
400.06 MHz. The chemical shifts are expressed as δ values
(parts per million) relative to tetramethylsilane (TMS) as the
internal standard. High-resolution mass spectrometric analy-
ses were performed on a Finnigan MAT900XP high-resolution
double-focusing mass spectrometer using electron impact.
Elemental analyses were performed by NuMega Resonance
Labs, San Diego, CA. Thin-layer chromatography was per-
formed on silica gel 60 F-254 plates (EM Reagents). E Merck
silica gel (230-400 mesh) was used for flash column chroma-
tography.
mediated death pathway. Similar compounds were
previously tested by Zhu12 and Pietrancosta11 for their
neuroprotective ability. Recently, Zhu and co-workers
found that oxazole analogues of PFT-R as well as
compound 17 improved p53 inactivation by 2- to 4-fold12
in a neuroprotection assay. Moreover, Pietrancosta and
co-workers found that an analogue of 17 with a cyano
group adjacent to the ketone, as well as compound 20,
had a 10-fold greater potency in protecting mouse
embryo cortical neurons against DNA damage than
PFT-R.11 Interestingly, no mention was made of the
tendency of these PFT-R derivatives to cyclize, even
during recrystallization.
Biological Materials. Mice. C57Bl/6 and p53-/- mice were
purchased from The Jackson Laboratories (Bar Harbor, ME).
The mice were bred and maintained under standard conditions
at the University of California San Diego Animal Facility that
is accredited by the American Association for Accreditation of
Laboratory Animal Care. All animal protocols receive prior
approval by the institutional review board.
Reagents. Dexamethasone (DEX) was purchased from
Sigma-Aldrich (St. Louis, MO). Other chemicals were pur-
chased from Maybridge plc (Trevillett, Tintagel, Cornwall,
U.K.) and Lancaster Synthesis (Windham, NH).
The mechanism of action of these compounds reported
here is still unknown. PFT-R has been reported to
diminish p53 transactivation and disrupt other death-
inducing pathways. While cell death induced by irradia-
tion is p53-dependent, dexamethasone-induced cell death
is not.6 Thus, it is unlikely that the compounds directly
target either the glucocorticoid receptor or p53 directly.
Alternative candidates for the molecular target of
these compounds would include the proapoptotic BH3-
only proteins, which converge on mitochondrial mech-
anisms to initiate cell death.22 Dexamethasone and p53
both induce the transcription of one of these proteins,
namely, PUMA/bbc3.7,23,24 In addition, PUMA deficiency
has been reported to protect thymocytes from apoptosis
induced by a variety of stimuli including irradiation,
cytokine withdrawal, staurosporine, and dexametha-
sone.25 Indeed, dexamethasone-treated p53 deficient
thymocytes have augmented intracellular PUMA levels
(Figure 4). Prior treatment of these thymocytes with
either 1 or 60 restricted the levels of PUMA expression
(Figure 4), which correlated with enhanced survival as
assayed by DiOC6 and PI staining (data not shown).
Apoptosis Assays. Thymocytes were harvested from young
C57Bl/6 mice and cultured at 37 °C in 5% CO2 in RPMI 1640
containing 10% FBS, 1% penicillin/streptomycin (Gibco BRL,
Rockville, MD). Thymocytes were plated at a density of 107
cells/mL and preincubated with 5-10 µM of each compound
(from 10 mM stock in DMSO) for 30 min before induction of
apoptosis. Apoptosis was induced with 5 µM dexamethasone
or by exposure to 6 Gy of γ-radiation. After 6 h, cell apoptosis
was assayed by propidium iodide (PI) and 3,3′-dihexyloxacar-
bocyanine iodide (DiOC6) staining. The cells were removed
from the plate and incubated for 30 min in medium with 40
nM DiOC6 and 5 µg/mL PI and then analyzed by flow
cytometry in a FACS caliber (Beckton-Dickinson, San Jose,
CA). Viable cells had high DiOC6 (FL-1) and low PI (FL-3),
whereas apoptotic cells had low DiOC6 (FL-1) and low PI (FL-
3). To evaluate the EC50 values, the thymocytes were pre-
exposed to graded concentrations of selected compounds for
30 min and then apoptosis was induced with 5 µM dexa-
methasone. After 6 h the cells were harvested and stained as
above. This time point was selected on the basis of previously
reported assay conditions5 and confirmed for our own system
(data not shown).
EC50 Determination. The concentration (EC50) of each
compound that inhibited dexamethasone-induced cell death
by 50% was determined by nonlinear regression fitting of the
data to a one-site model. Pseudo Hill slopes were determined
by nonlinear regression fit of the data to a sigmoidal dose
response equation (variable slope):
These data suggest that the regulation of cell death
by the compounds described here is not restricted to p53
as originally described. Regulation of other independent
proapoptotic mechanisms such as the BH3-only proteins
should also be examined. The divergence in the struc-
tures presented here also suggests that a variety of
signaling intermediates may be influenced before the
convergence on the mitochondrial death cascade.
max - min % viability
1 + 10(log EC50 - X)n
% viability ) min % viability +

6418 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Barche´chath et al.
where X is the logarithm of inhibitor concentration and n is
the pseudo Hill slope. The maximum % viability and minimum
% viability were experimentally determined after dexametha-
sone exposure and drug treatment. EC50 values and 95%
confidence intervals (CI) were derived from the sigmoid fits
to the percent control transformed data shown using GraphPad
Prism, version 4.0b, for Macintosh (GraphPad Software, San
Diego, CA).
2.55 (s, 2H), 5.79 (s, 2H), 8.14 (d, 2H), 8.20 (d, 2H), 9.55 (s,
2H); HR-MS (EI) m/z 297.0934 (M+).
1-(3,4-Difluorophenyl)-2-(2-imino-4,5,6,7-tetrahydroben-
zothiazol-3-yl)ethanone Hydrobromide Salt (7). was pre-
pared from 3,4-difluorophenacyl bromide (Maybridge) and
2-amino-4,5,6,7-tetrahydrobenzothiazole using method A in
71% yield: mp 270 °C (dec); 1H NMR (DMSO-d6) δ 1.72 (s,
4H), 2.40 (t, 2H), 2.49 (t, 2H), 5.73 (s, 2H), 7.74 (dd, 1H), 7.96
(s, 1H), 8.13 (dt, 1H), 9.52 (s, 2H); HR-MS (EI) m/z 308.0794
(M+).
1-(3-Fluorophenyl)-2-(2-imino-4,5,6,7-tetrahydroben-
zothiazol-3-yl)ethanone Hydrobromide Salt (9). 9 was
prepared from 3-fluorophenacyl bromide (Maybridge) and
2-amino-4,5,6,7-tetrahydrobenzothiazole using method A in
72% yield: mp 265-365 °C; 1H NMR (DMSO-d6) δ 1.72 (s, 4H),
2.34 (m, 2H), 2.49 (m, 2H), 5.75 (s, 2H), 7.64 (t, 1H), 7.69 (m,
1H), 7.88 (dd, 1H), 7.91 (dd, 1H), 9.52 (s, 2H). Anal. (C15H16-
BrFN2OS) C, H, N.
1-(4-Azidophenyl)-2-(2-imino-4,5,6,7-tetrahydroben-
zothiazol-3-yl)ethanone Hydrobromide Salt (10). was
prepared from p-azidophenacyl bromide (Sigma) and 2-amino-
4,5,6,7-tetrahydrobenzothiazole using method A in 67%
yield: mp 179-182 °C; 1H NMR (DMSO-d6) δ 1.73 (s, 4H),
2.36 (s, 2H), 2.55 (m, 2H), 5.71 (s, 2H), 7.37 (t, 2H), 8.08 (t,
2H), 9.51 (s, 2H). Anal. (C15H16BrN5OS) C, H, N.
1-(3,4-Dichlorophenyl)-2-(2-imino-4,5,6,7-tetrahydroben-
zothiazol-3-yl)ethanone Hydrobromide Salt (11). 11 was
prepared from 3,4-dichlororophenacyl bromide (Maybridge)
and 2-amino-4,5,6,7-tetrahydrobenzothiazole using method A
in 71% yield: mp 294-299 °C (dec); 1H NMR (DMSO-d6) δ
1.73 (s, 4H), 2.35 (s, 2H), 2.55 (s, 2H), 5.76 (s, 2H), 7.96 (m,
2H), 8.29 (d, 1H), 9.52 (s, 2H); HR-MS (EI) m/z 340.0201 (M+).
Immunoblotting. After removal of medium, cells were
disrupted in lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl,
0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.6% NP-40) on ice. The
nuclei were separated by centrifugation, and the cytoplasmic
sample was removed. The nuclear pellets were resuspended
in 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1.0 mM EDTA, 1.0
mM EGTA, 1 mM dithiothreitol, and 0.1 mM phenylmethyl-
sulfonyl fluoride, and the insoluble material was removed by
centrifugation. The samples were heated to 70 °C for 10 min
in loading buffer with 10 mM DTT. Each lane of an SDS-
PAGE gel was loaded with 20 µg of protein. After electro-
phoresis, the proteins were transferred to a polyvinylidene
difluoride (PVDF) membrane, blocked with 2% I-blockTM
(Tropix Inc, Bedford, MA) containing 0.05% Tween-20 in PBS,
and then incubated with anti-PUMA (AbCam, Inc., Cambridge,
MA). Horseradish peroxidase conjugated anti-IgG (Santa Cruz
Laboratories, Santa Cruz, CA) was used as the secondary
antibody. The membranes were developed using a chemilu-
minescence system (ECL detection reagent: Amersham Life
Science, Aylesbury, U.K.). The membranes were reprobed with
anti-actin (Sigma, St Louis, MO) to ensure equivalent loading.
Method A: General Procedure for Synthesis of PFT-r
Derivatives and Aromatic Analogues: Compounds 1-19
and 42-47. Following a procedure reported by Singh,10
a
1-(2-Chlorophenyl)-2-(2-imino-4,5,6,7-tetrahydroben-
zothiazol-3-yl)ethanone Hydrobromide Salt (15). 15 was
prepared from 2-chlororophenacyl bromide (Maybridge) and
2-amino-4,5,6,7-tetrahydrobenzothiazole using method A in
mixture of 2-amino-4,5,6,7-tetrahydrobenzothiazole (1 equiv)
and the appropriate phenacyl bromide (1.1 equiv) was stirred
in toluene at room temperature for 24-48 h. The product
crystallized out and was collected by filtration. The same
procedure was used to synthesize aromatic analogues starting
from commercially available 2-aminobenzothiazole, some bear-
ing a substituent on C-6.
1
49% yield: mp 205-209 °C; H NMR (DMSO-d6) δ 1.74 (m,
4H), 2.40 (m, 2H), 2.56 (d, 2H), 5.64 (s, 2H), 7.68 (d, 2H), 8.09
(d, 2H), 9.56 (s, 2H); HR-MS (EI) m/z 306.0578 (M+).
1-(4-Diethylaminophenyl)-2-(2-imino-4,5,6,7-tetrahy-
drobenzothiazol-3-yl)ethanone Hydrobromide Salt (18).
18 was prepared from R-bromo-4-(diethylamino)acetophenone
(Lancaster) and 2-amino-4,5,6,7-tetrahydrobenzothiazole using
method A in 45% yield: mp 189-191 °C; 1H NMR (DMSO-d6)
δ 1.13 (t, 6H), 1.72 (s, 4H), 2.30 (s, 2H), 2.54 (m, 2H), 3.47 (q,
4H), 5.55 (s, 2H), 6.78 (d, 2H), 7.83 (d, 2H), 9.42 (s, 2H). Anal.
(C19H26BrN3OS) C, H, N.
2-(2-Imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-(4-
pyrrolidin-1-yl-phenyl)ethanone Hydrobromide Salt (19).
19 was prepared from R-bromo-4-(1-pyrrolidino)acetophenone
(Lancaster) and 2-amino-4,5,6,7-tetrahydrobenzothiazole using
method A in 57% yield: mp 224-226 °C; 1H NMR (DMSO-d6)
δ 1.73 (s, 4H), 1.99 (m, 4H), 2.29 (m, 2H), 2.54 (s, 2H), 3.36 (s,
4H), 5.56 (s, 2H), 6.66 (d, 2H), 7.86 (d, 2H), 9.43 (s, 2H). Anal.
(C19H24BrN3OS) C, H, N.
General Procedure for Synthesis of IBT Derivatives
and Aromatic Analogues: Compounds 20-41 and 48-
60. Method B: One-Step Ring Closure. A mixture of
2-amino-4,5,6,7-tetrahydrobenzothiazole (1 equiv) and the
appropriate phenacyl bromide (1.1 equiv) was refluxed in
ethanol for 90 min. The ring-closed product crystallized out
upon cooling and was separated by filtration.
Method C: Two-Step Ring Closure. 2-Imino-3-phenacyl-
4,5,6,7-tetrahydrobenzothiaole hydrobromide or analogue was
refluxed in ethanol for 90 min. The ring-closed product
crystallized out upon cooling and was separated by filtration.
The aromatic analogues were made using the same proce-
dures.
Benzoic Acid 4-[2-(2-Imino-4,5,6,7-tetrahydrobenzothi-
azol-3-yl)acetyl]phenyl Ester Hydrobromide Salt (2). 2
was prepared from 4-(bromoacetyl)phenyl benzoate and
2-amino-4,5,6,7-tetrahydrobenzothiazole using method A in
65% yield: mp 175-177 °C; 1H NMR (DMSO-d6) δ 1.73 (s, 4H),
2.34 (s, 2H), 2.54 (s, 2H), 5.74 (s, 2H), 5.59 (d, 2H), 7.63 (t,
2H), 7.81 (t, 1H), 8.16 (d, 4H), 9.49 (s, 2H); HR-MS (EI) m/z
392.1197 (M+).
2-(2-Imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-(4-
trifluoromethylphenyl)ethanone Hydrobromide Salt (5).
5 was prepared from 4-(trifuoromethyl)phenacyl bromide (May-
bridge) and 2-amino-4,5,6,7-tetrahydrobenzothiazole using
method A in 79% yield: mp 272-282 °C; 1H NMR (DMSO-d6)
δ 1.73 (s, 4H), 3.42 (s, 4H), 5.81 (s, 2H), 8.03 (d, 2H), 8.25 (d,
2H), 9.55 (s, 2H); HR-MS (EI) m/z 340.0856 (M+).
4-(5,6,7,8-Tetrahydroimidazo[2,1-b]benzothiazol-2-yl)-
phenylamine (21).11 A mixture of 20 (239 mg, 0.63 mmol),
NaO2CH (136 mg, 2.00 mmol), and KH2PO4 (142 mg, 1.04
mmol) was refluxed for 6 h in 1-methyl-2-pyrrolidinone (5 mL).
The mixture was filtered, and the filtrate was dissolved in
EtOAC and washed with brine. Preparative TLC (5% MeOH/
CH2Cl2) was used to separate the product in 33% yield: mp
1
>150 °C (dec); H NMR (DMSO-d6) δ 1.87 (m, 4H), 2.66 (m,
4H), 6.57 (d, 2H), 7.48 (d, 2H), 7.85 (s, 1H); HR-MS (EI) m/z
269.0984 (M+). Anal. Calcd for C15H18N3O4PS: C, 49.04; H,
4.94; N, 11.44. Found: C, 49.23; H, 5.24; N, 13.59.
4-(5,6,7,8-Tetrahydroimidazo[2,1-b]benzothiazol-2-yl)-
benzoic Acid Hydrochloride Salt (23). A solution of 25 (359
mg, 1.0 mmol) in methanol (25 mL) was refluxed with 25%
aqueous NaOH (8.5 mL) for 3.5 h. The solvent was evaporated
under vacuum, and the solution was made acidic by addition
of concentrated HCl. A precipitate dropped out and was filtered
4-[2-(2-Imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)acetyl]-
benzonitrile Hydrobromide Salt (6).12 was prepared from
4-cyanophenacyl bromide (Maybridge) and 2-amino-4,5,6,7-
tetrahydrobenzothiazole using method A in 75% yield: mp
1
1
322-325 °C; H NMR (DMSO-d6) δ 1.73 (s, 4H), 2.36 (s, 2H),
to give a white solid in 47% yield: mp 315-319 °C; H NMR

Inhibitors of Apoptosis in Lymphocytes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6419
(DMSO-d6) δ 1.89 (d, 4H), 2.73 (s, 5H), 7.95 (m, 4H), 8.51 (d,
1H); HR-MS (EI) m/z 298.0779 (M+).
and R-bromo-4-(1-pyrrolidinyl)acetophenone (Lancaster) (1.109
g, 4.14 mmol) was refluxed in methoxyethanol (30 mL) for 2
h. The solvent was removed under vacuum, and the residue
was purified by flash chromatography on silica gel (1% MeOH
in CH2Cl2) to give 38 as a solid in 25% yield: mp 255-257 °C;
1H NMR (DMSO-d6) δ 1.86 (m, 4H), 2.50 (m, 4H), 2.66 (s, 4H),
3.24 (s, 4H), 6.54 (d, 2H), 7.62 (d, 2H), 7.90 (s, 1H). Anal.
(C19H22BrN3S‚1/5H2O) C, H, N.
2-Naphthalen-2-yl-5,6,7,8-tetrahydroimidazo[2,1-b]ben-
zothiazole Hydrobromide Salt (40). 40 was prepared from
2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-naphthalen-
2-ylethanone hydrobromide salt using method C in 73%
yield: mp >300 °C (dec); 1H NMR (DMSO-d6) δ 1.91 (m, 4H),
2.76 (m, 4H), 7.55 (q, 2H), 7.94 (m, 3H), 8.02 (d, 1H), 8.37 (s,
1H), 8.57 (s, 1H). Anal. (C19H17BrN2S) C, H, N.
4-(5,6,7,8-Tetrahydroimidazo[2,1-b]benzothiazol-2-yl)-
phenol Hydrobromide Salt (24). To a solution of 26 (248
mg, 0.55 mmol) in dry methanol (6 mL) was added a catalytic
amount of sodium methoxide. The mixture was stirred over-
night. A cation-exchange resin (H+ form) was added to the
mixture and stirred for 10 min. The resin was removed by
filtration, and the solvent was evaporated to give a white solid
in 39% yield: mp 239-343 °C (dec); 1H NMR (DMSO-d6) δ
1.89 (d, 4H), 2.76 (s, 4H), 6.88 (d, 2H), 7.62 (d, 2H), 8.38 (s,
1H), 9.86 (s, 1H); HR-MS (EI) m/z 270.0823 (M+). Anal. Calcd
for C15H15BrN2OS: C, 51.29; H, 4.30; N, 7.98. Found: C, 48.38;
H, 4.55; N, 7.63.
4-(5,6,7,8-Tetrahydroimidazo[2,1-b]benzothiazol-2-yl)-
benzonitrile Hydrobromide Salt (25). 25 was prepared
1-(4-Cyclopentylphenyl)-2-(2-iminobenzothiazol-3(2H)-
yl)ethanone Hydrobromide Salt (44). 44 was prepared
from R-bromo-4-(1-cyclopentyl)acetophenone26,27 and 2-ami-
nobenzothiazole using method A in 16% yield: mp 186-190
1
from 6 using method C in 74% yield: mp >310 °C (dec); H
NMR (DMSO-d6) δ 1.87 (m, 4H), 2.70 (m, 4H), 7.86 (d, 2H),
8.00 (d, 2H), 8.49 (s, 1H). Anal. (C16H14BrN3S) C, H, N.
Benzoic Acid 4-(5,6,7,8-Tetrahydrobenzo[d]imidazo-
[2,1-b]thiazol-2-yl)phenyl Ester Hydrobromide Salt (26).
26 was prepared from 2 using method C in 76% yield: mp 255-
257 °C; 1H NMR (DMSO-d6) δ 1.88 (m, 4H), 2.73 (d, 4H), 7.40
(d, 2H), 7.62 (t, 2H), 7.76 (t, 1H), 7.91 (d, 2H), 8.14 (d, 2H),
8.45 (s, 1H). Anal. (C22H19BrN2O2S‚H2O) C, H, N.
1
°C (dec); H NMR (DMSO-d6) δ 1.59 (m, 2H), 1.69, (m, 2H),
1.81 (m, 2H), 2.07 (m, 2H), 3.12 (m, 1H), 6.07 (s, 2H), 7.44 (d,
2H), 7.52 (m, 2H), 7.69 (t, 1H), 7.84 (t, 1H), 8.03 (d, 2H), 9.13
(s, 1H); HR-MS (FAB) m/z 337.1376 (M+). Anal. Calcd for
C20H21BrN2OS: C, 57.56; H, 5.07; N, 6.71. Found: C, 50.82;
H, 4.21; N, 7.65.
2-(4-Trifluoromethylphenyl)-5,6,7,8-tetrahydrobenzo-
[d]imidazo[2,1-b]thiazole Hydrobromide Salt (27). 27 was
prepared from 5 using method C in 43% yield: mp 294-296
°C (dec); 1H NMR (DMSO-d6) δ 1.88 (d, 4H), 2.73 (d, 4H), 7.79
(d, 2H), 8.04 (d, 2H), 8.51 (s, 1H). Anal. (C16H14BrF3N2S) C,
H, N.
3-Bromo-2-p-tolyl-5,6,7,8-tetrahydroimidazo[2,1-b]ben-
zothiazole (28). To a suspension of 39 (372 mg, 1.07 mmol)
in DMF (40 mL) at room temperature was slowly added
dropwise a solution of N-bromosuccinimide (210 mg, 1.18
mmol) in DMF (5 mL). The solvent was removed under high
vacuum, and the residue was slurried in ice-water/ethanol
(50 mL, 50%). The solid that dropped out was collected by
filtration and rinsed with water to give 28 in 100% yield: mp
>165 °C (dec); 1H NMR (DMSO-d6) δ 1.85 (m, 4H), 2.33 (s,
3H), 2.68 (s, 2H), 3.04 (s, 2H), 7.25 (d, 2H), 7.79 (d, 2H); HR-
MS (EI) m/z 346.0138 (M+).
2-(3-Fluorophenyl)-5,6,7,8-tetrahydroimidazo[2,1-b]-
benzothiazole Hydrobromide Salt (31). 31 was prepared
from 9 using method C in 75% yield: mp 293-297 °C; 1H NMR
(DMSO-d6) δ 1.87 (m, 4H), 2.71 (t, 4H), 7.16 (dt, 1H), 7.49 (dt,
1H), 7.65 (m, 2H), 8.49 (s, 1H). Anal. (C15H14BrFN2S) C, H, N.
2-(3,4-Dichlorophenyl)-5,6,7,8-tetrahydroimidazo[2,1-
b]benzothiazole Hydrobromide Salt (34). 34 was prepared
from 11 using method C in 75% yield: mp 314-319 °C; 1H
NMR (DMSO-d6) δ 1.89 (d, 4H), 2.70 (s, 4H), 7.68 (d, 1H), 7.80
(dd, 1H), 8.07 (d, 1H), 8.47 (s, 1H). Anal. (C15H13BrCl2N2S) C,
H, N.
2-(2-Chlorophenyl)-5,6,7,8-tetrahydroimidazo[2,1-b]-
benzothiazole Hydrobromide Salt (35). 35 was prepared
from 15 using method C in 19% yield: mp 189-191 °C; 1H
NMR (DMSO-d6) δ 1.86 (q, 4H), 2.70 (m, 4H), 7.28 (dt, 1H),
7.39 (dt, 1H), 7.50 (dd, 1H), 8.12 (dd, 1H), 8.21 (s, 1H). Anal.
(C15H14BrClN2S‚1/2H2O) C, H, N. HR-MS (EI) m/z 288.0486
(M+).
2-(2-Iminobenzothiazol-3-yl)-1-(4-pyrrolidin-1-ylphenyl)-
ethanone Hydrobromide Salt (46). 46 was prepared from
R-bromo-4-(1-pyrrolidino)acetophenone and 2-amino-4,5,6,7-
tetrahydrobenzothiazole using method A in 43% yield: mp
285-290 °C (dec); 1H NMR (DMSO-d6) δ 2.00 (s, 4H), 3.38 (m,
4H), 5.93 (s, 2H), 6.68 (d, 2H), 7.43 (t, 1H), 7.50 (t, 1H), 7.57
(d, 1H), 7.90 (d, 2H), 8.03 (d, 1H). Anal. (C19H20BrN3OS) C, H,
N.
2-Furan-2-ylimidazo[2,1-b]benzothiazole Hydrobromide
Salt (48). 48 was prepared from 2-bromo-1-furan-2-ylethanone
and 2-aminobenzothiazole using method B in 30% yield: mp
>250 °C (dec); 1H NMR (DMSO-d6) δ 6.63 (s, 1H), 6.78 (s, 1H),
7.48 (t, 1H), 7.60 (t, 1H), 7.77 (s, 1H), 8.09 (d, 1H), 8.13 (d,
1H), 8.68 (s, 1H). Anal. (C13H9BrN2OS‚2/3H2O) C, H, N.
2-p-Tolylbenzo[d]imidazo[2,1-b]thiazol-7-ol (49). A mix-
ture of 54 (362 mg, 0.845 mmol) and copper(I) oxide (46 mg,
0.321 mmol) in a 30% aqueous solution of NaOH (4 mL) was
heated in a bomb for 4 h. The mixture was neutralized and
extracted with EtOAc. The solid recovered upon evaporation
was purified by preparative TLC to give 49 in 47% yield: mp
275-277 °C; 1H NMR (DMSO-d6) δ 2.31 (s, 3H), 6.95 (dd, 1H),
7.22 (d, 2H), 7.38 (d, 1H), 7.72 (d, 2H), 7.77 (d, 1H), 8.60 (s,
1H), 9.95 (s, 1H); HR-MS (EI) m/z 280.0662 (M+). Anal. Calcd
for C16H13BrN2OS‚1/2HBr: C, 59.90; H, 3.93; N, 8.73. Found:
C, 59.47; H, 4.43; N, 8.55.
3-Bromo-2-p-tolylimidazo[2,1-b]benzothiazole (51). To
a suspension of 58 (1.643 g, 4.76 mmol) in DMF (150 mL) at
room temperature was slowly added dropwise a solution of
N-bromosuccinimide (933 mg, 5.24 mmol) in DMF (6 mL). The
solvent was removed under high vacuum, and the residue was
slurried in ice-water/ethanol (100 mL, 50%). A solid dropped
out and was collected by filtration and rinsed with water to
give 51 in 82% yield: mp >180 °C (dec); 1H NMR (DMSO-d6)
δ 2.36 (s, 3H), 7.31 (d, 2H), 7.51 (t, 1H), 7.61 (t, 1H), 7.90 (d,
2H), 8.11 (d, 1H), 8.44 (d, 1H). Anal. (C16H11BrN2S‚1/3H2O) C,
H, N.
2-(3,4-Difluorophenyl)-5,6,7,8-tetrahydroimidazo[2,1-
b]benzothiazole Hydrobromide Salt (36). 36 was prepared
from 7 using method C in 68% yield: mp 301-305 °C (dec);
1H NMR (DMSO-d6) δ 1.89 (d, 4H), 2.72 (s, 4H), 7.54 (qt, 1H),
7.68 (m, 1H), 7.87 (m, 1H), 8.48 (d, 1H). Anal. (C15H13BrF2N2S)
C, H, N.
2-p-Tolylimidazo[2,1-b]benzothiazol-7-ylamine (52). To
a suspension of 7-nitro-2-p-tolylimidazo[2,1-b]benzothiazole
hydrobromide salt (128 mg, 0.328 mmol) in water at 80 °C were
added four portions (10 equiv) of sodium dithionite over 30
min whereupon the suspension become clear and decolorized
1
to give 52 in 22% yield: mp >210 °C (dec); H NMR (DMSO-
2-(4-Azidophenyl)-5,6,7,8-tetrahydro-imidazo[2,1-b]ben-
d6) δ 2.32 (s, 3H), 6.80 (dd, 1H), 7.13 (d, 1H), 7.23 (d, 2H),
7.65 (d, 1H), 7.72 (d, 2H), 8.53 (s, 1H); HR-MS (EI) m/z
279.0821 (M+). Anal. Calcd for C16H13N3S‚7/3HBr: C, 41.05;
H, 3.30; N, 8.98. Found: C, 41.81; H, 2.86; N, 6.10.
zothiazole Hydrobromide Salt (37). 37 was prepared from
1
10 using method C in 62% yield: mp >200 °C (dec); H NMR
(DMSO-d6) δ 1.88 (m, 4H), 2,73 (s, 4H), 7.23 (d, 2H), 7.86 (d,
2H), 8.43 (s, 1H). Anal. (C15H14BrN5OS) C, H, N.
2-(4-Pyrrolidin-1-ylphenyl)-5,6,7,8-tetrahydroimidazo-
[2,1-b]benzothiazole Hydrobromide Salt (38). A mixture
of 2-amino-4,5,6,7-tetrahydrobenzothiazole (584 mg, 3.79 mmol)
2-(4-Cyclopentylphenyl)imidzxo[2,1-b]benzothiazoles
Hydrobromide Salt (53). 53 was prepared from 44 using
method method C in 65% yield: mp 250 °C (dec); 1H NMR

6420 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Barche´chath et al.
(DMSO-d6) δ 1.57 (m, 2H), 1.66, (m, 2H), 1.79 (m, 2H), 2.03
(m, 2H), 3.00 (q, 1H), 7.33 (d, 2H), 7.45 (t, 1H), 7.59 (t, 1H),
7.78 (d, 2H), 8.01 (d, 1H), 8.06 (d, 1H), 8.77 (s, 1H); HR-MS
(FAB) m/z 319.1267 (M+). Anal. Calcd for C20H18N2S‚1/4HBr:
C, 70.93; H, 5.43; N, 8.27. Found: C, 70.01; H, 5.82; N, 10.97.
7-Bromo-2-p-tolylimidazo[2,1-b]benzothiazole Hydro-
bromide Salt (54). 54 was prepared from 2-bromo-4′-methyl-
acetophenone and 2-amino-6-bromobenzothiazole using method
yield: mp 76-78 °C; 1H NMR (DMSO-d6) δ 2.42 (s, 3H), 5.15
(s, 2H), 7.36 (t, 1H), 7.41 (d, 2H), 7.44 (t, 1H), 7.78 (d, 1H),
8.00 (m, 3H). Anal. (C16H14BrNOS2‚1/2H2O) C, H, N.
(2-Imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)acetic Acid
Ethyl Ester Hydrobromide Salt (66).12 A mixture of
2-amino-4,5,6,7-tetrahydrobenzothiazole (2.296 g, 14.9 mmol)
and ethyl bromoacetate (2.747 g, 16.4 mmol) was refluxed in
ethanol (50 mL) containing three drops of Et3N for 90 min.
The reaction mixture was concentrated to half volume in
vacuo, and the resulting precipitate was filtered and rinsed
with cold ethanol and then ether to give a white powder in
79% yield: mp 222-224 °C; 1H NMR (DMSO-d6) δ 1.25 (t, 3H),
1.74 (d, 4H), 2.39 (s, 2H), 2.52 (m, 2H), 4.22 (q, 2H), 4.95 (s,
2H), 9.67 (s, 2H). Anal. (C11H17BrlN2O2S) C, H, N. HR-MS (EI)
m/z 240.0931 (M+).
1
B in 24% yield: mp 205-208 °C; H NMR (DMSO-d6) δ 2.31
(s, 3H), 7.23 (d, 2H), 7.73 (m, 3H), 7.91 (d, 1H), 8.32 (s, 1H),
8.70 (s, 1H); HR-MS (EI) m/z 341.9822 (M+).
7-Methoxy-2-p-tolylimidazo[2,1-b]benzothiazole Hydro-
bromide Salt (56). 56 was prepared from 2-bromo-4′-methyl-
acetophenone and 2-amino-6-bromobenzothiazole using method
1
B in 62% yield: mp 274-276 °C (dec); H NMR (DMSO-d6) δ
4-Phenyl-1H-imidazole-2-thiol (69) was synthesized fol-
2.33 (s, 3H), 3.84 (s, 3H), 7.18 (dd, 1H), 7.26 (d, 2H), 7.72 (m,
3H), 7.92 (d, 1H), 8.71 (s, 1H). Anal. (C17H15BrN2OS) C, H, N.
2-(4-Pyrrolidin-1-ylphenyl)benzo[d]imidazo[2,1-b]thi-
azole Hydrobromide Salt (60). 60 was prepared from
R-bromo-4-(1-pyrrolidino)acetophenone (Lancaster) and 2-ami-
nobenzothiazole using method B and 10 h of reflux in 16%
lowing the procedure described by Maeda et al.16
3-Phenyl-5,6,7,8-tetrahydroimidazo[2,1-b]benzothi-
azole Hydrochloride Salt (70). A solution of 4-phenyl-1H-
imidazole-2-thiol 6916 and 2-chlorocyclohexanone was refluxed
in butanol for 3 h. After the mixture was cooled, the resulting
precipitate was filtered to give 70 in 75% yield: mp 274-278
1
yield: mp 283-287 °C; H NMR (DMSO-d6) δ 1.98 (m, 4H),
1
°C; H NMR (DMSO-d6) δ 1.89 (m, 4H), 2.76 (m, 4H), 7.38 (t,
3.31 (m, 4H), 6.68 (m, 2H), 7.51 (d, 1H), 7.66 (d, 3H), 8.11 (m,
2H), 8.71 (s, 1H). Anal. (C19H18BrN3S) C, H, N.
1H), 7.49 (t, 2H), 7.88 (d, 2H), 8.51 (s, 1H); HR-MS (EI) m/z
254.0874 (M+).
3-Benzyl-3H-benzothiazol-2-ylideneamine Hydrochlo-
ride Salt (61).12 A mixture of 2-aminobenzothiazole (2.116 g,
13.7 mmol), benzyl chloride (2.1 mL, 18.2 mmol), and sodium
iodide (200 mg) was refluxed for 8 h in methoxyethanol (25
mL). Crystallization occurred upon cooling. The crystals were
filtered off and thoroughly washed with ether to give yellow
crystals in 39% yield: mp 275-278 °C; 1H NMR (DMSO-d6) δ
5.65 (s, 2H), 7.28, (d, 2H), 7.36 (m, 4H), 7.50 (t, 1H), 7.55 (d,
1H), 8.02 (s, 1H); HR-MS (EI) m/z 240.0719 (M+).
2-(2-Iminothiazol-3-yl)-1-p-tolylethanone Hydrobromide
Salt (73). 73 was prepared from 2-bromo-4′-methylacetophe-
none and 2-aminothiazole using method A in 64% yield: mp
>220 °C (dec); 1H NMR (DMSO-d6) δ 2.42 (s, 3H), 5.79, (s,
2H), 7.06 (d, 1H), 7.35 (d, 1H), 7.44 (d, 2H), 7.92 (d, 2H), 9.55,
(s, 2H). Anal. (C12H13BrN2OS‚1/3H2O) C, H, N.
6-p-Tolylimidazo[2,1-b]thiazole Hydrobromide Salt (74).
74 was prepared from 73 using a modification of method C
with methoxyethanol instead of ethanol. The solvent was
evaporated, and the residue was recrystallized from ethanol
in 49% yield: mp 260-262 °C; 1H NMR (DMSO-d6) δ 2.35 (s,
3H), 7.31 (d, 2H), 7.55 (d, 1H), 7.72 (d, 2H), 8.16 (d, 1H), 8.41
(s, 1H). Anal. (C12H11BrN2S) C, H, N.
3-(4-Methylbenzyl)-3H-benzothiazol-2-ylideneamine Hy-
drochloride Salt (62). A mixture of 2-aminobenzothiazole
(2.104 g, 13.6 mmol) and 4-methylbenzyl chloride (2.25 mL,
12.5 mmol) was refluxed for 8 h in methoxyethanol in the
presence of a catalytic amount of sodium iodide (220 mg, 1.46
mmol). After the mixture was cooled, a precipitate formed that
was filtered and rinsed with ether to give 35% yield: mp 243-
3-(2-Oxo-2-p-tolylethyl)benzothiazol-2(3H)-one (75). To
a solution of 2-hydroxybenzothiazole (1.17 g, 7.58 mmol) in
THF (10 mL) was added Et3N (1.1 mL, 7.89 mmol). A solution
of 2-bromo-4′-methylacetophenone (1.67 g, 7.06 mmol) in THF
(10 mL) was added dropwise. Another equivalent of Et3N was
added after 24 h. Purification by flash chromatography on
silica gel (CH2Cl2) gave a white powder in 67% yield: mp 168-
1
248 °C; H NMR (DMSO-d6) δ 2.26 (s, 3H), 5.65 (s, 2H), 7.20
(dd, 4H), 7.39 (t, 1H), 7.48 (t, 1H), 7.55 (d, 1H), 8.02 (d, 1H);
HR-MS (EI) m/z 254.0870 (M+).
N-(3H-Benzothiazol-2-ylidene)-4-methyl-benzenesul-
fonamide (63). To a solution of 2-aminobenzothiazole (1.018
g, 6.78 mmol) in dry pyridine (4 mL) was added portionwise
p-toluenesulfonyl chloride (1.42 g, 7.48 mmol). The solution
turned yellow upon addition. After 5 min of stirring at room
temperature, the mixture was heated (70-80 °C) for 5 min.
The mixture was poured on a bed of ice, and the resulting
precipitate was filtered off and dried overnight in a desiccator
under vacuum to give a yellow powder in 93% yield: mp 246-
249 °C; 1H NMR (DMSO-d6) δ 2.36 (s, 3H), 7.26, (dt, 1H), 7.30
(d, 1H), 7.37 (d, 2H), 7.40 (dt, 1H), 7.75 (d, 2H), 7.81 (d, 1H).
Anal. (C14H12N2O2S2) C, H, N.
4-Methyl-N-[3-(2-oxo-2-p-tolylethyl)-3H-benzothiazol-
2-ylidene]benzenesulfonamide (64). To a solution of 63
(930 mg, 3.01 mmol) in DMF (15 mL) at room temperature
was added NaH. After the effervescence subsided, 2-bromo-
4′-methylacetophenone (775 mg, 3.36 mmol) was added. The
reaction mixture was first stirred at room temperature for an
hour and then heated at 80 °C until completion (by TLC). The
mixture was allowed to cool before being poured on ice (400
mL). A precipitate dropped out. It was filtered and washed
with cold water and dried to give a pale-yellow powder in
quantitative yield: mp 180-182 °C; 1H NMR (DMSO-d6) δ 2.35
(s, 3H), 2.43 (s, 3H), 5.86 (s, 2H), 7.33 (m, 3H), 7.42 (m, 3H),
7.57 (d, 1H), 7.64 (d, 2H), 7.91 (d, 1H), 7.97 (d, 2H). Anal.
(C23H20N2O3S2) C, H, N.
1
170 °C; H NMR (DMSO-d6) δ 2.43 (s, 3H), 5.62 (s, 2H), 7.21
(t, 1H), 7.27 (d, 1H), 7.32 (t, 1H), 7.42 (d, 2H), 7.70 (d, 1H),
8.01 (d, 2H). Anal. (C16H13NO2S) C, H, N.
3-(2-Oxo-2-p-tolylethyl)-4,5,6,7-tetrahydrobenzothiazol-
2(3H)-one (76). To a solution of 2-hydroxy-4,5,6,7-tetrahy-
drobenzothiazole (335 mg, 2.16 mmol) in DMF (3 mL) was
added NaH (101 mg, 2.52 mmol). After the mixture was stirred
for 10 min at room temperature, a solution of 2-bromo-4′-
methylacetophenone (668 mg, 2.82 mmol) in DMF (5 mL) was
added and the mixture was stirred overnight. Water was
added, and the mixture was extracted with EtOAc and dried
over MgSO4. The solvent was evaporated. The oil recovered
was purified by flash chromatography on silica gel (CH2Cl2)
1
to give 76 as a solid in 65% yield: mp 108-110 °C; H NMR
(DMSO-d6) δ 1.73 (s, 4H), 2.19 (s, 2H), 2.40 (m, 5H), 5.23 (s,
2H), 7.39 (d, 2H), 7.95 (d, 2H). Anal. (C16H17NO2S) C, H, N.
N-(4-Methylbenzylidene)-N′-quinazolin-4-yl-hydrazine
(77). A solution of quinazolin-4-ylhydrazine28 (265 mg, 1.65
mmol) was refluxed with tolualdehyde (400 µL, 3.39 mmol) in
methanol for 1 h. The solvent was evaporated and the resulting
solid was triturated with water and filtered to give a yellow
powder in 87% yield: mp 211-214 °C; 1H NMR (DMSO-d6) δ
2.37 (s, 3H), 7.29 (d, 2H), 7.47 (dt, 2H), 7.67 (t, 1H), 7.88 (d,
3H), 8.20 (m, 1H), 8.51 (s, 1H), 11.65 (s, 1H); HR-MS (EI) m/z
262.1223 (M+).
2-(Benzothiazol-2-ylsulfanyl)-1-p-tolylethanone Hydro-
bromide Salt (65). A mixture of 2-mercaptobenzothiazole (570
mg, 3.34 mmol) and 2-bromo-4′-methylacetophenone (870 mg,
3.67 mmol) was refluxed in ethanol (15 mL) for 90 min. The
solution was cooled in the fridge to give a yellow solid in 18%
1-(2-p-Tolyl-2H-[1,2,4]triazolo[1,5-c]quinazolin-3-yl)-
ethanone (78). A solution of 77 (273 mg, 1.04 mmol) in acetic
anhydride (5 mL) was refluxed for 12 h. After cooling, the
mixture was poured on ice and extracted with CH2Cl2, washed

Inhibitors of Apoptosis in Lymphocytes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6421
with aqueous Na2CO3, and dried over MgSO4. The solvent was
removed under vacuum, and the product was cooled to promote
crystallization. The solid was triturated with methanol to give
47% yield: mp 166-169 °C; 1H NMR (DMSO-d6) δ 2.20 (s, 3H),
2.29 (s, 3H), 7.22 (m, 3H), 7.34 (d, 2H), 7.47 (m, 2H), 7.67 (dt,
1H), 7.79 (s, 1H), 7.88 (dd, 1H). Anal. (C18H16N4O) C, H, N.
2-p-Tolyl[1,2,4]triazolo[1,5-c]quinazoline (79). A mix-
ture of 78 (124 mg, 0.407 mmol) and ferric chloride (260 mg,
0.961 mmol) in water (5 mL) was heated under reflux for 4 h.
The insoluble salts were filtered off while hot, and the solution
was cooled. The resulting crystals were filtered to give 50%
yield: mp 249-251 °C; 1H NMR (DMSO-d6) δ 2.41 (s, 3H),
7.40 (d, 2H), 7.86 (t, 1H), 7.96 (t, 1H), 8.09 (d, 1H), 8.19 (d,
2H), 8.53 (d, 1H), 9.65 (s, 1H). Anal. (C16H12N4‚H2O) C, H, N.
Anal. Calcd for C17H13Cl2N3: C, 61.83; H, 3.97; N, 12.73.
Found: C, 40.84; H, 2.90; N, 8.58.
2-(5-Chloro-2-iminobenzooxazol-3-yl)-1-p-tolylethanone
Hydrobromide Salt (86). A solution of 2-amino-5-chlorobenz-
oxazole 1.172 g, 6.74 mmol) and 2-bromo-4′-methylacetophe-
none (1.81 g, 7.64 mmol) was refluxed in ethanol (50 mL) for
90 min. A precipitate formed. It was filtered and rinsed with
ether to give 33% yield: mp 288-290 °C (dec); 1H NMR
(DMSO-d6) δ 2.44 (s, 3H), 5.86 (s, 2H), 7.47 (d, 3H), 7.81 (d,
1H), 7.97 (m, 3H). Anal. (C16H14BrClN2O2) C, H, N.
Acknowledgment. We thank Michael Rosenbach
and Kathy Pekny for their assistance and Nancy Noon
for secretarial support. We thank the Rheumatic Dis-
eases Core Center for breeding our mice and the UCSD
NMR and MS facilities. This work was supported in part
by a research agreement with Salmedix, Inc., Grant
GM23200 from the National Institutes of Health, and
the UC Biotechnology Strategic Targets for Alliances
in Research Project (BioSTAR).
2-(Quinazolin-4-ylamino)-1-p-tolylethanone Hydro-
bromide Salt (80) and 2-p-Tolylimidazo[1,2-c]quinazo-
line Hydrobromide Salt (81). To a hot solution of 4-amino-
quinazoline (420 mg, 2.89 mmol) in ethanol (50 mL) was added
2-bromo-4′-methylacetophenone (931 mg, 3.93 mmol). The
mixture was refluxed for 4 h. After the mixture was cooled,
the precipitate was filtered to give 80 in 44% yield: mp 312-
1
313 °C (dec); H NMR (DMSO-d6) δ 2.44 (s, 3H), 6.30 (s, 2H),
References
7.47 (d, 2H), 7.86 (m, 2H), 8.05 (m, 3H), 8.54 (d, 1H), 8.90 (s,
1H), 10.12 (s, 2H). Anal. (C17H16BrN3O) C, H, N. Flash
chromatography on silica gel (3:7 EtOAc/hex) separated the
ring closed 81 in 20% yield: mp 215-218 °C; 1H NMR (DMSO-
d6) δ 2.35 (s, 3H), 7.29 (d, (2H), 7.74 (m, 2H), 7.94 (t, 3H), 8.47
(m, 2H), 9.27 (s, 1H); HR-MS (EI) m/z 259.1106 (M+). Anal.
Calcd for C16H13N3S‚1/4H2O: C, 77.40; H, 5.16; N, 15.93.
Found: C, 77.74; H, 6.18; N, 15.31.18
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(500 mg, 12.5 mmol) in small portions. After the effervescence
subsided, 4′-methyl-4-bromoacetophenone (2.64 g, 12.4 mmol)
was added, and the solution was stirred overnight at room
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1
184 °C; H NMR (DMSO-d6) δ 2.44 (s, 3H), 5.71 (s, 2H), 7.37
(m, 2H), 7.44 (d, 2H), 7.80 (t, 1H), 8.05 (d, 2H), 8.09 (d, 1H);
HR-MS (EI) m/z 295.0842 (M+).
Methyl-1-[2-(2-oxo-2-p-tolylethylamino)benzoyl]isothio-
urea (83). A suspension of 82 (2.317 g, 7.85 mmol), Na2CO3
(915 mg, 8.63 mmol), and 2-methyl-2-thiopseudourea sulfate
(1.092 g, 3.92 mmol) was refluxed in acetonitrile (30 mL) for
1
30 min. TLC showed starting material left and /3 more Na2-
CO3 and 2-Methyl-2-thiopseudourea sulfate was added, and
the mixture was refluxed for an additional 30 min. The solvent
was removed under vacuum, and the residue was triturated
in methylene chloride. The insoluble salts were removed by
filtration, and the filtrate was concentrated under vacuum.
The residue was recrystallized from methanol to give 17%
yield: mp 305 °C (dec); 1H NMR (DMSO-d6) δ 2.41 (s, 3H),
2.50 (s, 3H), 4.84 (d, 2H), 6.59 (t, 1H), 6.79 (d, 1H), 7.33 (t,
1H), 7.40 (d, 2H), 8.01 (d, 2H), 8.28 (d, 1H), 9.20 (m, 3H); HR-
MS (EI) m/z 341.1239 (M+). Anal. Calcd for C18H19N3O2S: C,
63.32; H, 5.61; N, 12.31. Found: C, 68.94; H, 5.17; N, 13.88.
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refluxed for 2 h with a catalytic amount of NaOH. A precipitate
formed upon cooling and was collected by filtration and rinsed
with EtOAC to give 84 in 65% yield: mp 325-328 °C (dec);
1H NMR (DMSO-d6) δ 2.33 (s, 3H), 3.50 (br, 1H), 7.24 (d, 2H),
7.49 (t, 1H), 7.74 (d, 2H), 7.90 (t, 1H), 8.03 (d, 1H), 8.19 (d,
1H), 8.50 (d, 1H); HR-MS (EI) m/z 275.1069 (M+).
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5-Chloro-2-p-tolylimidazo[1,2-a]quinazoline (85). A so-
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90 min. The mixture was poured onto ice, stirred for 10 min,
and filtered. The residue was recrystallized to give 85 in 20%
yield: mp 190 °C (dec); 1H NMR (DMSO-d6) δ 2.37 (s, 3H),
7.35 (d, 2H), 7.86 (t, 1H), 7.94 (d, 2H), 8.24 (t, 1H), 8.41 (d,
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