Discovery of N-(4-methoxy-7-methylbenzo[d]thiazol-2-yl)isonicatinamide, ML293, as a novel, selective and brain penetrant positive allosteric modulator of the muscarinic 4 (M4) receptor

Article abstract of DOI:10.1016/j.bmcl.2012.05.109

Herein we describe the discovery and development of a novel class of M ₄ positive allosteric modulators, culminating in the discovery of ML293. ML293 exhibited modest potency at the human M4 receptor (EC₅₀ = 1.3 μM) and excellent efficacy as noted by the 14.6-fold leftward shift of the agonist concentration-response curve. ML293 was also selective versus the other muscarinic subtypes and displayed excellent in vivo PK properties in rat with low IV clearance (11.6 mL/min/kg) and excellent brain exposure (PO PBL, 10 mg/kg at 1 h, [Brain] = 10.3 μM, B:P = 0.85).

Full text of DOI:10.1016/j.bmcl.2012.05.109

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5084–5088
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-(4-methoxy-7-methylbenzo[d]thiazol-2-yl)isonicatinamide,
ML293, as a novel, selective and brain penetrant positive allosteric modulator
of the muscarinic 4 (M4) receptor
James M. Salovich a,c,d, Paige N. Vinson a,c,d, Douglas J. Sheffler a,c,d, Atin Lamsal a,c,d, Thomas J. Utley a,c,d
Anna L. Blobaum a,c,d, Thomas M. Bridges a,c,d, Uyen Le a,c,d, Carrie K. Jones a,c,d,e, Michael R. Wood a,b,c,d
J. Scott Daniels a,c,d, P. Jeffrey Conn a,c,d, Colleen M. Niswender a,c,d, Craig W. Lindsley a,b,c,d
,
,
,
Corey R. Hopkins a,b,c,d,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
c Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
e US Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the discovery and development of a novel class of M4 positive allosteric modulators,
culminating in the discovery of ML293. ML293 exhibited modest potency at the human M4 receptor
(EC50 = 1.3 lM) and excellent efficacy as noted by the 14.6-fold leftward shift of the agonist concentra-
tion–response curve. ML293 was also selective versus the other muscarinic subtypes and displayed excel-
lent in vivo PK properties in rat with low IV clearance (11.6 mL/min/kg) and excellent brain exposure (PO
Received 14 May 2012
Revised 22 May 2012
Accepted 29 May 2012
Available online 6 June 2012
PBL, 10 mg/kg at 1 h, [Brain] = 10.3 lM, B:P = 0.85).
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Positive allosteric modulator
M4
ML293
CNS penetration
Muscarinic receptor 4
The neurotransmitter acetylcholine (ACh) regulates a diverse set
of physiological actions through the activation of two classes of
cell-surface receptors. These receptors, the nicotinic ACh receptors
(nAChRs)1 are ACh-gated cation channels and the muscarinic ACh
receptors (mAChRs) are G protein-coupled receptors (GPCRs).2,3
Both sets of receptors are involved in numerous physiological pro-
cesses. Molecular cloning has identified five separate subtypes of
the mAChRs (M1–M5) which are subclassified due to their coupling
patterns to different G proteins.2–4 One class couples to Gq, activat-
ing phospholipase C (M1,3,5), and the second class couples to Gi/o
(M2,4), regulating cAMP levels and various ion channels.2–4 Two
mAChRs are widely distributed in the CNS (M1 and M4) and have
been shown in pre-clinical experiments to be potentially viable
therapeutic targets for Alzheimer’s disease and schizophrenia.5 In
addition to pre-clinical validation, these mechanisms have been
shown to be clinically effective utilizing the non-selective M1/M4-
preferring agonist xanomeline.6 Finally, significant work utilizing
knockout mice has shown the importance of the role of M1 and
M4 in cognition and psychosis, respectively.2,4
Unfortunately, due to high sequence homology and conserva-
tion of the ACh binding site among the mAChRs, development of
Me
NH2
Me
N
NH2
O
O
S
HN
Me
N
S
HN
F
O
N
F
ML108
OMe
Me
ML173
NH2
Cl
O
S
HN
Me
N
N
ML253
⇑
Corresponding author. Tel.: +1 615 936 6892; fax: +1 615 936 4381.
E-mail address: corey.r.hopkins@vanderbilt.edu (C.R. Hopkins).
Figure 1. Previously reported M4 probe molecules ML108, ML173, ML253.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.109

J. M. Salovich et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5084–5088
5085
Figure 2. Initial ACh EC20 triage screen of M4 analogs. Assay was performed at 10 lM using hM4-Gqi5-CHO cells; activity was monitored using an intracellular calcium
mobilization assay. Values represent a single screening experiment performed in triplicate; numbered compounds were then reassessed using full concentration–response
curves.
selective orthosteric modulators has been difficult. Due to this, we
Table 1
SAR around the eastern amide moiety8
and others have been investigating the use of allosteric modulators
in order to overcome the homology of the orthosteric site and de-
velop selective tools for these receptors. As part of the NIH-
sponsored Molecular Libraries Probe Production Center Network,
we have recently reported three selective M4 positive allosteric
modulator probe molecules (Fig. 1). These molecules have been
important tool compounds and have shown efficacy in pre-clinical
in vivo animal models of schizophrenia; however, these compounds
suffer from less than ideal pharmacokinetic properties.7
Due to these shortcomings, we were still interested in the discov-
ery of novel M4 tool compounds that offer improved in vivo PK prop-
erties. To this end, we performed a screen at Vanderbilt which
revealed a novel structural class of compounds as potential M4
positive allosteric modulators (Fig. 2 and 1). A small library was gen-
erated around this molecule and from this effort there were many
compounds that potentiated a submaximal (EC20) concentration
of ACh (Fig. 2). Based on these promising initial results, a lead opti-
mization campaign was initiated in our laboratories.
O
O
R
N
S
NH
Entry
1
R
hM4 pEC50
SEM
hM4 EC50
(lM)
%AChmax
SEM
O
∗
∗
5.74 0.03
1.8
55.7 4.6
52.5 3.1
O
O
2
3
<5*
>10
F
Inactive
∗
∗
4
5.87 0.02
1.3
51.8 2.6
43.7 2.9
5
6
<5
>10
∗
The first SAR library kept the left-hand benzothiazole portion
constant and looked at a variety of amide analogs (Table 1). These
compounds were synthesized by utilizing the commercially avail-
able 4-methoxy-7-methylbenzo[d]thiazol-2-amine and coupling
with an appropriate acid chloride under basic conditions. The ori-
ginal molecule, 1, reconfirmed and exhibited micromolar potency
Me
Inactive
7
8
ND**
ND
Weak
∗
∗
Inactive
9
<5
<5
>10
>10
61.5 3.1
43.2 2.7
∗
∗
(EC50 = 1.8 lM). Addition of a methyl group, 18, led to an inactive
compound; in addition, replacement of the dioxine moiety with a
saturated pyran was deleterious to activity (16 and 17). Replace-
ment with other heteroaryl groups proved more fruitful with the
10
O
∗
11
Inactive
2-furyl group (4, 1.3
In addition, 3-pyridyl (20, 2.33
pyrazine (23, 1.1 M) modifications were all well tolerated. How-
lM) showing a slight improvement in activity.
∗
∗
l
M), 4-pyridyl (21, 1.3 M) and 2-
l
12
13
14
<5
>10
66.5 2.2
l
Inactive
<5
ever, these analogs showed significant differences in efficacy
(%ACh Max) with the 4-pyridyl analog, 20, proving to be the most
efficacious (65% vs <40% for 20 and 23). Replacement of the hetero-
cyclic moiety with alkyl, cycloalkyl or aromatic groups led to inac-
tive compounds.
Next, we turned our attention to amide replacements—an exer-
cise which proved to be less informative and highlights the shallow
nature of allosteric modulator SAR (Table 2).9 Moving from the 4-
pyridyl amide to the 4-pyridyl urea (29, inactive) led to a complete
loss of activity. In fact, all ureas that were analyzed were inactive,
with the lone exception being 35, which was weak in terms of both
∗
∗
>10
>10
52.2 4.6
45.0 1.8
15
16
<5
<5
∗
∗
>10
>10
64.1 1.4
44.0 2.4
O
O
17
18
<5
O
O
Inactive
∗
potency (7.1 lM) and efficacy (36%). Other amide replacements
(sulfonamide/reverse amides) were not tolerated, resulting in
(continued on next page)
either inactive or weakly active compounds.

5086
J. M. Salovich et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5084–5088
Table 1 (continued)
Table 2 (continued)
Entry
R
hM4 pEC50
SEM
hM4 EC50
M)
%AChmax
SEM
Entry
R
hM4
pEC50 SEM
hM4 EC50
M)
%AChmax SEM
43.5 2.3
(l
(l
O
∗
∗
33
34
<5**
>10
N
19
Inactive
N
NH
O
∗
∗
20
21
22
5.63 0.04
5.89 0.01
Inactive
2.3
1.3
37.6 3.1
65.2 2.7
N
Inactive
N
N
NH
N
∗
∗
5.15 0.15*
7.1
36.5 2.3
O
35
NH
∗
∗
NH
Cl
∗
∗
O
S
23
24
5.95 0.09
Inactive
1.1
38.2 1.3
36
37
Inactive
Inactive
O
N
NH
F
F
O
S
O
∗
∗
∗
NH
O
S
F
N
N
O
NH
38
39
Inactive
Inactive
25
26
Inactive
Inactive
F
F
∗
∗
∗
O
O
S
CF3
F
NH
O
S
O
40
Inactive
F
F
∗
∗
NH
N
O
S
27
28
Inactive
Inactive
O
NH
F
41
42
43
44
45
Inactive
Inactive
Inactive
ND***
F
∗
O
S
O
NH
O
∗
∗
For all tables, means are determined from at least 3 experimentsperformed in
triplicate on separate days.
Compounds that did not exhibit a plateau in their concentration-responsewere
given a potency value of >10 lM and therefore no error was calculated for this
parameter.
N
*
HN
O
**
ND: not determined. This compound showed weak (low %AChmax) potentiation
in two runs and did not meet criteria for PAM categorization in the third run.
HN
N
ND
Weak
∗
∗
O
HN
Inactive
O
N
Table 2
Amide replacements8
HN
46
47
48
Inactive
5.11 0.11*
ND***
∗
∗
O
O
N
HN
R1
S
7.7
ND
35.6 2.0
Weak
O
HN
Entry
29
R
hM4
pEC50 SEM
hM4 EC50
M)
%AChmax SEM
∗
∗
O
(l
HN
O
N
49
ND***
ND
Weak
O
Inactive
O
NH
NH
*
∗
∗
NH
O
Average of two low potency estimates (10
Compounds that did not exhibit a plateau in their concentration–response were
l
M) and one potency measure.
**
N
given a potency value of >10 lM and therefore no error was calculated for this
30
31
Inactive
<5**
parameter.
NH
***
ND: Not determined. These compounds showed weak (low %AChmax) potenti-
ation and did not meet criteria for PAM categorization in one or two of the three
runs.
O
>10
39.3 3.0
NH
NH
NH
∗
∗
O
Lastly, we investigated replacements of the benzothiazole moi-
ety (Table 3). Removal of both the 4-methoxy and 7-methyl groups
were not tolerated (50). In addition, replacement of the benzothi-
32
Inactive
NH

J. M. Salovich et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5084–5088
5087
Table 3
(14-fold) showing a potentiation of the ACh potency ( Fig. 3B).
Based on the hM4 potency, fold shift and selectivity profile, 21
was declared an MLPCN probe molecule and redesignated
ML293.10
Benzothiazole replacements8
R2
X
O
R
N
S
NH
ML293 was next profiled in a number of Tier 1 in vitro pharma-
cokinetic assays (Table 4). ML293 was assessed using an intrinsic
clearance assay (CLINT) in hepatic microsomes to evaluate the oxi-
dative metabolism potential. This assay also provides an in vitro
prediction for eventual assessment of clearance values in in vivo
PK studies (CLHEP). ML293 was predicted to display moderate clear-
ance in both human and rat microsomes (14.9 and 48.5 mL/min/
kg). In addition to intrinsic clearance, we determined the plasma
protein binding (human and rat equilibrium dialysis studies) of
ML293. ML293 showed high plasma protein binding in human
plasma (1.0%, unbound); however, in rat plasma, ML293 showed
a more favorable free fraction (3.2%, unbound). Lastly, ML293
was evaluated in a rat brain homogenate binding study to predict
the fraction of unbound compound in brain. ML293 showed higher
brain homogenate binding profile versus the plasma binding (BHB,
0.9% unbound vs. 3.2% unbound, respectively).
R3
Entry
R
hM4
pEC50 SEM EC50
M)
hM4 %AChmax SEM
(l
O
O
N
S
50
51
Inactive
Inactive
NH
O
N
N
S
NH
N
N
N
N
N
N
O
N
N
S
52
53
54
55
Inactive
Inactive
Inactive
Inactive
NH
O
N
S
NH
O
Table 4
N
S
In vitro and in vivo properties of ML29312
NH
O
O
O
N
N
N
N
S
21, ML293 (VU0409524)
NH
NH
S
O
O
N
O
MW
tPSA (Å)
cLogP
299.35
64.1
1.76
N
N
S
56
5.55 0.04
2.8
36.5 1.3
NH
hM4 EC50
hM4 FS
(l
M)
1.30
14.9 1.5
For all tables, means are determined from at least three experiments performed in
triplicate on separate days.
Intrinsic Clearance (mL/min/kg)
Human CLHEP
Rat CLHEP
14.9
48.5
Plasma Protein Binding (PPB)
human (%fu)
rat (%fu)
azole with thiazolo[5,4-b]pyridine groups also resulted in inactive
compounds (51–55). However, deletion of the 7-methyl group
(while maintaining the 4-methoxy) was tolerated, resulting in
1.0
3.2
0.9
BHB (rat,%fu)
PK parameters (rat)
compound 56 (2.8 lM).
IV dose (1 mg/kg)a
We determined that compound 21 possessed the appropriate
balance between in vitro potency and efficacy, thus, this compound
was further evaluated in our muscarinic selectivity panel as well as
in a fold-shift assay (an evaluation of a compound’s ability to pro-
duce a left-ward shift of the ACh curve). Compound 21 was exam-
ined at the other four muscarinic subtypes (M1,2,3,5) and was
inactive against each of these receptors at a concentration up to
t
(min)
57
85
0.97
1445
½
MRT (min)
Vss (L/kg)
AUC-IV (ng/mL)
PBL (p.o., 10 mg/kg @ 1 h), ng/mL or g
Plasma Systemic
3740
3070
3093
0.85
HPV
Brain
B/P
30
30
l
M. In our fold-shift assay (at
a constant concentration,
lM), 21 displayed a robust left-ward shift of the ACh curve
A
Potency
B
Efficacy
100
75
50
25
0
125
Vehicle
ML293
100
75
50
25
0
-25
-13.0
-10
-9
-8
-7
-6
-5
-4
-10.5
-8.0
-5.5
-3.0
Log ML293, [M]
Log ACh, [M]
Figure 3. (A) Concentration–response curve of ML293 in human M4 cells in the presence of an EC20 concentration of ACh. (B) ACh fold-shift assay for ML293 at a 30 lM
concentration. Data represent three independent determinations performed in triplicate.

5088
J. M. Salovich et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5084–5088
Gqi5 (Conklin et al., 1993). All transfections utilized Lipofectamine 2000. hM1,
hM3, and hM5 CHO cells were cultured in Ham’s F-12; 10% heat-inactivated
fetal bovine serum (FBS), 20 mM HEPES, and 500 g/mL G418 (Mediatech, Inc.,
Herndon, VA). hM2-Gqi5 and hM4-Gqi5 were grown in the same medium
supplemented with 500 g/mL hygromycin B. All cell culture reagents were
purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Calcium Mobilization Assays—Potency determinations. Assays were performed
within the Vanderbilt Center for Neuroscience Drug Discovery’s Screening
Center. CHO cell lines expressing muscarinic acetylcholine receptors were
As a follow-up to the in vitro studies, a standard rat IV PK exper-
iment was performed to determine the in vivo clearance of ML293.
In this experiment (1 mg/kg, IV), ML293 exhibited low clearance
(CLp = 11.5 mL/min/kg) with a modest half-life (t1/2 = 57 mins) and
l
l
significant plasma levels (AUC = 1445 h ng/mL, 4.8 lM). These val-
ues are in contrast to the previously reported M4 probe molecules (
Fig. 1), as each of those molecules exhibit high clearance in rat when
assessed using IV PK experiments (CLp >100 mL/min/kg).7b,11 Lastly,
we evaluated the CNS exposure of ML293 in a PBL (Plasma:Brain le-
vel) experiment after a single dose and at a single time point. ML293
was dosed orally at 10 mg/kg and after 1 hour the Brain:Plasma (B/
plated (15,000 cells/20 ll/well) in black-walled, clear-bottomed, TC treated,
384 well plates (Greiner Bio-One, Monroe, North Carolina) in Ham’s F-12, 10%
FBS, 20 mM HEPES. The cells were grown overnight at 37 °C in the presence of
5% CO2. The following day, plated cells had their medium exchanged to Assay
Buffer (Hank’s balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid
(Sigma–Aldrich, St. Louis, MO)) using an ELX405 microplate washer (BioTek),
P) ratio was 0.85 with absolute brain levels of 3093 ng/g (ꢀ10
lM).
leaving 20 lL/well, followed by addition of with 20 lL of 4.5 lM Fluo-4, AM
In addition, in this experiment we assessed the concentrations in
the hepatic portal vein as well as plasma. The levels found in the
HPV and plasma can be used as another assessment of the extent
of oxidative metabolism, as a ratio at or close to 1:1 indicates a
low level of metabolism; ML293 exhibits a roughly 0.8 ratio in these
studies. Based on these experiments, ML293 is a potent and selec-
tive M4 PAM with favorable pharmokinetics that demonstrates sig-
nificant CNS penetration following oral administration.
(Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a
1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer for
45 min at 37 oC. The dye was then exchanged to Assay Buffer using an ELX405,
leaving 20
lL/well and the plates were incubated at room temperature for
10 min prior to assay. Test compounds were transferred to daughter plates
using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA) and then
diluted into Assay Buffer to generate a 2Â stock in 0.6% DMSO (0.3% final).
Acetylcholine (ACh) EC20 and EC80 were prepared at a 5X stock solution in assay
buffer prior to addition to assay plates. Calcium mobilization was measured at
37 oC using a Functional Drug Screening System 6000 or 7000 (FDSS6000 or
FDSS7000, Hamamatsu, Japan) kinetic plate reader according to the following
protocol. Cells were preincubated with test compound (or vehicle) for 144
seconds prior to the addition of an EC20 concentration of the agonist, ACh. 86
seconds after this addition, an EC80 concentration of ACh was added. Control
wells also received a maximal ACh concentration (1 mM) for eventual response
normalization. The signal amplitude was first normalized to baseline and then
In conclusion, we have discovered an additional M4 tool com-
pound (ML293) with superior in vivo PK properties compared to
previously published molecules. ML293 represents a novel chemi-
cal scaffold as other reported M4 PAMs have been derived from
a
common 3-amino-N-(aryl)-4,6-disubstitutedthieno[2,3-b]pyri-
as
a percentage of the maximal response to ACh. Microsoft XLfit (IDBS,
dine-2-carboxamide moiety, and have all suffered from similar
poor in vivo PK properties. ML293 is a potent and selective small
molecule M4 positive allosteric modulator that significantly left-
ward shifts the ACh curve (14.9 Fold Shift). In addition, ML293,
in contrast to the previously reported compounds, displays low
clearance when evaluated in an in vivo rat IV clearance assay
Bridgewater, NJ) was utilized for curve fitting and EC50 value determination
using a four point logistical equation.
Calcium Mobilization Assays
– Fold-Shift Assays. Assays were performed
within the Vanderbilt Center for Neuroscience Drug Discovery’s Screening
Center. CHO cell lines expressing muscarinic acetylcholine receptors were
plated (15,000 cells/20 lL/well) in black-walled, clear-bottomed, TC treated,
384 well plates (Greiner Bio-One, Monroe, North Carolina) in Ham’s F-12, 10%
FBS, 20 mM HEPES. The cells were grown overnight at 37 °C in the presence of
5% CO2. The following day, plated cells had their medium exchanged to Assay
Buffer (Hank’s balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid
(Sigma–Aldrich, St. Louis, MO)) using an ELX405 microplate washer (BioTek),
and is highly brain penetrant (B:P = 0.85, [brain] = ꢀ10
lM).
Acknowledgments
leaving 20
lL/well, followed by addition of with 20 lL of 4.5 lM Fluo-4, AM
(Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a
1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer for 45
minutes at 37 oC. The dye was then exchanged to Assay Buffer using an ELX405,
The authors would like to thank Tammy Santomango, Frank
Byers, and Ryan Morrison for technical assistance with the PK
experiments and Nathan Kett and Sichen Chang for the purification
of compounds. Vanderbilt is a member of the MLPCN and houses
the Vanderbilt Specialized Chemistry Center for Accelerated Probe
Development (U54MH084659).
leaving 20
10 min prior to assay. Test compounds were prepared in Assay Buffer to
generate
2Â stock in 0.6% DMSO (0.3% final). Acetylcholine (ACh)
concentration responses were prepared at a 5X stock solution in assay buffer
prior to addition to assay plates. Calcium mobilization was measured at 37 o
using Functional Drug Screening System 6000 or 7000 (FDSS6000 or
lL/well and the plates were incubated at room temperature for
a
C
a
FDSS7000, Hamamatsu, Japan) kinetic plate reader according to the following
protocol. Cells were preincubated with test compound (or vehicle) for 144
seconds prior to the addition of a concentration response of the agonist ACh
and the fluorescence was monitored for a total of 5 min. The signal amplitude
was first normalized to baseline and then as a percentage of the maximal
response to acetylcholine. Microsoft XLfit (IDBS, Bridgewater, NJ) was utilized
for curve fitting and EC50 value determination using a four point logistical
equation. Fold-Shift values were calculated by dividing the ACh EC50 in the
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Probe Production Centers Network (MLPCN) and is freely available through the
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8. Human muscarinic acetylcholine receptor cell line creation and culture. Human
12. N-(4-methoxy-7-methylbenzo[d]thiazol-2-yl)isonicotinamide, ML293: To
a
stirred solution of 4-methoxy-7-methylbenzo[d]thiazol-2-amine (0.050 g,
0.26 mmol) and DIEA (0.112 mL, 0.64 mmol) in DCM (1.3 mL) at 0 oC was
added isonicotinoyl chloride hydrochloride (0.050 g, 0.28 mmol). The reaction
was allowed to warm to room temperature and stir overnight. The reaction
was concentrated under vacuum and the residue purified by reverse-phase
HPLC to give 55 mg (71%) of the pure product. 1H NMR (400 MHz, DMSO-d6): d
8.82 (d, J = 6.0 Hz, 2H), 8.02 (d, J = 6.1 Hz, 2H), 7.11 (d, J = 8.1 Hz, 1H), 6.95 (d,
J = 8.1 Hz, 1H), 3.90 (s, 3H), 2.45 (s, 3H). LC/MS: RT = 0.60 min., m/z = 300
[M+H]+.
M1 (hM1) cDNA in pcDNA3.1(+) was purchased from www.cDNA.org and stably
transfected into Chinese hamster ovary (CHO K1) cells purchased from the
ATCC (www.atcc.org). CHO cells stably expressing hM2, hM3, and hM5 were
generously provided by A. Levey (Emory University, Atlanta, GA). hM4 cDNA in
pcDNA3.1 (+) was purchased from www.cDNA.org and stably transfected into
CHOK1 cells. To make stable hM2 and hM4 cell lines for use in calcium
mobilization assays, cell lines were cotransfected with the chimeric G protein