2-Amino-4-benzyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridines: Novel selective β3-adrenoceptor agonists

Article abstract of DOI:10.1021/jm990012z

Trimetoquinol (TMQ, 7) is a potent nonselective β-adrenoceptor (AR) agonist. Replacement of the catechol moiety of TMQ with a 2-aminothiazole group resulted in novel thiazolopyridine derivatives 9-11 which have been synthesized and evaluated for biologica

Full text of DOI:10.1021/jm990012z

J . Med. Chem. 1999, 42, 2287-2294
2287
2-Am in o-4-ben zyl-4,5,6,7-tetr a h yd r oth ia zolo[5,4-c]p yr id in es: Novel Selective
â3-Ad r en ocep tor Agon ists
Weiping Zheng,† Victor I. Nikulin,† Anish A. Konkar,‡,§ Sandeep S. Vansal,§ Gamal Shams,‡
Dennis R. Feller,‡ and Duane D. Miller*,†
Department of Pharmaceutical Sciences, University of Tennessee, Memphis, Tennessee 38163, Division of Pharmacology,
College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, and Department of Pharmacology and National
Center for the Development of Natural Products, School of Pharmacy, University of Mississippi, University, Mississippi 38677
Received J anuary 11, 1999
Trimetoquinol (TMQ, 7) is a potent nonselective â-adrenoceptor (AR) agonist. Replacement of
the catechol moiety of TMQ with a 2-aminothiazole group resulted in novel thiazolopyridine
derivatives 9-11 which have been synthesized and evaluated for biological activity on human
â1-, â2-, and â3-AR. The Bischler-Napieralski reaction has been employed as a novel approach
to construct the 2-amino-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine ring system. Although in
radioligand binding studies analogues 9 and 10 did not show selectivity toward â3-AR, they
exhibited a high degree of selective â3-AR agonist activity in functional assays. Moreover, the
â3-AR agonist activity of the 2-aminothiazole derivatives is abolished by N-acetylation (analogue
11) or ring opening (analogue 25). This illustrates the importance of the intact 2-amino-4,5,6,7-
tetrahydrothiazolo[5,4-c]pyridine ring for â3-AR activity.
Ch a r t 1
In tr od u ction
Following the classification of â-adrenergic receptors
(AR) into â1- and â2-subtypes in 1967,1 a third member
of this family was pharmacologically identified in 19842
and was designated as the â3-AR. The subsequent
cloning and characterization of â3-AR from genomic
libraries of several species including human,3 mouse,4
and rat5 provided conclusive evidence for its existence.
Human â3-AR and its mRNA have been found in the
GI tract (e.g., small intestine, gall bladder) and adipose
tissue (e.g., omental, retroperitoneal, mammary subcu-
taneous, abdominal subcutaneous fat).6-10
The â3-AR has been shown to mediate various phar-
macological and physiological effects such as lipolysis,
thermogenesis, and intestinal smooth muscle relaxation
in rodents.11 It has also been thought to play an
important role in glucose homeostasis and energy bal-
ance in humans.12 Additional evidence for this role was
provided by the discovery of a Trp64Arg mutation in
the human â3-AR that is associated with the early onset
of non-insulin-dependent diabetes mellitus (NIDDM) in
Pima Indians,13 a greater dynamic capacity to gain
weight in French obese patients,14 and an increased
tendency to develop NIDDM in Finnish obese patients.15
Therefore, a number of laboratories12,16 have pursued
selective â3-AR agonists that have the potential for
treating obesity, NIDDM, and intestinal hypermotility
disorders. These agonists shall also be able to help
further define the physiological roles played by the â3-
AR. The current â3-AR agonists belong to two general
structural classes (Chart 1): i.e., phenethanolamines,
e.g., BRL compounds (1-4) and CL 316243 (5), and
aryloxypropanolamines, e.g., CGP-12177 (6). Despite
these reported endeavors, none of the compounds in-
vestigated have been reported to produce good thera-
peutic effects in humans due to a lack of selectivity or
potency.17 Hence, efforts are underway in our and other
laboratories to synthesize novel, potent, selective â3-AR
agonists.
Trimetoquinol (TMQ, 7; Chart 2) is a known potent,
nonselective â-AR agonist, used clinically in J apan as
a bronchorelaxant.18-21 Our laboratory has focused on
TMQ structural modifications in order to obtain selec-
tive â-AR agonists. Our previous structural modifica-
tions of TMQ have generated potent, selective â2-AR
agonists.22 In this study, we report novel structural
modifications of TMQ, with the goal of synthesizing
potent and selective â3-AR agonists. It has been discov-
ered that the catechol moiety of TMQ is indispensable
for the agonist activities at both human â1- and â2-AR,23
whereas its degree of contribution to TMQ’s â3-AR
† University of Tennessee.
‡ The Ohio State University.
§ University of Mississippi.
10.1021/jm990012z CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/27/1999

2288 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Brief Articles
Ch a r t 2
Sch em e 1a
a
(a) Methyl vinyl ketone, NaOEt, EtOH, EtOAc, rt f reflux;
(b) (i) Br2, MeOH, 0 °C f rt, (ii) 10.0 M H2SO4, rt; (c) thiourea,
acetone, rt; (d) 30% HBr, reflux.
â-AR subtypes to determine the effects of N-acetylation
or ring opening, respectively, on biological activities.
Analogue 12 was designed to determine the effect of
switching positions of S and N of the thiazole ring in
analogue 9. However, several attempts to prepare 12
were unsuccessful.
activity has not been evaluated. On the basis of the
observation that many previously claimed â3-AR ago-
nists lack a catechol moiety, we hypothesized that the
catechol of TMQ is not necessary for the observed
activity at â3-AR, and selective â3-AR agonists will thus
be achieved by modifying this catechol group. Compound
9 (Chart 2) has been designed by replacing catechol with
2-aminothiazole. It is also expected that this replace-
ment will lead to chemically and enzymatically more
stable compounds, because the stability problems as-
sociated with the catechol have been circumvented.
Indeed, 2-aminothiazole has been employed to replace
the catechol or phenolic group to afford orally active
dopamine autoreceptor agonists.24 We have previously
found that substitution of the 3′- and 5′-methoxy groups
of TMQ with iodine atoms (i.e., 8, Chart 2) is well-
tolerated at â1- and â2-AR.25 Furthermore, compound 8
exhibits greater binding affinities at all three â-AR
subtypes (especially at â3-AR) than TMQ (Table 1); a
similar modification was thus applied to analogue 9 to
give analogue 10. Compounds 11 and 25 were also
synthesized and their activities evaluated at the three
Ch em istr y
The preparation of 2-amino-4,5,6,7-tetrahydrothia-
zolo[5,4-c]pyridines has been well-documented in
the literature.26-28 They are constructed by two ap-
proaches: (a) Pictet-Spengler reactionsthe condensa-
tion and subsequent cyclization between an aldehyde
and 2-(2′-amino-4′-thiazolyl)ethylamine derivatives; (b)
Hantzsch thiazole synthesissthe condensation and
subsequent cyclization between an R-bromopiperidone
derivative and thiourea (Chart 3). However, it was
envisioned that both of these approaches would not meet
our synthetic challenge. The Pictet-Spengler reaction
would necessitate the use of an inherently unstable
substituted phenylacetaldehyde, whereas the required
R-bromopiperidone for Hantzsch thiazole synthesis is
not readily accessible synthetically. Alternatively, the
Bischler-Napieralski reaction (Chart 3) is an attractive
Ch a r t 3

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2289
Sch em e 2a
approach for the synthesis of our designed TMQ ana-
logues, because the required substituted phenylacetic
acids are stable and readily accessible. The Bischler-
Napieralski reaction is routinely used to prepare 3,4-
dihydroisoquinolines;29 however, its application to the
preparation of 2-amino-4,5,6,7-tetrahydrothiazolo[5,4-
c]pyridines via the corresponding dihydro intermediate
is novel.
According to Timmerman’s synthetic scheme,30 com-
pound 15, which is the starting amine for the Bischler-
Napieralski approach, has been prepared (Scheme 1).
However, in our hands, the transformation from 13 to
14 did not proceed in good yield as described. Instead,
the procedure described by Sprague et al.31 converted
13 to 14 almost quantitatively.
Scheme 2 depicts the synthesis of analogues 9 and
10. Under the Schotten-Baumann conditions, substi-
tuted phenylacetic acid 16 or 1732 was allowed to react
with compound 15. The amide precursors 18 and 19
were obtained, and each was treated with POCl3 in
refluxing acetonitrile. The dihydro intermediate was
reduced in situ with NaBH4, giving final compounds 9
and 10 which were purified by crystallization or column
chromatography on silica gel.
a
(a) Oxalyl chloride, dry benzene, 0 °C f rt f reflux; (b) 14,
NaOH, CHCl3, H2O, rt; (c) POCl3, CH3CN, reflux; (d) NaBH4,
MeOH, 0 °C f rt; (e) 1.0 M HCl in Et2O.
Sch em e 3a
The acetamido analogue 11 was prepared according
to Scheme 3. Acetylation of 19 with acetic anhydride
gave precursor 20 that was then subjected to POCl3 and
NaBH4. Compound 11 was isolated as its maleic acid
salt. Attempts to make the salt of 11 with stronger acids
(HCl, oxalic acid) failed because of spontaneous deacety-
lation.
Analogue 12 (Scheme 4) is a derivative of 2-amino-
4,5,6,7-tetrahydrothiazolo[4,5-c]pyridine that is an un-
known heterocyclic system. Amide precursor 22 has
been prepared from compound 21 and 3,4,5-trimethoxy-
phenylacetic acid under Schotten-Baumann conditions.
Compound 21 was synthesized according to a litera-
ture scheme (Scheme 5);30 however, the conversion from
the R-bromoaldehyde 27 to 2-aminothiazole 28 was
again achieved effectively by using the procedure of
Sprague et al.31 Using various conditions29,33,34 of the
a
(a) Acetic anhydride, dry benzene, dry CH3CN, reflux; (b)
POCl3, CH3CN, reflux; (c) NaBH4, MeOH, 0 °C f rt; (d) maleic
acid, CH3CN.
Sch em e 4a
a
(a) 3,4,5-Trimethoxyphenylacetyl chloride, NaOH, CHCl3, H2O, rt; (b) (i) POCl3, CH3CN, reflux, (ii) NaBH4, MeOH, 0 °C f rt; (c)
P2O5, celite, dry CHCl3, dry benzene, reflux; (d) acetic anhydride, dry benzene, dry CH3CN, reflux; (e) (i) BH3‚THF, THF, 0 °C f rt f
reflux, (ii) 1.0 M HCl in Et2O.

2290 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Brief Articles
Sch em e 5a
a
(a) 4-Chloro-1-butanol, dry DMF, 70 ˚C; (b) (i) oxalyl chloride, dry CH2Cl2, dry DMSO, -60 ˚C f -50 °C, (ii) Et3N, H2O, -50 °C f
rt; (c) Br2, dry CCl4, rt; (d) thiourea, acetone, rt; (e) 30% HBr, reflux.
Ta ble 1. Binding Affinities of TMQ Analogues on Human â-AR
Subtypes
pKi ( SEMa
human â1-AR
human â2-AR
human â3-AR
ISO
TMQ
8
9
10
5.80 ( 0.07
6.49 ( 0.06
7.10 ( 0.06
5.21 ( 0.08
6.14 ( 0.08
6.17 ( 0.12
7.36 ( 0.23
8.69 ( 0.16
6.21 ( 0.12
6.37 ( 0.08
4.73 ( 0.25
5.43 ( 0.28
7.67 ( 0.24
4.17 ( 0.05
5.83 ( 0.15
a
Human â1-, â2-, and â3-AR were expressed in CHO cells.
[
125I]ICYP was used as the radioligand. Ki values were calculated
using the following equation: Ki(M) ) IC50(1 + [L]/Kd), wherein
IC50 is the molar concentration of an analogue at which the
radioligand binding was reduced by 50%, [L] is the radioligand
concentration used, and Kd is the radioligand equilibrium dis-
sociation constant. pKi ) -log Ki; SEM, standard error of mean;
n ) 3-9.
F igu r e 1. Concentration-response curves of compound 10
on cAMP accumulations in CHO cells expressing human â1-,
â2-, and â3-AR. Drug-induced changes in cellular cAMP were
determined by the CRE-LUC assay (see Experimental Sec-
tion). Data are the mean ( SEM of n ) 8.
Bischler-Napieralski reaction, attempts to cyclize the
amide precursor 22 proved to be unsuccessful. On the
basis of the published mechanism35 of the Bischler-
Napieralski reaction, in which the cyclization step is
achieved by aromatic electrophilic substitution via a
nitrilium intermediate, the failed cyclizations in our
studies could be due to the low negative charge density
at C4 of precursors 22 and 23, which has been predicted
by semiempirical charge density calculations (W. Zheng,
J . E. De Los Angeles, D. D. Miller, unpublished results).
However, 22 has been reduced by BH3 to give the open-
chain analogue 25 that has also been examined in all
three human â-AR subtypes. Similarly, the acetamido
amide precursor 23 also failed to give the cyclized
product under the reaction conditions described.
Biologica l Resu lts a n d Discu ssion
Chinese hamster ovary (CHO) cells expressing human
â1-, â2-, or â3-AR were used to evaluate the newly
designed and synthesized TMQ analogues. Table 1
compares the binding affinities of analogues 9 and 10
at human â1-, â2-, or â3-AR to those of the reference
compounds, (R)-(-)-isoproterenol (ISO), TMQ, and 8.
Receptor activation potencies and intrinsic activities of
the novel TMQ analogues, together with those for
reference compounds (ISO, TMQ, 8), at human â1-, â2-,
or â3-AR are given in Table 2. Both cAMP radioimmu-
noassay and cAMP response element (CRE)-luciferase
Ta ble 2. Functional Activities of TMQ Analogues on Human â-AR Subtypes
human â1-AR human â2-AR
human â3-AR
pKact ( SEMa
IA ( SEMb
pKact ( SEM
IA ( SEM
pKact ( SEM
IA ( SEM
A. cAMP-RIA Assayc
8.40 ( 0.17
8.33 ( 0.24d
8.47 ( 0.12
NA
ISO
TMQ
8
9
10
8.75 ( 0.14
8.70 ( 0.11
8.11 ( 0.13
NAe
NA
NA
100
100
7.37 ( 0.11
8.60 ( 0.15
8.76 ( 0.2
5.06 ( 0.01
6.95 ( 0.11
NA
100
109 ( 10
103 ( 4
<10
<5
<10
95 ( 3
56 ( 9
20 ( 1
<5
95 ( 3
120 ( 9
54 ( 1
67 ( 3
<10
NA
NA
11
<10
B. CRE-LUC Assayc
NA
NA
10
25
NA
NA
<10
<10
<15
<10
6.71 ( 0.18
NA
62 ( 3
<10
a
Human â1-, â2-, and â3-AR were expressed in CHO cells. Kact is the molar drug concentration which produces a cAMP response equal
b
to 50% of its maximal response, pKact ) -log Kact
.
IA, intrinsic activity, expressed as the percentage of a maximal analogue response
relative to the maximal response (100%) of (R)-(-)-isoproterenol (ISO). c See Experimental Section. Data for S-(-)-TMQ. e NA, not active
d
at 100 µM; n ) 4-12.

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2291
(LUC) reporter gene (CRE-LUC) assays (see Experi-
mental Section) were employed to determine the recep-
tor activities of the compounds.
We were also interested in examining the possible
effects of functionalizing the 2-aminothiazole moiety of
10 on its activities at â-AR. Introduction of an acetyl
group on 10 resulted in compound 11 that is inactive
(up to 100 µM) at â1- and â2-AR. Surprisingly, this
modification of 10 completely abolishes â3-AR activity
(up to 100 µM) (Table 2). This result indicates that an
intact 2-aminothiazole moiety is essential for maintain-
ing functional activity at â3-AR. This phenomenon is
likely due to steric perturbation at the receptor site
corresponding to the 2-amino group of 10, and hence a
functionally active â3-AR conformation cannot be
achieved.
The open-chain compound 25 was also evaluated at
human â1-, â2-, and â3-AR by the CRE-LUC assay
(Table 2). It is functionally inactive (up to 100 µM) at
all three human â-AR subtypes. Similar biological
results have been previously reported37 on some other
TMQ open-chain analogues.
Replacement of the catechol of TMQ with a 2-ami-
nothiazole group (analogue 9) resulted in a moderate
decrease (14-19-fold) in receptor binding affinities for
all three human â-AR subtypes (Table 1). However,
while 9 is inactive (up to 100 µM) at human â1- and â2-
AR, it was observed to be a partial agonist for human
â3-AR despite a 3467-fold decrease in potency as com-
pared to TMQ (Table 2). These results suggest that the
catechol group of TMQ is essential for its functional
activity at human â1- and â2-AR, but not for its binding
affinity at these two â-AR subtypes. Findings consistent
with this observation were provided by previous site-
directed mutagenesis studies on human â1- and â2-AR.23
A similar moderate decrease in affinity observed for the
human â3-AR suggests that the catechol moiety is also
not critical for binding to â3-AR. Perturbation of catechol
interactions with all the â-AR subtypes may well have
been compensated for by a strong binding interaction
between the 1-trimethoxybenzyl group of TMQ and
â-AR, and this provides a possible explanation for lack
of loss of receptor affinity. However, our results from
functional assays suggest that, unlike at â1- and â2-AR,
the catechol moiety of TMQ is not indispensable for â3-
AR activation.
Con clu sion s
In this study, we have shown that replacing the
catechol moiety of TMQ with the 2-aminothiazole group
completely abolished the â1- and â2-AR activities while
retaining activity at â3-AR. Thus, highly selective â3-
AR agonists (9, 10) have been identified. It has been
demonstrated that the â3-AR activity of the 2-ami-
nothiazole analogues is abolished by N-acetylation or
ring opening. A parallel increase in â3-AR binding
affinity and functional activity has also been observed
in proceeding from analogue 9 to 10. This information
should prove to be very useful for identifying highly
selective and more potent â3-AR agonists that could be
useful in the treatment of obesity, NIDDM, and intes-
tinal hypermotility disorders. These agonists, including
10 identified in this study, shall also be helpful in
further defining the physiological roles of â3-AR.
The 2-aminothiazole group has been demonstrated to
be a viable bioisostere for catechol/phenolic groups in
developing orally active dopamine agonists.24,36 How-
ever, this replacement in our studies with TMQ led to
dichotomous results. While a moderate decrease in
receptor binding affinity was observed at all three
human â-AR subtypes, the functional activity was
abolished (up to 100 µM) at both human â1- and â2-AR,
but functional activity was retained at â3-AR, thus
providing the desired selectivity for â3-AR.
Replacing the 3′- and 5′-methoxy group of 9 with
iodine atoms (analogue 10) resulted in a significant
increase (46-fold) in binding affinity at â3-AR, compared
to minor increases (8.5- and 1.4-fold) at â1- and â2-AR,
respectively (Table 1). Similar observations were noted
for compound 8 which exhibited significant (174-fold)
and moderate increases (4- and 21-fold) in binding
affinities at â3- and â1-, and â2-AR, respectively, as
compared to TMQ (Table 1). Interestingly, while com-
pound 10 is inactive (up to 100 µM) at human â1- and
â2-AR, it activates â3-AR with a 78-fold greater potency
than 9, and its intrinsic activity (67%) is also greater
than that of 9 (54%) (Table 2). These results indicate
that the decreases in potency and intrinsic activity at
human â3-AR following replacement of the catechol
moiety of TMQ with a 2-aminothiazole group can be
reversed to a certain extent by increasing the receptor
binding affinity resulting from modification of the
4-trimethoxybenzyl group of analogue 9. In fact, while
9 is >3000-fold less potent than TMQ at activating â3-
AR, 10 is only 45-fold less potent than TMQ. However,
in contrast to TMQ, compound 10 displays a very high
selectivity for activation of â3-AR. Consistent functional
activities for 10 were obtained by both the cAMP
radioimmunoassay and the CRE-LUC assays (Table 2,
Figure 1).
Exp er im en ta l Section
Ch em istr y. A Thomas-Hoover capillary melting point ap-
paratus was used to measure all melting points that are
uncorrected. 1H and 13C NMR spectra were recorded on a
Bruker AX 300 spectrometer (300 and 75.5 MHz, respectively),
and chemical shift values (δ) are expressed as parts per million
(ppm) relative to tetramethylsilane (TMS). Peaks are abbrevi-
ated as follows: s, singlet; d, doublet; t, triplet; q, quartet; bs,
broad singlet. IR spectra were recorded on a Perkin-Elmer
System 2000-FTIR. Elemental analyses were performed by
Atlantic Microlab, Inc. (Norcross, GA 30091), and the values
found are within (0.4% of the theoretical values. Silica gel
(Merck, 230-400 mesh, 60 Å) was used for flash column
chromatography. THF was dried by distillation from sodium
benzophenone ketyl, anhydrous benzene was from Aldrich, and
anhydrous ethyl ether was from Fischer. All starting materials
were from Aldrich and used without further purification.
2-Am in o-4-(2-p h t h a lim id oet h yl)t h ia zole H yd r ob r o-
m id e (14). To a solution of 1330 (2.08 g, 7.0 mmol) in acetone
(45 mL) was added a solution of thiourea (0.535 g, 7.0 mmol)
in acetone (25 mL) with rapid rate at room temperature. J ust
after the addition was complete, a precipitate appeared; the
suspension was stirred overnight at room temperature and
filtered to afford 2.45 g (99%) of 14 as a colorless powder: mp
258-260 °C dec (lit.30 mp 195 °C dec starting point); 1H NMR
(DMSO-d6) δ 2.82 (t, J ) 6.3 Hz, 2H), 3.83 (t, J ) 6.2 Hz, 2H),
6.56 (s, 1H), 7.81-7.88 (m, 4H), 9.01 (bs, 2H); IR
(KBr) 3214, 3092, 1767, 1721, 1633, 1573, 1402 cm-1. Anal.
(C13H11N3O2S‚HBr) C, H, N.

2292 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Brief Articles
N-2-[(2-Am in o-4-th ia zolyl)eth yl]-3,4,5-tr im eth oxyp h e-
n yla ceta m id e (18). (a ) To a suspension of 3,4,5-trimethoxy-
phenylacetic acid (4.52 g, 0.02 mol) in dry benzene (200 mL)
was added dropwise oxalyl chloride (20 mL, 0.23 mol) at 0 °C.
After the addition was complete, the reaction mixture was
stirred at room temperature until a clear solution was obtained
(about 1 h). The solution was then heated at reflux for 2.5 h.
The reaction mixture was cooled to room temperature and
evaporated to give a yellow oil. It was dissolved in benzene
(∼50 mL) and evaporated again (repeated for two more times);
4.9 g (100%) of the acid chloride was obtained as a viscous
yellow oil after drying in vacuo: 1H NMR (CDCl3) δ 3.83 (s,
3H), 3.84 (s, 6H), 4.05 (s, 2H), 6.45 (s, 2H); IR (neat) 300, 2941,
ing acid 1732 with oxalyl chloride as described above for 3,4,5-
trimethoxyphenylacetic acid. Recrystallization from EtOAc
gave 642.8 mg (61%) of 19 as a colorless crystal: mp 175-176
°C; 1H NMR (CD3OD) δ 2.63 (t, J ) 6.8 Hz, 2H), 3.35 (s, 2H),
3.41 (t, J ) 6.8 Hz, 2H), 3.79 (s, 3H), 6.05 (t, J ) 0.8 Hz, 1H),
7.72 (s, 2H); 13C NMR (CD3OD) δ 31.9, 39.7, 41.8, 61.1, 90.9,
103.4, 137.1, 141.6, 149.9, 159.4, 171.5, 172.9; IR (KBr)
3431, 3272, 3083, 2933, 1643, 1623, 1579, 1524 cm-1. Anal.
(C14H15I2N3O2S) C, H, N.
2-Am in o-4-(3,5-diiodo-4-m eth oxyben zyl)-4,5,6,7-tetr ah y-
d r oth ia zolo[5,4-c]p yr id in e Dih yd r och lor id e (10). In the
same manner as 9, the title compound was prepared from 19
(380.1 mg, 0.7 mmol). After flash column chromatography on
silica gel, eluting with MeOH/CHCl3 (1:30), 72 mg (20%) of
the free base form of the product was obtained as a white solid,
and it was treated with HCl (1.0 M solution in dry Et2O) to
afford 10 as a pale-yellow solid: mp 201-203 °C dec; 1H NMR
(DMSO-d6) δ 2.67-2.77 (m, 2H), 3.02-3.11 (m, 2H), 3.15-3.35
(m, 2H), 3.74 (s, 3H), 4.74 (bs, 1H), 7.90 (s, 2H), 8.31 (bs, 2H),
9.59 (bs, 1H), 9.79 (bs, 1H); 13C NMR δ 22.5, 38.0, 41.5, 54.9,
61.2, 91.9, 112.6, 135.1, 135.5, 142.6, 160.7, 171.4; IR (KBr)
3421, 2964, 2937, 2775, 1628, 1577 cm-1. Anal. (C14H15I2N3-
OS‚2HCl) C, H, N.
2840, 1799, 1593 cm-1
.
(b) To a well-stirred suspension of 1530 (976 mg, 3.2 mmol),
NaOH (512 mg, 12.8 mmol) in CHCl3 (7 mL), and H2O (5 mL)
was added slowly a solution of above-obtained 3,4,5-trimethoxy-
phenylacetyl chloride (784 mg, 3.2 mmol) in CHCl3 (6 mL) at
room temperature. After the addition was complete, the
reaction mixture was stirred vigorously at room temperature
for 1.5 h. The CHCl3 layer was separated, and the H2O layer
was extracted with CHCl3. The combined organics were dried
over anhydrous Na2SO4 and concentrated under reduced
pressure. The oily residue was dissolved in CHCl3 (40 mL), to
which HCl (1.0 M solution in dry Et2O) (10 mL) was added at
0 °C. The whole mixture was concentrated; the oily residue
was dissolved in H2O (10 mL). The aqueous solution was
washed successively with EtOAc, Et2O, and CHCl3 and basified
with 20% NaOH. The product was extracted with CHCl3, the
combined organics were washed with brine, dried over anhy-
drous Na2SO4, and concentrated, and the resulting solid
residue was crystallized from EtOAc to afford 803 mg (71%)
of 18 as a colorless crystal: mp 148-149.5 °C; 1H NMR (CDCl3)
δ 2.57 (t, J ) 6.2 Hz, 2H), 3.42 (q, J ) 5.8 Hz, 2H), 3.46 (s,
2H), 3.79 (s, 3H), 3.80 (s, 6H), 5.16 (bs, 2H), 5.96 (s, 1H), 6.43
(s, 2H), 6.50 (bs, 1H); 13C NMR (CDCl3) δ 30.6, 38.9, 44.1, 56.1,
60.8, 103.1, 106.8, 130.5, 136.9, 150.1, 153.3, 167.9, 170.7; IR
(KBr) 3433, 3266, 3080, 2939, 1646, 1624, 1590 cm-1. Anal.
(C16H21N3O4S) C, H, N.
N-2-[(2-Acetam ido-4-th iazolyl)eth yl]-3,5-diiodo-4-m eth -
oxyp h en yla ceta m id e (20). To a stirred suspension of 19
(434.5 mg, 0.8 mmol) in dry CH3CN (1.8 mL) was added
dropwise a solution of acetic anhydride (o.16 mL, 1.7 mmol)
in dry benzene (0.6 mL). After the addition was complete, the
reaction mixture was heated at reflux for 2.5 h. After the
reaction mixture was cooled to room temperature and concen-
trated under reduced pressure to remove solvents completely,
H2O (5 mL) was added to the residue and the mixture was
basified with saturated NaHCO3 aqueous solution to pH 7.5-
8.0. The solid material was filtered off and crystallized from
CH3CN to afford 430 mg (92%) of 20 as a colorless crystal:
1
mp 231-232 °C; H NMR(DMSO-d6) δ 2.10 (s, 3H), 2.69 (t, J
) 7.0 Hz, 2H), 3.29-3.32 (m, 4 H), 3.71 (s, 3H), 6.70 (s, 1H),
7.67 (s, 2H), 8.08 (t, J ) 5.4 Hz, 1H), 12.04 (s, 1H); 13C NMR
(DMSO-d6) δ 22.5, 31.1, 38.2, 40.1, 60.2, 90.9, 108.0, 136.7,
139.9, 148.3, 157.0, 157.5, 168.2, 169.3; IR (KBr) 3429, 3273,
3062, 1644, 1554, 1537 cm-1. Anal. (C16H17I2N3O3S) C, H, N.
2-Am in o-4-(3,4,5-t r im e t h oxyb e n zyl)-4,5,6,7-t e t r a h y-
d r oth ia zolo[5,4-c]p yr id in e Dih yd r och lor id e (9). A mix-
ture of 18 (70.3 mg, 0.2 mmol) and phosphorus oxychloride
(0.27 mL, 2.9 mmol) in CH3CN (4 mL) was stirred and heated
at reflux for 5 h. After the reaction mixture was cooled and
concentrated under reduced pressure, the residue was dis-
solved in MeOH (2 mL), and the solution was heated at reflux
for 30 min. After evaporation, the residue was dissolved in
MeOH and evaporated again (repeated three more times). To
the stirred solution of the resulting residue in MeOH (10 mL)
was added NaBH4 (757 mg, 20 mmol) in portions cautiously
at 0 °C. After the addition was complete, the reaction mixture
was stirred overnight at room temperature. After the reaction
mixture was evaporated to dryness under reduced pressure,
the residue was dissolved in H2O (5 mL), cooled with an ice-
water bath, and basified with 20% NaOH. The basic solution
was extracted with EtOAc; the combined organics were washed
with brine, dried over anhydrous Na2SO4, and concentrated
to give a viscous yellow oil. The oily residue was dissolved in
CHCl3 (5 mL), to which HCl (1.0 M solution in dry Et2O) (2
mL) was added at 0 °C. The precipitate was filtered off, washed
successively with Et2O, EtOAc, and CHCl3, and crystallized
from MeOH-Et2O to afford 36.5 mg (45%) of 9 as a pale-yellow
powder: mp 230-231 °C dec; 1H NMR (DMSO-d6) δ 2.71-
2.90 (m, 2H), 2.96-3.03 (m, 1H), 3.17-3.23 (m, 3H), 3.64 (s,
3H), 3.75 (s, 6H), 4.77 (bs, 1H), 6.67 (s, 2H), 8.48 (bs, 1H), 9.68
(bs, 1H), 9.85 (bs, 1H); 13C NMR (CD3OD) δ 21.8, 39.8, 41.0,
54.8, 56.8, 61.2, 108.3, 112.9, 130.7, 133.9, 139.2, 155.2, 171.5;
IR (KBr) 3392, 2940, 2839, 2771, 1632, 1593 cm-1. Anal.
(C16H21N3O3S‚2HCl‚0.5H2O) C, H, N.
2-Acet a m id o-4-(3,5-d iiod o-4-m et h oxyb en zyl)-4,5,6,7-
tetr a h yd r oth ia zolo[5,4-c]p yr id in e Ma lea te (11). In the
same manner as 9, the title compound was prepared from 20
(300 mg, 0.51 mmol). After flash column chromatography on
silica gel, eluting with acetone/hexane (1:3), 150 mg (52%) of
the free base form of 11 was obtained as a white powder: mp
170-172 °C; 1H NMR (DMSO-d6) δ 2.09 (s, 3H), 2.53-2.60
(m, 2H), 2.66-2.73 (m, 1H), 2.77-2.86 (m, 1H), 2.89-2.95 (m,
1H), 3.09-3.15 (m, 1H), 3.72 (s, 3H), 4.12-4.13 (m, 1H), 7.80
(s, 2H), 11.91 (bs, 1H); 13C NMR (DMSO-d6) δ 22.5, 27.0, 40.4,
40.8, 53.9, 60.2, 90.9, 124.2, 139.3, 140.4, 143.7, 155.5, 156.7,
168.0; IR (KBr) 3428, 3252, 2914, 1675, 1636, 1565. Anal.
(C16H17N3SO2I2) C, H, N.
11 was obtained as a white powder by treating the above-
obtained free base with maleic acid in CH3CN: mp 121 °C dec;
1H NMR (CD3OD) δ 2.18 (s, 3H), 2.93-3.09 (m, 4H), 3.38-
3.44 (m, 1H), 3.63-3.71 (m, 1H), 4.93-4.96 (m, 1H), 6.26 (s,
8/3H), 7.85 (s, 2H); 13C NMR (DMSO-d6) δ 22.4, 23.4, 37.7,
40.4, 53.7, 60.3, 91.6, 118.2, 134.7, 135.4, 140.7, 142.1, 157.5,
157.8, 167.1, 168.6; IR (KBr) 3436, 3049, 2968, 2946, 1700,
1686, 1624, 1571 cm-1. Anal. (C16H17I2N3O2S‚4/3C4H4O4‚1/3Et2O)
C, H, N.
2-Am in o-5-(2-p h t h a lim id oet h yl)t h ia zole H yd r ob r o-
m id e (28). In the same manner as 14, the title compound was
prepared from thiourea (2.75 g, 36.1 mmol) and 2730 (crude,
10.69 g, 36.1 mmol). Recrystallization from MeOH/EtOH (1:
10) gave 6.19 g (46% based on aldehyde 2630) of 28 as colorless
plates: mp 244-246 °C dec (lit.30 mp 180 °C dec starting
point); 1H NMR (DMSO-d6) δ 2.96 (t, J ) 6.2 Hz, 2H), 3.77 (t,
J ) 6.3 Hz, 2H), 7.08 (s, 1H), 7.82-7.89 (m, 4H), 9.01 (bs, 2H);
IR (KBr) 3308, 3222, 3108, 2975, 1764, 1711, 1618, 1608, 1554,
1402 cm-1. Anal. (C13H11N3O2S‚HBr) C, H, N.
N-2-[(2-Am in o-4-th ia zolyl)eth yl]-3,5-d iiod o-4-m eth oxy-
p h en yla ceta m id e (19). In the same manner as 18, the title
compound was prepared from 15 (590.3 mg, 1.94 mmol) and
3,5-diiodo-4-methoxyphenylacetyl chloride (844.8 mg, 1.94
mmol) which in turn was obtained by treating its correspond-

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2293
N-2-[(2-Am in o-5-th ia zolyl)eth yl]-3,4,5-tr im eth oxyp h e-
n yla ceta m id e (22). In the same manner as 18, the title
compound was prepared from 2130 (1.95 g, 6.4 mmol) and 3,4,5-
trimethoxyphenylacetyl chloride (1.57 g, 6.4 mmol). Recrys-
tallization from CHCl3/hexanes gave 1.42 g (63%) of 22 as
by the method of Lowry et al.,41 using bovine serum albumin
as the standard.
3. cAMP Resp on se Elem en t (CRE)-Lu cifer a se (LUC)
Rep or ter Gen e (CRE-LUC) Assa y. CHO cells stably ex-
pressing human â1-, â2-, or â3-AR subtypes were transfected
with a 6 CRE-LUC plasmid (gift from Dr. A. Himmler,
Vienna, Austria) using electroporation with a single 70-ms,
150-V pulse.42 The transfected CHO cells were seeded at a
density of 40 000/well in 96-well microtiter plates (Culturplate,
Packard) and allowed to grow for 20 h. After 20 h, the cells
were treated with varying drug concentrations (10-11-10-4 M)
for 4 h. Following drug exposures, the cells were lysed, and
luciferase activity was measured using the LucLite assay kit
(Packard). Changes in light production were measured by a
Topcount luminometer (Packard).
1
colorless crystals: mp 121-122 °C; H NMR (CDCl3) δ 2.74
(t, J ) 6.0 Hz, 2H), 3.34 (q, J ) 6.3 Hz, 2H), 3.43 (s, 2H), 3.79
(d, 9H), 5.22 (bs, 2H), 5.78 (t, J ) 5.6 Hz, 1H), 6.39 (s, 2H),
6.59 (s, 1H); 13C NMR (CDCl3) δ 26.8, 40.4, 44.0, 56.1, 60.8,
106.4, 124.4, 130.3, 135.8, 137.1, 153.5, 167.3, 170.9; IR
(KBr) 3294, 3114, 2995, 2936, 1654, 1636, 1588 cm-1. Anal.
(C16H21N3O4S) C, H, N.
N-2-[(2-Am in o-5-th iazolyl)eth yl]-3,4,5-tr im eth oxyph en -
eth yla m in e Dih yd r och lor id e (25). To a suspension of 22
(176 mg, 0.5 mmol) in dry THF (0.5 mL) was added dropwise
slowly BH3‚THF (1.0 M in THF, 3.5 mL). After the addition
was complete, the reaction mixture was stirred at room
temperature for 30 min and then was heated at reflux for 1 h.
The reaction mixture was cooled to room temperature and
treated cautiously with 10% HCl aqueous solution (3 mL), and
the solution was heated at reflux for 30 min. After removal of
THF, H2O (10 mL) was added to the residue. The acidic
solution was basified with 10% NaOH aqueous solution at 0
°C. The product was extracted with CHCl3 (20, 20, 10 mL);
the combined organic was washed with brine (20 mL) and
dried over anhydrous Na2SO4. After filtration and evaporation,
a viscous oil was obtained, and it was dissolved in CHCl3 (8
mL) and treated with HCl (1.0 M in dry Et2O). The whole
mixture was evaporated to dryness, and the resulting white
solid was crystallized in MeOH/Et2O to give 109 mg (53%) of
25 as white crystals: mp 235 °C dec; 1H NMR (CD3OD) δ 2.98-
3.03 (m, 2H), 3.11-3.16 (m, 2H), 3.29-3.34 (m, 4H), 3.72 (s,
3H), 3.84 (s, 6H), 6.63 (s, 2H), 7.18 (s, 1H); 13C NMR (CD3OD)
δ 24.6, 33.5, 48.4, 50.3, 56.7, 61.1, 107.3, 121.7, 125.3, 133.7,
138.3, 154.9, 172.0; IR (KBr) 3427, 2947, 2767, 1630, 1590
cm-1. Anal. (C16H23N3O3S‚2HCl) C, H, N.
P h a r m a cologica l St u d ies. 1. R a d ioliga n d Bin d in g
Stu d ies. Following digestion with trypsin, CHO cells express-
ing human â1-, â2-, or â3-AR were harvested into Ham’s F-12
solution. Cells were pelleted and washed three times with Tris-
EDTA buffer (pH 7.4; TRIZMA HCl, 50 mM; NaCl, 150 mM;
disodium EDTA‚2H2O, 20 mM). Cells were then suspended
in Tris-EDTA buffer after centrifugation. Competition binding
assays were performed at 37 °C by incubating cells with
varying concentrations of drugs in the presence of [125I]-
iodocyanopindolol ((1.5-5) × 104 cells/18-70 pM for human
â1- or â2-AR, (3-5) × 105 cells/200-500 pM for human â3-AR).
Nonspecific binding was determined in the presence of (-)-
propranolol (1 µM for human â1- or â2-AR, 100 µM for human
â3-AR). The incubations were terminated by rapid filtration
over Whatman GF/B (for human â1- or â2-AR) or Whatman
GF/C (for human â3-AR, presoaked in 0.1% poly(ethylenimine))
glass fiber filters using a Brandel model 12-R cell harvester.
The filters were washed three times with the Tris-EDTA buffer
(4 °C) and dried under cell harvester vacuum. The radioactivity
in the filters was measured by gamma scintillation counting
using a Beckman gamma counter model 8000. Ki values were
calculated from the obtained IC50 values by the method of
Cheng and Prusoff.38
Ack n ow led gm en t. We thank the National Institute
of Health (USPHS NHLBI Grant HL-22533) and Mo-
lecular Design International (MDI) for their support of
this research. We also thank Dr. Richard Fertel (De-
partment of Pharmacology, The Ohio State University,
Columbus, OH) for providing radiolabel and antibody
for the cAMP radioimmunoassay.
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