Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor

Article abstract of DOI:10.1016/j.bmcl.2012.08.105

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ～330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.

Full text of DOI:10.1016/j.bmcl.2012.08.105

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6656–6660
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl
4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor
Patrick R. Maloney a, Pasha Khan a, Michael Hedrick b, Palak Gosalia b, Monika Milewski b, Linda Li b,
Gregory P. Roth a, Eduard Sergienko b, Eigo Suyama a, Eliot Sugarman a, Kevin Nguyen a, Alka Mehta a,
Stefan Vasile a, Ying Su b, Derek Stonich b, Hung Nguyen b, Fu-Yue Zeng b, Arianna Mangravita Novo a,
Michael Vicchiarelli a, Jena Diwan b, Thomas D. Y. Chung b, Layton H. Smith a,c, Anthony B. Pinkerton b,
⇑
a Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, 6400 Sanger Road, Orlando, FL 32827, USA
b Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
c Cardiopathobiology Program, Diabetes and Obesity Research Center, Sanford Burnham Medical Research Institute at Lake Nona, 6400 Sanger Road, Orlando, FL 32827, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeo-
stasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy
metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and char-
acterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent
APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angioten-
sin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ꢀ330,600 compound MLSMR collec-
Received 22 June 2012
Revised 23 August 2012
Accepted 28 August 2012
Available online 7 September 2012
Keywords:
GPCRs
HTS
APJ
tion. This antagonist showed no significant binding activity against 29 other GPCRs, except to the
j-
opioid and benzodiazepinone receptors (<50/<70%I at 10 M). The synthetic methodology, development
l
of structure–activity relationship (SAR), and initial in vitro pharmacologic characterization are also
presented.
Apelin
Ó 2012 Elsevier Ltd. All rights reserved.
Cardiovascular disease
Antagonists
SAR
Apelin is a circulating peptide hormone, synthesized and se-
creted by a number of cell types including those of the cardiovas-
cular, endocrine, gastrointestinal and nervous systems. Apelin was
recently identified as the endogenous ligand of the APJ, a formerly
orphaned G-protein coupled receptor (GPCR) with a similarly
broad distribution of expression.1 The tissue distribution of both
apelin and APJ suggests an involvement of this system in a range
of physiological functions. Indeed, apelin has been shown to play
a role in mediating gastrointestinal function,2–6 food and water
consumption,7–11 energy metabolism,12–16 and cardiovascular
homeostasis.17–21 In addition to normal physiological function,
apelin has been associated with the pathogenesis of cardiovascular
and metabolic diseases including atherosclerosis,22,23 hyperten-
sion,24–26 heart failure27,28 and both type 129 and type 2 diabetes
mellitus.30,31 Despite this abundance of work, several unanswered
questions regarding the role apelin and APJ in normal physiology
and pathology remain. Small molecule probes of the apelin/APJ
system would advance apelin research significantly. In particular
an APJ antagonist would be a useful tool for determining the
function and pharmacology of APJ, and ultimately to validate the
importance of this system in animal models.
To date, there have been no reports of small molecule antago-
nists of APJ. Therefore we undertook a high throughput screen of
the NIH’s small molecule collection (MLSMR) as part of the NIH
sponsored Molecular Libraries Program. Approximately 330,600
compounds were tested in the APJ DiscoveRx b-arrestin primary
assay that has been described in PubChem (AID 2766),32 Chemin-
formatics analysis revealed 1064 hits with activity >50% at a single
concentration point of 10 lM. Liquid samples were then ordered
through the MLSMR and 948 compounds were received. The com-
pound solutions resupplied by the MLSMR were first confirmed in
10
mary assay. Of these, 622 compounds were confirmed to have at
least 50% activity at a 10 M assay concentration. These were fur-
lM single-point duplicate in the APJ DiscoveRx b-arrestin pri-
l
ther triaged for direct b-galactosidase inhibition and an additional
237 compounds were eliminated. The remaining 385 confirmed
compounds were next tested in dose response in the primary APJ
DiscoveRx b-arrestin primary assay to obtain IC50 values and these
were rank ordered for potency: 67 compounds met probe criteria
(IC50 = 1–5
lM), 62 additional compounds had IC50 = 5–10 lM,
⇑
Corresponding author. Tel.: +1 858 795 5320.
E-mail address: apinkerton@sanfordburnham.org (A.B. Pinkerton).
and 86 compounds were significantly less potent (IC50 = 10–
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.105

P. R. Maloney et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6656–6660
6657
Figure 1. General SAR strategy around Kojic acid scaffold.
Scheme 1. Synthesis of APJ antagonists, conditions: (a) neat SOCl2 (17 equiv); (b) R1SH (1 equiv), NaOMe (1 equiv), MeCN; (c) R2COCl (1.4 equiv), Cs2CO3 (1 equiv), MeCN.
20
l
M). To eliminate compounds acting through non-specific
for APJ. Ultimately, only a single scaffold that was potent and selec-
tive against AT1 (MLS-0224164, Fig. 1) was identified.
inhibition of the assay reporter, the activity of the best scaffolds
was assessed in a b-galactosidase counterscreen assay. The best
scaffolds were also subjected to a counterscreen of the closely re-
lated angiotensin II type 1 (AT1) receptor to determine selectivity
The general method for preparation of the compounds de-
scribed herein is shown in the scheme below (Scheme 1). Starting
from kojic acid (A), chlorination using neat thionyl chloride gave B.
This was then used to alkylate a thiol to give C. Reaction with an
acid chloride then provided the desired APJ antagonists D. In gen-
eral yields were reasonable for this sequence and gram scale quan-
tities of materials could be produced.33
Table 1
SAR analysis of APJ antagonists: Kojic acid scaffold—left region R3
Table 2
SAR analysis of APJ antagonists: Kojic acid scaffold—left region R3
Entry
R3
APJ
AT1
SI
1
2
3
4
H
>79
6.20 0.77
7.25 0.75
75.70 3.30 (2)
54.50 3.12 (2)
1.75 0.19
6.59 2.87
29.00 4.94 (3)
18.20 3.36 (7)
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
ND
>13
>11
ND
ND
>45
>12
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3-NO2-4-Cl
3-NO2-4-Me
3-NO2
4-Cl
4-NO2
4-CN
4-CF3
4-Br
4-Me
4-OMe
4-OEt
4-OiPr
4-OBu
4-tBu
4-F
4-SO2NMe2
4-SO2N(CH2)
3,4-DiMe
2-NO2-5-Cl
2-Cl-5-NO2
2-Me-3-NO2
4-Ph
4-SO2NEt2
4-SO2N(CH2)
4-SO2morpholine
2-NO2-4,5-diOMe
Entry
R1
APJ
AT1
SI
28
29
30
31
32
33
H
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
>79
ND
>45
>17
ND
ND
>25
>23
>9
5
3-NO2-4-Cl
3-NO2-4-Me
3-NO2
4-Cl
4-NO2
1.75 0.14
4.70 0.91 (3)
59.20 4.77 (2)
>79
3.11 0.68
4.32 1.29
8.51 1.22
11.50 1.35
20.6 (1)
>79
6 (ML221)
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
34
4-CN
>7
35
36
37
38
39
40
41
42
43
44
45
46
47
48
4-CF3
4-Me
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4-OMe
4-F
4-SO2N(CH2)
2-NO2-5-Cl
2-Cl-5-NO2
2-Cl
3-OMe
3-Me
3-Cl
>79
>79
>79
>79
>79
>79
39.90 8.65
58.80 6.30 (2)
74.10 (1)
>59
>79
>79
>79
>79
>79
>79
4
4
51.00 4.07 (4)
72.2 (1)
>79
>79
>79
60.10 1.35 (3)
>79
>79
>79
>79
>79
>79
2-Me
4-OCF3
3-CN
5
>79
>79
Potency IC50 (lM), Ave. SEM (stdv/sqrt (n)) (n = 4) if number of replicates is dif-
ferent than the default it is noted in parentheses. SI = selectivity index: (IC50 AT1)/
(IC50 APJ).
Potency IC50 (lM), Ave. SEM (stdv/sqrt (n)) (n = 4) if number of replicates is dif-
ferent than the default it is noted in parentheses. SI = selectivity index: (IC50 AT1)/
(IC50 APJ).

6658
P. R. Maloney et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6656–6660
Table 3
Table 3 (continued)
SAR analysis of APJ antagonists: Kojic acid scaffold—left region R4 and right region R5
Entry
R4
R5
APJ
AT1
>79
SI
69
>79
ND
Entry
49
R4
R5
APJ
AT1
>79
SI
70
71
>79
>79
>79
>79
ND
ND
HO–
>79
ND
50
51
52
HO–
HO–
HO–
>79
>79
>79
>79
>79
>79
ND
ND
ND
72
3.21 0.46
>79
>25
Potency IC50 (lM), Ave. SEM (stdv/sqrt (n)) (n = 4) if number of replicates is dif-
ferent than the default it is noted in parentheses. SI = selectivity index: (IC50 AT1)/
(IC50 APJ).
53
HO–
>79
>79
ND
Table 4
ADMET properties for ML221
54
55
56
67
58
8.27 1.75
24.00 5.45
4.53 0.66
12.80 0.35
9.61 3.02
>79
>79
>79
>79
>79
>9
Compound characteristic
Aqueous solubility pH 7.4
Plasma protein binding (mouse)
Permeability (PAMPA) pH 7.4
Hepatic microsome Stability (mouse)
Plasma stability
25.5
99.1%
lg/mL
>3
271.0 Â 10À6 cm/s
4.9%
42.2%
>50 lM
>17
>6
Cytotoxicity
125
100
75
50
25
0
>8
59
60
>79
>79
>79
>79
ND
ND
61
>79
>79
ND
-10
-9
-8
-7
-6
-5
-4
62
63
>79
>79
>79
>79
ND
ND
Log [ML221], M
Figure 2. Representative dose response curve for ML221. The compound antago-
nized Ap13-mediated activation of APJ in a concentration-dependent manner in
both a cAMP assay (j), and a b-arrestin recruitment assay (d). Data plotted are the
mean SEM% inhibition of Ap13. Curves represent the best fit of a four parameter
logistic generated using GraphPad Prism5.
64
>79
>79
ND
The general SAR strategy we pursued around the kojic acid scaf-
fold is depicted in Figure 1. The structure represented by the
screening hit MLS-0224164 was the only scaffold that exhibited
selectivity over the AT-1 receptor. Efforts immediately focused on
replacing the undesirable 3-nitro-4-chloro motif due to the inher-
ent electrophilic nature, subsequent exploration of alternatives to
the ester linkage to address potential hydrolytic instability, and
substitution/replacement of the thiopyrimidine moiety. The results
are summarized in the four tables below (Tables 1–4). The first
three tables cover changes around the benzoate group (in green)
with either unsubstituted, monomethyl, or dimethyl thiopyrimi-
dine groups.
65
66
67
>79
>79
>79
>79
>79
>79
ND
ND
ND
68
>79
>79
ND

P. R. Maloney et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6656–6660
6659
Figure 3. GPCR profiling panel for ML221.
Table 1 depicts the SAR of the scaffold with an unsubstituted
thiopyrimidine. In general, we found that an electron-withdrawing
group on the benzoate was necessary for APJ activity. Interestingly,
while 3-nitro substitution was tolerated with a 4-chloro or methyl
group (entries 2 and 3), an analog with only 3-nitro substitution
was only weakly active (entry 4). Singly substituted analogs with
electron-withdrawing groups in the 4-position were the most ac-
tive with the exception of sulfonamides (for example, entry 17).
The, 4-nitro analog, ML221, (entry 6) was the most potent of this
series and was selected for further characterization. The 4-nitro
group could be replaced with a 4-cyano group (entry 7), albeit with
a 4-fold loss in potency. Unsubstituted benzoates (entry 1) as well
as a broad range of other para-substituted benzoates were all inac-
tive or weakly active (entries 5 and 8–29), with the exception of 4-
bromobenzoate analog (entry 9) and 4-trifluoromethyl analog (en-
try 8). Similar trends were seen with monomethyl substituted
pyrimides (Table 2) as well as 4,6-dimethylpyrimidines (data not
shown). In both cases potency was typically lower.
In addition, an expanded range of substituents was explored
around the scaffold (Table 3). Early evaluation of MLS-0224164 (en-
try 29) found the ester bond to be readily hydrolyzed in aqueous
acetonitrile to produce the truncated analog depicted as entry 52
(data not shown). To confirm the observed activity of MLS-
0224164 was due to the intact molecule and not the product of
hydrolysis, the compounds represented by entries 49–53 were pre-
pared and found to be inactive. Interestingly, replacing the thiopy-
rimidine with simple thiophenols led to a number of active analogs,
indicating that a range of groups is tolerated in that region. In par-
ticular a 4-chlorothiophenol (entry 56) and unsubstituted thiophe-
Antagonism of apelin-13-mediated activation of APJ by ML221
was assessed using two complimentary assays of APJ function; inhi-
bition of cAMP and recruitment of b-arrestin. Increasing concentra-
tions of ML221 antagonized a fixed concentration of Ap13 (EC80
10 nM) in both assays, with a calculated IC50 equal to 0.70 M in
the cAMP assay, and 1.75 M in the b-arrestin assay (Fig. 2).
=
l
l
The drug-like and ADME/T properties of ML221 were evaluated
in a detailed in vitro pharmacology panel (Table 4). ML221 is
poorly soluble in aqueous media at pH 7.4. We note that the aque-
ous solubility obtained at physiological pH is 14-fold higher than
the obtained potency of the probe. In a PAMPA permeability assay,
ML221 exhibits moderate permeability. ML221 displays moderate
plasma and poor microsomal stability, as it is rapidly metabolized
in both human and mouse liver homogenates (4.2% and 4.9%
remaining at 60 min). Neither the plasma nor the microsomal sta-
bility assay results are surprising given the ester linkage in this
probe. Ultimately this limits the utility of this probe to in vitro
studies or apelin receptor or in vivo studies using acute intrave-
nous doses to avoid metabolism. Lastly, ML221 shows no toxicity
(>50 lM) toward human hepatocytes.
Profiling against other GPCRs: ML221 was submitted to the Psy-
choactive Drug Screening Program (PDSP) at the University of
North Carolina and the data against a GPCR binding assay panel
is shown in Figure 3. Overall the compound shows a relatively
clean binding profile, with the only significant activity at the kappa
opioid and the benzodiazepinone receptors.
In conclusion, we have discovered the first reported APJ antag-
onist, ML221, which represents a selective tool compound to fur-
ther explore the function of the apelin/APJ system. ML221
displays limited cross reactivity against a range of GPCRs. Current
efforts to optimize this scaffold to explore the in vivo effects of APJ
antagonists are underway.
nol (entry 61) were both <10 lM. Attempts to replace the ester
linkage of the benzoate were less successful. A simple benzyl link-
age (entry 60) was inactive, as were a range of aliphatic esters (en-
tries 61–63). Sulfonates were also inactive (entries 64 and 65) as
were amides (entries 66 and 67). A range of heteroaryl esters also
showed no activity (entries 68–71). Lastly, oxidation of the sulfur
of the thiopyrimidine to a sulfone gave a compound that was of
comparable activity (entry 72 as compared to entry 29 in Table
2), although we did not further pursue this finding due to the poten-
tial reactivity of the sulfone-pyrimidine motif.
Acknowledgments
This work was supported by NIH Grants 1R21NS059422-01 to
L.H.S. and an NIH Molecular Libraries Grant (U54 HG005033-03)
to the Conrad Prebys Center for Chemical Genomics at the
Sanford-Burnham Medical Research Institute, one of the compre-

6660
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32. Cells (angiotensin II receptor-like 1 (AGTRL-1) cell line (DiscoveRx, Cat# 93-
0250C2)) were seeded at 1000 cell/well (1536 plate, Corning) in 4 lL and
grown overnight (16–18 h) at 37 °C, 5% CO2, 100% humidity, then 60 nL of
either DMSO control or 2 mM stock test compounds in DMSO were transferred
to each well, followed by 2
compound wells, and
supplemented with 10% hi-FBS, 1X penicillin/streptomycin) to positive
control wells. This yielded a final concentration of test compound of 20
and 1% final DMSO. Assay was incubated for 90 min at room temperature, and
then developed with 3 L of detection reagent (PathHunter Detection Reagents
lL of 30 nM Apelin-13 to negative control and test
2 lL of assay media (F12 nutrient mix HAMs
l
M
l
(DiscoveRx, Cat# 93-0001)) for 60 min and luminescence read on a Perkin
Elmer ViewLux.
33. For the synthesis of ML221: Step 1:
A
mixture of 5-hydroxy-2-
(hydroxymethyl)-4H-pyran-4-one (Kojic acid) (0.55 g, 3.87 mmol) was
dissolved in thionyl chloride (5 mL, 68.5 mmol) and was stirred at ambient
temperature for 3 h. Excess reagent was removed in vacuo to provide 0.61 g.
(98%) of 2-(chloromethyl)-5-hydroxy-4H-pyran-4-one as an off-white solid. 1
H
NMR. (500 MHz, DMSO-d6): d (ppm) 8.13 (s, 1H), 6.57 (s, 1H), 4.66 (s, 2H). Step
2: A mixture of pyrimidine-2-thiol (161 mg, 1.433 mmol) in 2 ml methanol
was treated with sodium methoxide solution (310 mg, 1.433 mmol) and
stirred until dissolved. Acetonitrile (10 mL) was added followed by 2-
(chloromethyl)-5-hydroxy-4H-pyran-4-one (230 mg, 1.433 mmol) and the
mixture was stirred at ambient temperature for 3 h at which time analysis
by LC/MS indicated the reaction to be complete. The solvent was removed in
vacuo to provide 406 mg (96%) of a yellow solid containing crude 5-hydroxy-2-
((pyrimidin-2-ylthio)methyl)-4H-pyran-4-one and an equimolar amount of
sodium chloride which was used without further purification. 1H NMR.
(500 MHz, CDCl3): d (ppm) 8.52 (d, 2H, J = 4.9 Hz), 7.80 (s, 1H), 7.02 (t, 1H,
16. Yue, P.; Jin, H.; Aillaud, M.; Deng, A. C.; Azuma, J.; Asagami, T.; Kundu, R. K.;
Reaven, G. M.; Quertermous, T.; Tsao, P. S. Am. J. Physiol. Endocrinol. Metab.
2010, 298, E59.
17. Hashimoto, T.; Kihara, M.; Ishida, J.; Imai, N.; Yoshida, S.; Toya, Y.; Fukamizu,
A.; Kitamura, H.; Umemura, S. Arterioscler. Thromb. Vasc. Biol. 2006, 26,
1267.
J = 4.8 Hz), 6.63 (s, 1H), 4.23 (s, 2H). Step 3:
A mixture of 5-hydroxy-2-
((pyrimidin-2-ylthio)methyl)-4H-pyran-4-one (200 mg, 0.847 mmol), cesium
carbonate (276 mg, 0.847 mmol), and 4-nitrobenzoyl chloride (220 mg,
1.185 mmol) in acetonitrile (8 mL) was stirred at ambient temperature
overnight. The solvent was removed in vacuo to provide a pale yellow solid,
which was partitioned with approximately 20 ml of 1:1 ethyl acetate and
water. The desired product remained insoluble in the biphase and was
collected by filtration. The solid was dried in vacuo to yield 202 mg (62%) as a
tan solid. 1H NMR. (500 MHz, DMSO-d6): d (ppm) 8.69 (d, 2H, J = 4.8 Hz), 8.68
(s, 1H), 8.40 (d, 2H, J = 8.8 Hz), 8.29 (d, 2H, J = 8.8 Hz), 7.29 (t, 1H, J = 4.9 Hz),
6.65 (s, 1H), 4.45 (s, 2H). 13C NMR. (125 MHz, DMSO-d6): d (ppm) 171.2, 168.8,
165.9, 161.7, 158.1, 150.8, 149.9, 140.3, 133.0, 131.4, 124.2, 118.0, 114.6, 31.2.
18. Jia, Y. X.; Lu, Z. F.; Zhang, J.; Pan, C. S.; Yang, J. H.; Zhao, J.; Yu, F.; Duan, X. H.;
Tang, C. S.; Qi, Y. F. Peptides 2007, 28, 2023.
19. Katugampola, S. D.; Maguire, J. J.; Kuc, R. E.; Wiley, K. E.; Davenport, A. P. Can. J.
Physiol. Pharmacol. 2002, 80, 369.
20. Lee, D. K.; Cheng, R.; Nguyen, T.; Fan, T.; Kariyawasam, A. P.; Liu, Y.; Osmond, D.
H.; George, S. R.; O’Dowd, B. F. J. Neurochem. 2000, 74, 34.
21. Tatemoto, K.; Takayama, K.; Zou, M. X.; Kumaki, I.; Zhang, W.; Kumano, K.;
Fujimiya, M. Regul. Pept. 2001, 99, 87.
22. Chun, H. J.; Ali, Z. A.; Kojima, Y.; Kundu, R. K.; Sheikh, A. Y.; Agrawal, R.; Zheng,
L.; Leeper, N. J.; Pearl, N. E.; Patterson, A. J.; Anderson, J. P.; Tsao, P. S.; Lenardo,
M. J.; Ashley, E. A.; Quertermous, T. J. Clin. Invest. 2008, 118, 3343.