Identification of substituted benzothiazole sulfones as potent and selective inhibitors of endothelial lipase

Article abstract of DOI:10.1016/j.bmcl.2019.05.048

A low level of high density lipoprotein (HDL) is an independent risk factor for cardiovascular disease. HDL reduces inflammation and plays a central role in reverse cholesterol transport, where cholesterol is removed from peripheral tissues and atheroscle

Full text of DOI:10.1016/j.bmcl.2019.05.048

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of substituted benzothiazole sulfones as potent and selective
inhibitors of endothelial lipase
Soong-Hoon Kim⁎, James A. Johnson, Ji Jiang, Brandon Parkhurst, Monique Phillips, Zulan Pi,
Jennifer X. Qiao, George Tora, Alice Ye Chen, Eddie Liu, Xiaohong Yin, Richard Yang, Lei Zhao,
David S. Taylor, Michael Basso, Kamelia Behnia, Joelle Onorato, Xue-Qing Chen, Lynn M. Abell,
Hao Lu, Gregory Locke, Christian Caporuscio, Leonard P. Adam, David Gordon, Ruth R. Wexler,
Heather J. Finlay
Research and Development, Bristol-Myers Squibb, Princeton, NJ 08543, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
A low level of high density lipoprotein (HDL) is an independent risk factor for cardiovascular disease. HDL
reduces inflammation and plays a central role in reverse cholesterol transport, where cholesterol is removed
from peripheral tissues and atherosclerotic plaque. One approach to increase plasma HDL is through inhibition
of endothelial lipase (EL). EL hydrolyzes phospholipids in HDL resulting in reduction of plasma HDL. A series of
benzothiazole sulfone amides was optimized for EL inhibition potency, lipase selectivity and improved phar-
macokinetic profile leading to the identification of Compound 32. Compound 32 was evaluated in a mouse
pharmacodynamic model and found to show no effect on HDL cholesterol level despite achieving targeted
plasma exposure (Ctrough > 15 fold over mouse plasma EL IC50 over 4 days).
Endothelial lipase
HDL cholesterol
Cardiovascular diseases
Phospholipids
Hepatic lipase
HDL-C is an independent risk factor for cardiovascular disease
(CVD) and routine screening for HDL-C is recommended for all adults.1
The plasma level of HDL-C inversely correlates with cardiac events, and
low levels are present in patients who have CVD.2 The cardio-protective
function of HDL and apo A-I (the major protein constituent of HDL) is
believed to be related to anti-oxidant, anti-thrombotic and anti-in-
flammatory activity.3 In addition, HDL-C has a central role in reverse
cholesterol transport where cholesterol is removed from atherosclerotic
plaque and peripheral tissues for removal from the body via the liver.4
The potential benefit of increasing HDL-C has encouraged multiple
therapeutic approaches, including inhibition of endothelial lipase (EL).5
EL is a 69-kD phospholipase that is structurally related to lipoprotein
lipase (LPL), pancreatic lipase (PL) and hepatic lipase (HL). EL is a
serine hydrolase with a catalytic triad of serine, aspartic acid and his-
tidine, and the active form is a homodimer with head to tail con-
formation.6The primary substrates for EL are the phospho-lipids in
HDL, a complex composed of apo A-I/A-II proteins, triglycerides,
phospholipids and cholesterol. This selectivity for HDL places EL as a
key modulator of HDL metabolism and associated HDL-C plasma level.7
Homozygote EL KO (-/-) mice show an increase in HDL-C plasma
level,8 while transgenic expression of EL in mice result in a reduction of
HDL-C levels.9 These findings from genetic models are further sup-
ported by direct inhibition of EL by neutralizing polyclonal anti-
bodies.10 In addition, a small molecule EL inhibitor, XEN455 (Fig. 1),
has been disclosed.11 This approach has been reported to elevate HDL-C
plasma concentration in vivo, further supporting EL as a potential target
for HDL modulation.
As previously described, compound 2 (EL IC50 0.15 μM) did not
elevate HDL-C level in mice when dosed for 6 days.12 This observation
was attributed to a disconnect between the in vitro assays, which were
run in the absence of plasma or serum, and the potency in physiological
conditions for EL inhibition. When evaluated in an assay where lipid
hydrolysis was measured in HDL, and in the presence of human serum,
2 had an IC50 value of > 476 μM. Evaluating multiple series for target
potency in the plasma and serum assays led to the optimization of
sulfonylated benzothiazole-oxadiazole 3, a potent inhibitor in the pre-
sence of mouse plasma (mPlasma IC50 0.02 μM) with selectivity versus
HL (45 fold).13 Selectivity versus HL was important since HL inhibition
has been reported to elevate low density lipoprotein (LDL), which is a
known risk factor for CVD.14 Despite having achieved Cmin ∼ 7 fold
above mPlasma IC50 over a period of 24 h, an increase in HDL-C was not
observed with 3 in C57BL/6 wild-type (WT) mouse model. The
⁎ Corresponding author.
E-mail address: soonghoon.kim@bms.com (S.-H. Kim).
https://doi.org/10.1016/j.bmcl.2019.05.048
Received 29 March 2019; Received in revised form 22 May 2019; Accepted 24 May 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Soong-HoonKim,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.05.048

S.-H. Kim, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Scheme 1. (a) 1N NaOH, THF, RT, 100%; (b) For Compound 7:
NH2CH2CH2NHBoc, PyBOP, TEA, DMF, RT then TFA, DCM, 47% for two steps;
NH2SO2NH2,
dioxane,
100 °C,
53%;
(c)
For
Compound
8:
Figure 1. Endothelial Lipase inhibitors.
NH2CH2CH2CH2SO2NH2 hydrochloride, PyBOP, TEA, DMF, RT, 34%; (d) For
Compound 10: NaHMDS, MeSO2Cl, −20 °C, 22%; (e) For Compound 11:
NaHMDS, MeSO2Cl, −20 °C, 18%; (f) DBU, MeSO2Cl, DMF, RT, 32%; (g)
Compound 12: NH2CH2CH2SO2NH2 hydrochloride, TEA, dioxane, 145 °C, 15%.
possibility that a pharmacodynamic increase in HDL-C required sus-
tained concentration at even higher multiples above mPlasma IC50 over
a longer period (> 24 h) could not be explored with 3 because of its
dose limiting solubility. We sought to maintain EL potency and se-
lectivity versus HL, concomitant with a p.o. PK profile,that would en-
able evaluation of PD at higher plasma concentrations of the compound
in vivo.
Our strategy focused on improving ADME properties to increase oral
PK exposure. Heterocycle bio-isosteres of the central oxadiazole were
significantly less potent. Previously disclosed compounds in this series
suggested that replacing the oxadiazole with an amide would retain
potency.15 In addition, SAR optimization of benzothiazole-linked ox-
adiazole series13 demonstrated that polar groups, such as the thiazoli-
dinedione in 3 and sulfonyl urea 4 (EL IC50 0.02 μM), in the terminal
position were required for potency. Based on these observations, ben-
zothiazole sulfones with terminal amide groups were targeted to
maintain potency and improve ADME properties. Synthesis of sulfones
10–12 (Table 1) is shown in Scheme 1. Compound 17 was prepared as
shown in Scheme 2. The route closely followed Scheme 1, with the
added flexibility of late-stage Suzuki coupling (step c) to introduce the
aryl ring.
Scheme 2. (a) 1N NaOH, THF, RT, 99%; (b) 2-amino-N-cyclopropylacetamide
hydrochloride, PyBOP, TEA, DMF, RT, 66%; (c) PhB(OH)2, Pd(PPh3)4, dioxane,
2 M Na2CO3, 90 °C, 30%; (d) NaHMDS, MeSO2Cl, −20 °C, 7.8%.
prepared. A series of terminal amides were explored and cyclopropyl
amide 17 was identified some potency for EL inhibition.
However, compounds 10–12 and 17 in Table 1 all showed poor
selectivity versus HL. This was consistent with the earlier SAR work13
on benzothiazole-linked oxadiazole sulfones where selectivity was im-
proved by incorporating polar substituents on the terminal ring (for
example, compound 3 compared to compound 4). The SAR for se-
lectivity versus HL proved to be consistent, and synthesis of polar-
substituted aryl and heterocyclic analogs led to compounds with im-
proved selectivity (compounds 21 and 22 shown in Table 2). Additional
heterocycles were explored at the C6 position and compound 23 was
identified with single digit nanomolar potency for EL and excellent
selectivity versus HL. In addition to improved selectivity, 21–23
showed good potency in the mPlasma assay (IC50 190–320 nM) and
The direct amide sulfone analog of oxadiazole 4, compound 10,
showed only modest EL inhibition (∼20 fold loss compared to 4).
Further modification of the terminal sulfamide to a sulfonamide,
compound 11, further reduced potency. However, we were gratified to
see that the one carbon homologue of 11, sulfonamide 12, showed a
significant improvement in potency (EL IC50 10 nM). Importantly, 12
also demonstrated potency in the mPlasma assay (IC50 210 nM). To
target compounds which maintained or enhanced potency and im-
proved ADME properties, additional non-sulfonamide groups were
Table 2
Table 1
Selectivity Versus HL.
Amide SAR.
Cmpd
12
R
EL IC50 (nM)a
HL IC50 (nM)a
mPlas (nM)a
100
Cmpd
R
EL IC50 (nM)a
450
HL IC50 (nM)a
81
mPlas (nM)a
10
10
15
10
21
280
320
11
12
7700
10
52
15
210
22
23
10
5
310
190
200
17
150
47
4400
a
a
Inhibition was measured in duplicate, and the mean values were used to
calculate IC50 values
Inhibition was measured in duplicate, and the mean values were used to
calculate IC50 values
2

S.-H. Kim, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 3
Mouse p.o. PK (10 mg/kg)a.
Cmpd
Cmax (nM)
C7hr (nM)
C24hr (nM)
Tmax (hr)
AUCtotal (nM*hr)b
Stability (Mouse, %)c
PAMPA (pH 5.5/7.4, nm/s)
cLog P
12
21
22
23
3,500
36
430
8
130
5
0.5
2.6
3
14,000
160
91
365/368
12/13
0/0
0.52
100
74
−0.42
−2.10
−0.21
15
4
1
100
30
1
50
96
17/78
a
PK studies were carried out in fasting balb/c male mice. Compounds were formulated in ethanol / PEG 400 / water (10:60:30). bAUCtotal was calculated based on
compound concentration to 24 h. cPercentage of compound (0.5 μM) remaining after 10 min incubation in mouse liver microsomes.
Scheme 4. (a) PdCl2(dppf), K3PO4, (6-fluoropyridin-3-yl)boronic acid, dioxane,
110 °C, 83%; (b) 1 N NaOH, THF, RT; tert-butyl 2-aminoacetate hydrochloride,
HATU, DIEA, DMF, 34% for two steps; (c) TFA/DCM, RT then cyclopropyl
amine, HATU, DIEA, DMF, 88% yield for two steps; (d) for 27 (R = Me),
NaHMDS, MeSO2Cl, DMF, −20 °C, 56%; (e) for 28 (R = CH2CH2CF3),
NaHMDS, CF3CH2CH2SO2Cl, DMF, −20 °C, 8%; (f) for 29 (R = CH2Ph),
NaHMDS, PhCH2SO2Cl, THF, −20 °C, 41%
Scheme 3. (a) NH2CH2CH2SO2NH2 hydrochloride, PyBOP, TEA, DMF, RT,
66%; (b) PdCl2(dppf), morpholino(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)methanone, K3PO4, dioxane, 105 °C, 67%; NaHMDS, MeSO2Cl,
THF, −20 °C, 24%; (c) NaHMDS, MeSO2Cl, THF, −20 °C, 21%; (d) 4-(2-(4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)ethyl) morpho-
line, Pd(PPh3)4, dioxane/2M Na2CO3, 100 °C, 47%; (e) DBU, MeSO2Cl, DMF,
RT, 49%; (f) PdCl2(dppf), 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)pyridine, K3PO4, dioxane, 105 °C, 56%; NH2CH2CH2SO2NH2 hydrochloride,
TEA, HOBT, ZrCl4, dioxane, 145 °C, 9%.
Scheme 5. (a) NaHMDS, MeOCH2CH2SO2Cl, THF, −20 °C, 35% (b) TFA/DCM,
RT, 98%; (b) cyclopropyl amine, HATU, DIEA, DMF, 91%
were selected for mouse oral PK evaluation (Table 3). The synthesis of
compounds 21–23 is shown in Scheme 3.
As shown in Table 3, compound 12 exhibited good exposure (Cmax
3,500 nM, AUCtotal 14,000 nM*hr). Unfortunately, EL selective com-
pounds 21–23 showed poor oral exposure in mice. The low absorption
(Cmax < 36 nM for 21–23) was likely related to poor permeability.
Compounds 21–23 were stable in mouse liver microsomes, but PAMPA
(parallel artificial membrane permeability assay) permeability values
were low at pH 5.5 and 7.4 (< 78 nm/s). The striking difference in oral
exposure for 21–23 and 12 was likely due to the difference in perme-
ability. Since polarity in the benzothiazole aryl portion was critical for
selectivity (21–23 in Table 2), structural changes to improve perme-
ability were sought elsewhere. Cyclopropyl amide 17 (Table 1) had a
clogP value of 1.24 compared to 0.52 for sulfonamide 12 and demon-
strated higher permeability (PAMPA 824 nm/s and 1,054 nm/s at pH
5.5 and 7.4 respectively). We therefore focused on synthesis of cyclo-
propyl amide sulfones 27–29 with C5 and C6 polar groups to increase
selectivity while maintaining permeability (Schemes 4 and 5).
Table 4
Sulfone SAR.
Cmpd
R
EL
HL
Selectivity mPlasma
IC50(nM)
hSerum
IC50
(nM)
IC50
(nM)
IC50 (nM)
27
28
47
29
130
79
2.8
2.7
290
600
7
660
29
32
25
7
0.3
84
94
65
83
The PAMPA permeability values (pH 5.5/7.4) for cyclopropyl amide
27 showed significant improvement over corresponding sulfonamide
23 (252/389 nm/s vs 17/78 nm/s, respectively). However, as shown in
Table 4, improved permeability was accompanied by loss of selectivity
versus HL. Cyclopropyl amide 27 was only ∼3 fold selective versus HL
compared to 880 fold selectivity observed for 23. In an effort to further
improve selectivity versus HL, additional sulfone substitutions were
explored while keeping the cyclopropyl amide and C6 substituent
constant (representative compounds include 28–29 and 32). Tri-
fluoropropyl analogue 28 and benzyl analogue 29 were less potent and
less selective versus HL compared to 23. Polar methoxy ethyl sulfone,
32 showed improved selectivity (50 fold) with good potency in the
4.6
230
120
Inhibition was measured in duplicate, and the mean values were used to cal-
culate IC50 values.
mPlasma assay. The selectivity was confirmed using EL (HL KO IC50
310 nM) and HL (EL KO IC50 26,000 nM) mPlasma assays derived from
KO mice, an approach more representative of physiological inhibition.
Inhibition of EL in the presence of added human serum (hSerum IC50
)
was comparable to mPlasma IC50. Cyclopropyl amide 32 had increased
3

S.-H. Kim, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 5
on day 4 for HDL-C or LDL-C (Fig. 2). This result was consistent with
our earlier observation13 for compound 3, however, in contrast with
studies where neutralizing EL antibody9 elevated HDL-C and (phos-
pholipids) PL in mice, and with HDL-C elevation observed with small
molecule EL inhibitor XEN455 1.11
Compound 32 mouse p.o. PK.a
Dose (mg/
Cmax (nM) C7hr (nM) C24hr
Tmax T1/
2(hr) /
AUCtotal
(nM)b
kg)
(nM)
10
20
30
39,000
1,700
7,400
2,420
14
45
25
1.0 / 2.2
0.5 / 2.3
0.25 / 2.3
111,000
400,000
240,000
In summary, further optimization of the benzothiazole sulfone series
led to the discovery of a potent, highly selective, and orally bioavailable
inhibitor, Compound 32. Compound 32 had an improved PK exposure
profile at 10 mg/kg compared to earlier compound 3 and could be dose
escalated to 20 mg/kg b.i.d. dosing to enable a Cmin concentration of
1,800 nM in the mouse PD study achieving > 15 fold over mouse IC50
at Cmin. Surprisingly, sustained EL inhibition over a 4 day period in a
mouse pharmacodynamic model failed to demonstrate an increase in
plasma HDL-C levels. This led us to evaluate in further detail the ki-
netics of EL inhibition and incorporation of an assay with pre-incuba-
tion which will be disclosed in due course.
143,000
142,000
a
PK studies were carried out in fasting balb/c male mice. Compound 32 was
formulated in ethanol / PEG 400 / water (10:60:30). bAUCtotal was calculated
based on compound concentration to 24 h
Cholesterol - Day 4
PL - Day 4
Vehicle
10
8
20
15
10
5
Vehicle
BMT-137275
BMT-137275
6
4
2
Acknowledgements
0
0
20
40
60
20
40
60
-2
fraction#
fraction#
-5
The authors would like to thank Dr. Arvind Mathur, Rick Rampulla
and colleagues at Department of Discovery Synthesis at Biocon Bristol-
Myers Squibb RD Center for the preparation of intermediates.
Figure 2. Effect of EL inhibitor 32 (red) and vehicle (blue) on HDL-cholesterol
and phospholipids (PL) plasma concen-tration on day 4.
Appendix A. Supplementary data
permeability (PAMPA at pH 5.5 = 94 nm/s) compared to sulfonamide
23 (PAMPA at pH 5.5 = 17 nm/s), while retaining good mouse liver
microsome stability (81% remaining after 10 min incubation).
Compound 32 was evaluated in mouse p.o. PK and results are shown
in Table 5. Compound 32 showed higher exposure (C7h 1,700 nM) than
compound 3 when dosed at 10 mg/kg, as well as significant improve-
ment over sulfonamides 21–23. As described previously, compound 3
had dose limiting solubility13 and could not be dosed higher than
10 mg/kg in vivo. When the dose was increased for compound 32, and
increase in exposure was observed at 20 mg/kg (∼3.6 fold increase in
AUC); however, no significant increase was observed from 20 to 30 mg/
kg. To target the highest feasible exposures at C24h, we elected to dose
b.i.d. with projected exposure based on the observed PK exposure at
20 mg/kg q.d., a Cmin concentration of 2,700 nM, which was 23 fold
over mPlasma IC50 of 120 nM in mouse.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.05.048.
References
1. Toth PP. Circulation. 2005;111:e89.
2. Castelli WP. Can J Cardiol. 1988;4(supple A):5A–10A.
3. Rader DJ. Am J Cardio. 2002;90(suppl):62i–70i.
4. Annema W, Tietge UJ. Curr Atheroscler Rep. 2011;13:257–265.
5. deGoma EM, Rader DJ. Nat Rev Cardiol. 2011;8:266–277.
6. Griffon N, Jin W, Petty TJ, et al. J Biol Chem. 2009;284:23322–23330.
7. McCoy MG, Sun G, Marchadier D, Maugeais C, Glick JM, Rader DJ. J Lipid Res.
2002;43:921–929.
8. Brown RJ, Lagor WR, Sankaranaravanan S, et al. Circ Res. 2010;107:357–364.
9. Ishida T, Choi S, Kundu RK, Hirata K, Rubin EM, Cooper AD, Quertermous T. J Clin
Invest. 2003;111:347–355.
10. Jin W, Millar JS, Broedl U, Click JM, Rader DJ. J Clin Invest. 2003;111:357–362.
11. Greco MN, Connelly MA, Leo GC, et al. Bioorg Med Chem Lett. 2013;23:2595–2597.
12. Hengeland J, Abell LM, Adam LP, et al. ACS Med Chem Lett. 2018;9(7):673–678.
13. Johnson JA, Tora G, Pi Z, et al. Sulfonylated benzothiazole based inhibitors of en-
dothelial lipase. ACS Med Chem Lett. 2018;9(12):1263–1268.
14. Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershatz H, Kannel B.
Circulation. 1998;97:1837–1847.
Wild-type C57BL/6 mice were dosed with compound 32 at 20 mg/
kg p.o. b.i.d. over 4 days. A satellite PK co-hort showed exposure con-
sistent with projected values (C8h = 5,200 nM, C24h = 1,800,
C
102h = 6,500 nM), with Cmin > 15 fold over the mPlasma IC50
throughout the 4 day period. The lipid profile was measured using fast
protein liquid chromatography (FPLC) on day 1 and day 4 from plasma
pooled from 8 mice. There were no differences observed between mice
treated with vehicle control (blue) and Compound 32 (red) on day 1 or
15. Masuda, Kouji; Kida, Shiro; Hayashi, Kyohei; Katou, Manabu; Yoshikawa, Naoki;
Kugimiya, Akira; Nakajima, Mado; Tanaka, Nobuyuki. WO2010044441, 2010.
4