Diamino-C,N-diarylpyridine positional isomers as inhibitors of lysophosphatidic acid acyltransferase-β

Article abstract of DOI:10.1016/j.bmcl.2005.07.055

2,6-Diamino-4,N-diarylpyridines were identified as potent, isoform selective inhibitors of the enzymatic activity of lysophosphatidic acid acyltransferase-β (LPAAT-β).

Full text of DOI:10.1016/j.bmcl.2005.07.055

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4703–4707
Diamino-C,N-diarylpyridine positional isomers as inhibitors
of lysophosphatidic acid acyltransferase-b
Feng Hong, David Hollenback, Jack W. Singer and Peter Klein*
Cell Therapeutics, Inc., 201 Elliott Ave. W., Suite 400, Seattle, WA 98119, USA
Received 2 June 2005; revised 14 July 2005; accepted 27 July 2005
Available online 6 September 2005
Abstract—2,6-Diamino-4,N-diarylpyridines were identified as potent, isoform selective inhibitors of the enzymatic activity of lyso-
phosphatidic acid acyltransferase-b (LPAAT-b).
Ó 2005 Elsevier Ltd. All rights reserved.
Lysophosphatidic acid acyltransferase (LPAAT) activi-
ties, detected in bacteria, yeast, plant, and animal cells,
catalyze the sn-2 acylation of lysophosphatidic acid (1-
acyl-sn-glycerol-3-phosphate, LPA) to phosphatidic acid
(1,2-diacyl-sn-glycerol-3-phosphate, PA). PA is a com-
ponent of cell membranes and a key intermediate in
the de novo synthesis of phosphoglycerides, which com-
prise the major components of cell membranes, and of
triacylglycerol, the major form of energy storage in
plants and animals. The majority of LPAAT activity
in mammalian cells has been attributed to two mem-
brane-associated isoforms, LPAAT-a and LPAAT-b.
These isoforms share about 34% sequence identity,
and contain putative transmembrane domains and two
highly conserved motifs, NHQSXXD and EGTR, essen-
tial for the catalytic activity of a family of acyltransfer-
ases.1,2 LPAAT-a and LPAAT-b also display similar
substrate preferences.3–7 While LPAAT-a is uniformly
expressed in all human tissues tested, LPAAT-b appears
to be expressed more prominently in liver, heart, and
pancreatic tissues, as well as in a wide variety of tumor
cells and their surrounding stroma.1,3,4,8,9 Both isoforms
are highly expressed in adipocytes.10 Curiously, the
LPAAT-b gene has been linked to a rare form of con-
genital, generalized lipodystrophy (CGL).11 These indi-
viduals have non-functional or missing LPAAT-b
genes and are characterized by a nearly complete ab-
sence of non-mechanical adipose tissue from birth. Pa-
tients also have high blood triacylglycerol levels and
develop extreme insulin resistance along with its compli-
cations. The relationship between LPAAT-b and CGL
has yet to be fully elucidated, particularly since patients
presumably still have functional LPAAT-a activity.
PA has also been implicated as a lipid cofactor in cell sig-
naling events including Raf translocation to membranes,
mTOR activation, epidermal growth factor receptor
(EGFR) internalization, and activation of PKCf.12–15
The production of PA associated with these cellular
events is often attributed to the catalytic activity of phos-
pholipase D. However, no small molecule inhibitor of this
enzyme has been reported to help confirm this hypothesis.
In contrast, ectopic overexpression of LPAAT-b has been
shown to cooperate in activation of the Ras/Raf/Erk
pathway in Xenopus oocytes and LPAAT-b appears to
play a role in tumor cell survival.16 RNAi knockdown
of LPAAT-b blocks tumor cell proliferation.9
In spite of the ubiquity of LPAATs in all life forms and
their potential importance in membrane homeostasis
and possibly lipid signaling, little progress has been
made to fully clarify their functions in cells. The synthe-
sis of potent, isoform-specific, membrane permeable,
small molecule inhibitors of LPAAT-b would greatly
aid in elucidating the biological role of LPAAT-b.
Groups of isoform-specific inhibitors of LPAAT-b
have been reported and include 2-arylbenzoxazoles 1,
2-arylbenzothiazoles 2, 2-arylbenzimidazoles 3,17 diami-
no-C,N-diaryltriazines 4,18 and diamino-C,N-diarylpyr-
imidine positional isomers 5, 6, and 7 (Fig. 1).19
Triazine 4 and diamino-C,N-diarylpyrimidine positional
isomer 5 emerged as potent LPAAT-b inhibitors.18,19
*
To clarify further the structural requirements for
LPAAT-b inhibitor activity developed with the triazines
Corresponding author. Fax: +1 206 284 6206; e-mail:
jpeterklein@yahoo.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.055

4704
F. Hong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4703–4707
NH2
NH2
Y
O
R3
R2
R2
H
R1
Cl
N
N
R1
X
N
X
N
N
N
Z
N
H
H
R1
R2
Cl
4
1 (X = O)
2 (X = S)
3 (X = NMe)
5 (X = Y = N, Z = CH)
6 (X = Z = N, Y = CH)
7 (X = CH, Y = Z = N)
8 (X = Y = CH, Z = N)
9 (X = N, Y = Z = CH)
10 (X = Z = CH, Y = N)
Figure 1.
and pyrimidines, we replaced these heterocyclic scaffolds
with a pyridine ring. Although substitution on vicinal
ring carbons is possible with pyridines, we chose to focus
on non-vicinal positional isomers 8, 9, and 10 based on
their structural similarities to the reported triazines and
pyrimidine inhibitors. This report presents the syntheses
of diamino-C,N-diarylpyridine positional isomers 8, 9,
and 10, and their ability to inhibit the enzymatic activity
of LPAAT-b.
heating with excess neat substituted aniline 14 at
180 °C yielded 8 after aqueous acid treatment to remove
unreacted 14 and column chromatography. For the syn-
thesis of 9, Cu(II) promoted N-arylation of intermediate
13 with arylboronic acid 15 afforded 2-chloropyridine
16.24–26 Surprisingly, 16 proved exceptionally resistant
to aminolysis procedures to produce 9. This conversion
was accomplished by a novel two-step approach. Heat-
ing a mixture of 16, acetamide, and copper powder pro-
vided acetamide 17. Amide hydrolysis of the acetamide
group in 17 by refluxing with hydrazine and ethanol
afforded 9.
Approaches for the synthesis of 2,4,6-trisubstituted pyri-
dines for use in drug discovery have been reported
recently.20–22 Our approach to synthesizing appropriate-
ly substituted pyridines as analogs of triazine 4 and
pyrimidines 5, 6, and 7 utilized methods involving C-
arylation of the pyridine ring and N-arylation of amino-
pyridines. The substitution patterns in the two aryl rings
(R1, R2, and Cl), optimized for the triazine and pyrimi-
dine series, were preserved in the pyridine series.18,19
2,6-Diamino-N2,4-diarylpyridine 10 was synthesized, as
described in Scheme 2. Treatment of chelidamic acid
(18) with oxalyl chloride yielded 4-chloropyridine dicar-
bonyl dichloride (19). Ethanolysis of 19 gave diester 20.
4-Arylation of 20 by palladium catalyzed Suzuki cou-
pling with arylboronic acid 12 afforded 4-arylpyridine
diester 21. Conversion of the two esters in 21 to amino
groups utilized a 4-step sequence involving Curtius rear-
rangement. Heating 21 with hydrazine and ethanol
yielded bis-acylhydrazide 22, which upon treatment with
nitrous acid gave bis-acylazide 23. Curtius rearrange-
ment proceeded upon heating 23 with t-butanol and tol-
uene affording bis-t-butylcarbamate 24, which was
deprotected with TFA to give diaminopyridine 25.
Syntheses of 2,4-diamino-N2,6-diarylpyridine 8 and 2,4-
diamino-N4,6-diarylpyridine 9 were carried out, as de-
scribed in Scheme 1. Both 8 and 9 were prepared from
4-amino-2-aryl-6-chloropyridine 13, which, in turn,
was prepared via a palladium catalyzed Suzuki coupling
of 4-amino-2,6-dichloropyridine (11) and aryl boronic
acid 12.23 Displacement of the chloro group in 13 by
NH2
R
OMe
R
b
N
N
H2N
H
NH2
N
14a (R = Cl)
14b (R = Me)
OMe
Cl
8a (R = Cl)
8b (R = Me)
+
+
NH2
Cl
OMe
Cl
B(OH)2
a
+
Cl
X
Cl
N
Cl
R
OMe N
R
c
13
11
12a
N
H
(OH)2B
15a (R = Cl)
15b (R = Me)
Cl
16a (R = Cl, X = Cl); 16b (R = Me, X = Cl)
17a (R = Cl, X = NHAc); 17b (R = Me, X = NHAc)
9a (R = Cl, X = NH2); 9b (R = Me, X = NH2)
d
e
˚
Scheme 1. Reagents and conditions: (a) Pd(OAc)2, PPh3, CsF, H2O, DME, 80 °C (40% yield); (b) 180 °C (75–80% yield); (c) Cu(OAc)2, Et3N, 4 A
mol sieve, CH2Cl2 (68–70% yield); (d) acetamide, Cu powder, 180 °C (35–40% yield); (e) NH2NH2, EtOH, reflux (74–80% yield).

F. Hong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4703–4707
4705
Y
NH2
N
R1
Cl
R1
N
R1
Y
R2
B(OH)2
c
g
N
+
+
Y
NH2
(OH)2B
15a (R2 = Cl)
X
Y
18 (X = O, Y = CO2H)
19 (X = Cl, Y = COCl)
20 (X = Cl, Y = CO2Et)
Cl
Cl
a
12a (R1 = OMe)
12b (R1 = OEt)
15b (R2 = Me)
25a (R1 = OMe)
25b (R1 = OEt)
21a,b (Y = CO2Et)
22a,b (Y = CONHNH2)
23a,b (Y = CON3)
b
d
15c (R2 = CHO)
e
h
f
24a,b (Y = NHCO2tBu)
NH2
N
R2
R1
Cl
N
H
10a (R1 = OMe, R2 = Cl)
10b (R1 = OMe, R2 = Me)
10c (R1 = OMe, R2 = CHO)
10d (R1 = OEt, R2 = Cl)
10e (R1 = OEt, R2 = Me)
10f (R1 = OEt, R2 = CHO)
10g (R1 = OEt, R2 = CH2OH)
i
Scheme 2. Reagents and conditions: (a) (COCl)2, DMF, CH2Cl2, reflux; (b) EtOH, pyridine (70% yield for 2 steps); (c) Pd(OAc)2, PPh3, Na2CO3,
H2O, DME (65–70% yield); (d) NH2NH2, EtOH, reflux; (e) NaNO2, 0.5 M aq HCl (85–90% yield for 2 steps); (f) t-BuOH, toluene, reflux (35–40%
˚
yield); (g) CF3CO2H, CH2Cl2 (88–90% yield); (h) Cu(OAc)2, Et3N, 4 A mol sieve, CH2Cl2 (20–35% yield); (i) NaBH4, MeOH, THF (40% yield).
Table 1. LPAAT-b inhibition of triazines, diamino-C,N-diarylpyrim-
idine positional isomers, and diamino-C,N-diarylpyridine positional
isomers
Cu(II) promoted N-arylation of 25 with 15 completed
the synthesis of 10a–f. Benzyl alcohol 10g was synthe-
sized by NaBH4 reduction of aldehyde 10f. Synthesized
compounds were purified either by flash chromatogra-
phy or preparative TLC to provide each compound to
be screened for LPAAT activity as single spot by
TLC. 1H NMR and MS data of synthesized compounds
are reported.27
NH2
Y
R2
R1
Cl
X
Z
N
H
Synthesized pyridines 8, 9, and 10 were tested for their
ability to inhibit the enzymatic activity of human
LPAAT-b and LPAAT-a, which were separately over-
expressed in SF9 insect cell membranes.28 Table 1 com-
pares LPAAT-b inhibition by pyridines 8, 9, and 10.
Examples of positional isomer 10 were consistently more
potent than examples of either 8 or 9. For example,
comparing inhibition by isomeric 8a, 9a, and 10a it is
evident that 10a is at least 100-fold more potent than
the other isomers. Of the pyridines listed in Table 1,
10a with IC50 = 0.04 lM was the most potent. This
potency preference for one of three positional isomers
mirrors observations with pyrimidine positional isomers
5, 6, and 7, where only isomer 5 displayed potent
LPAAT-b inhibition. Comparing the potency preferenc-
es for pyridines, pyrimidines, and triazines, it is tempting
to speculate that the presence of a nitrogen atom at the
ring position labeled Y in Table 1 may be an important
structural component influencing inhibitor potency.
Contrary to the LPAAT-b data, none of the compounds
measurably inhibited LPAAT-a activity up to a concen-
tration of 40 lM.
Compound
X
Y
Z
R1
R2
LPAAT-ba
(IC50, lM)
4a
4b
N
N
N
N
OMe Cl
OEt Cl
0.07, 0.045
0.14, 0.26
0.054
0.017
7
N
N
5a
5b
N
N
CH OMe Cl
CH OEt Cl
N
N
6a
7a
N
CH
CH
N
N
N
N
N
OMe Cl
OMe Cl
OMe Cl
2.0, 2.05
6.5
29
8a
8b
CH CH
CH CH
OMe Me
9a
9b
N
CH CH OMe Cl
CH CH OMe Me
18
N
35, > 40
0.04
0.24
10a
10b
10c
10d
10e
10f
10g
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
N
N
CH OMe Cl
CH OMe Me
CH OMe CHO
0.26
CH OEt
CH OEt
CH OEt
CH OEt
Cl
Me
0.050
0.49
0.050
CHO
CH2OH 0.099
a LPAAT assay is described in Ref. 28.
synthesized by novel approaches employing palladium
promoted C-arylation and Cu(II) promoted N-arylation
methodologies. These compounds potently inhibited
LPAAT-b activity but not LPAAT-a activity, in spite
In summary, we have synthesized pyridine ring analogs
of potent triazine and pyrimidine LPAAT-b inhibitors.
The three pyridine positional isomers 8, 9, and 10 were

4706
F. Hong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4703–4707
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Y.; Park, J. G.; Jeong, T. C.; Cho, M. J.; Choi, S. U.; Lee,
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of the two isoforms showing sequence similarities, con-
served residues at their catalytic sites, and similar sub-
strate preferences. New questions arise as to whether
the dramatic selectivity of the reagents is due to binding
to a unique site on LPAAT-b or whether the inhibitors
exploit subtle structural differences at the catalytic site.
These agents may prove useful as tools to study
LPAAT-b functions in cells, including the de novo syn-
thesis of phosphoglycerides and triacylglycerol, adipo-
cyte differentiation and metabolism, as well as events
involving PA signaling processes.
25. Lam, P. Y. S.; Clark, C. G.; Saubern, S.; Adams, J.;
Winters, M. P.; Chan, D. M. T.; Combs, A. Tetrahedron
Lett. 1998, 39, 2937.
26. Lam, P. Y. S.; Clark, C. G.; Saubern, S.; Adams, J.;
Averill, K.; Chan, D. M. T.; Combs, A. P. Synlett 2000,
674.
Acknowledgments
We thank Lisa Romero for conducting LPAAT assays,
and Man Shun Lai and Garland Bellamy for mass spec-
trometry analyses.
27. 1H NMR were recorded on a Bruker Avance 400
instrument and mass spectra were recorded on a Micro-
mass Quatro II electrospray mass spectrometer. 8a:
(acetone-d6) d 3.90 (s, 3H, CH3), 5.39 (s, 2H, NH2), 6.10
(d, 1H, J = 1.7 Hz, Ar), 6.90 (d, 1H, J = 1.7 Hz, Ar), 7.12
(d, 1H, J = 8.8 Hz, Ar), 7.24 (d, 2H, J = 8.9 Hz, Ar), 7.34
(dd, 1H, J = 8.8 Hz, J = 2.8 Hz, Ar), 7.73 (d, 2H,
J = 8.9 Hz, Ar), 7.91 (d, 1H, J = 2.8 Hz, Ar), 8.09 (s, 1H,
NH); EIMS m/z 360.0 (M+H)+. 8b: (acetone-d6) d 2.27 (s,
3H, CH3), 3.89 (s, 3H, CH3), 5.30 (d, 2H, J = 6.4 Hz,
NH2), 6.11 (d, 1H, J = 1.7 Hz, Ar), 6.89 (d, 1H,
J = 1.7 Hz, Ar), 7.07 (d, 2H, J = 8.3 Hz, Ar), 7.10 (d,
1H, J = 8.8 Hz, Ar), 7.32 (dd, 1H, J = 8.8 Hz, J = 2.8 Hz,
Ar), 7.51–7.54 (m, 2H, Ar), 7.68 (s, 1H, NH), 7.99 (d, 1H,
J = 2.8 Hz, Ar); EIMS m/z 339.9 (M+H)+. 9a: (acetone-d6)
d 3.90 (s, 3H, CH3), 5.27 (s, 2H, NH2), 6.22 (t, 1H,
J = 1.9 Hz, Ar), 7.09–7.12 (m, 2H, Ar), 7.26–7.36 (m, 5H,
Ar), 7.86 (s, 1H, NH), 7.96 (d, 1H, J = 2.8 Hz, Ar); EIMS
m/z 360.2 (M+H)+. 9b: (acetone-d6) d 2.31 (s, 3H, CH3),
3.90 (s, 3H, CH3), 5.40 (s, 2H, NH2), 6.16 (t, 1H,
J = 2.0 Hz, Ar), 7.04 (bs, 1H, Ar), 7.09 (t, 1H,
J = 8.8 Hz, Ar), 7.17 (bs, 4H, Ar), 7.32 (dd, 1H,
J = 8.8 Hz, J = 2.8 Hz, Ar), 7.72 (s, 1H, NH), 7.93 (d,
1H, J = 2.8 Hz, Ar); EIMS m/z 340.2 (M+H)+. 10a:
(acetone-d6) d 3.84 (s, 3H, CH3), 5.33 (d, 2H, J = 8.8 Hz,
NH2), 6.16 (d, 1H, J = 1.1 Hz, Ar), 6.27 (t, 1H, J = 1.1 Hz,
Ar), 7.12 (d, 1H, J = 8.8 Hz, Ar), 7.23 (d, 2H, J = 8.9 Hz,
Ar), 7.30 (d, 1H, J = 2.7 Hz, Ar), 7.36 (dd, 1H, J = 8.8 Hz,
J = 2.7 Hz, Ar), 7.76–7.79 (m, 2H, Ar), 8.05 (s, 1H, NH);
EIMS m/z 360.1 (M+H)+. 10b: (acetone-d6) d 2.26 (s, 3H,
CH3), 3.83 (s, 3H, CH3), 5.24 (bs, 2H, NH2), 6.10 (d, 1H,
J = 1.1 Hz, Ar), 6.26 (t, 1H, J = 1.1 Hz, Ar), 7.06 (d, 2H,
J = 8.5 Hz, Ar), 7.08 (d, 1H, J = 8.8 Hz, Ar), 7.29 (d, 1H,
J = 2.7 Hz, Ar), 7.35 (dd, 1H,J = 9.0 Hz, J = 3.3 Hz, Ar),
7.53–7.56 (m, 2H, Ar), 7.70 (s, 1H, NH); EIMS m/z 340.1
(M+H)+. 10c: (acetone-d6) d 3.85 (s, CH3), 5.51 (bs, 2H,
NH2), 6.27 (d, 1H, J = 1.1 Hz, Ar), 6.38 (d, 1H,
J = 1.1 Hz, Ar), 7.14 (d, 1H, J = 8.8 Hz, Ar), 7.32 (d,
1H, J = 2.7 Hz, Ar), 7.38 (dd, 1H, J = 8.8 Hz, J = 2.7 Hz,
Ar), 7.78 (d, 2H, J = 8.7 Hz, Ar), 7.96 (d, 2H, J = 8.7 Hz,
Ar), 8.59 (s, 1H, NH), 9.84 (s, 1H, CH).10d: (acetone-d6) d
1.35 (t, 3H, J = 7.0 Hz, CH3), 4.08 (q, 2H, J = 7.0 Hz,
CH2), 5.32 (bs, 2H, NH2), 6.21 (d, 1H, J = 1.2 Hz, Ar),
6.32 (t, 1H, J = 1.1 Hz, Ar), 7.09 (d, 2H, J = 8.6 Hz, Ar),
7.23 (d, 2H, J = 8.9 Hz, Ar), 7.31–7.78 (m, 3H, Ar), 8.04
(s, 1H, NH); EIMS m/z 374.1 (M+H)+. 10e: (acetone-d6) d
1.35 (t, 3H, J = 7.0 Hz, CH3), 2.27 (s, 3H, CH3), 4.08 (q,
2H, J = 7.0 Hz, CH2), 5.24 (bs, 2H, NH2), 6.14 (d, 1H,
J = 1.0 Hz, Ar), 6.32 (t, 1H, J = 0.9 Hz, Ar), 7.06–7.10 (m,
3H, Ar), 7.29–7.34 (m, 2H, Ar), 7.51–7.54 (m, 2H, Ar),
7.67 (s, 1H, NH); EIMS m/z 354.2 (M+H)+. 10f: (acetone-
d6) d 1.36 (t, 3H, J = 6.9 Hz, CH3), 4.11 (q, 2H, J = 7.0 Hz,
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F. Hong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4703–4707
4707
CH2), 5.51 (bs, 2H, NH2), 6.33 (d, 1H, J = 1.1 Hz, Ar),
6.44 (d, 1H, J = 1.0 Hz, Ar), 7.12 (d, 1H, J = 8.5 Hz, Ar),
7.31–7.37 (m, 2H, Ar), 7.78 (d, 2H, J = 8.8 Hz, Ar), 7.88–
7.97 (m, 2H, Ar), 8.57 (s, 1H, NH), 9.84 (s, 1H, CH);
EIMS m/z 368.1 (M+H)+. 10g: (acetone-d6) d 1.36 (t, 3H,
J = 6.9 Hz, CH3), 3.95 (bs, 1H, OH), 4.09 (q, 2H,
J = 7.0 Hz, CH2), 4.56 (s, 2H, CH2), 5.28 (bs, 2H, NH2),
6.16 (d, 1H, J = 1.1 Hz, Ar), 6.34 (d, 1H, J = 1.1 Hz, Ar),
7.09 (d, 1H, J = 8.5 Hz, Ar), 7.24 (d, 2H, J = 8.8 Hz, Ar),
7.30–7.34 (m, 2H, Ar), 7.60–7.63 (m, 2H, Ar), 7.80 (s, 1H,
NH); EIMS m/z 370.1 (M+H)+. 13: (acetone-d6) d 3.91 (s,
3H, CH3), 5.92 (s, 2H, NH2), 6.60 (d, 1H, J = 1.8 Hz, Ar),
7.14 (d, 1H, J = 8.8 Hz, Ar), 7.33 (d, 1H, J = 1.9 Hz, Ar),
7.37 (dd, 1H, J = 8.8 Hz,J = 2.8 Hz, Ar), 7.90 (d, 1H,
J = 2.8 Hz, Ar). 16a: (acetone-d6) d 3.92 (s, 3H, CH3), 6.86
(d, 1H, J = 8.9 Hz, Ar), 6.90 (t, 1H, J = 1.9 Hz, Ar), 7.18
(d, 1H, J = 8.9 Hz, Ar), 7.20 (d, 1H, J = 8.9 Hz, Ar), 7.36
(d, 2H, J = 8.9 Hz, Ar), 7.41 (dd, 1H, J = 8.8 Hz,
J = 2.8 Hz, Ar), 7.46 (d, 2H, J = 8.9 Hz, Ar), 7.72 (t, 1H,
J = 1.9 Hz, Ar), 7.95 (d, 1H, J = 2.8 Hz, Ar); EIMS m/z
379.0 (M+H)+. 16b: (DMSO-d6) d 2.32 (s, 3H, CH3), 3.91
(s, 3H, CH3), 6.73 (d, 1H, J = 1.9 Hz, Ar), 7.14 (d, 2H,
J = 8.4 Hz, Ar), 7.18 (d, 1H, J = 8.9 Hz, Ar), 7.22 (d, 2H,
J = 8.2 Hz, Ar), 7.44–7.48 (m, 2H, Ar), 7.75 (d, 1H,
J = 2.8 Hz, Ar), 9.1 (s, 1H, NH). 17b: (acetone-d6) d 2.19
(s, 3H, CH3), 2.32 (s, 3H, CH3), 3.91 (s, 3H, CH3), 7.12 (d,
1H, J = 8.8 Hz, Ar), 7.19–7.25 (m, 4H, Ar), 7.35 (dd, 1H,
J = 8.8 Hz, J = 2.8 Hz, Ar), 7.47–7.48 (m, 1H, Ar), 7.86 (s,
1H, NH), 7.89 (d, 1H, J = 2.8 Hz, Ar), 8.05 (s, 1H, NH);
EIMS m/z 382.2 (M+H)+. 20: (DMSO-d6) d 1.36 (t, 6H,
J = 7.1 Hz CH3), 4.40 (q, 4H, J = 7.1 Hz, CH2), 8.31 (s, 2H,
Ar). 21a: (acetone-d6) d 1.42 (t, 6H, J = 7.1 Hz, CH3), 3.92
(s, 3H, CH3), 4.45 (q, 4H, J = 7.1 Hz, CH2), 7.27 (d, 1H,
J = 8.9 Hz, Ar), 7.52 (dd, 1H, J = 8.8 Hz, J = 2.7 Hz, Ar),
7.56 (d, 1H, J = 2.7 Hz, Ar), 8.43 (s, 2H, Ar). 22a: (DMSO-
d6) d 3.92 (s, 3H, CH3), 4.68 (s, 4H, NH2), 7.24 (d, 1H,
J = 8.6 Hz, Ar), 7.53–7.56 (m, 2H, Ar), 8.20 (s, 2H, Ar),
10.72 (s, 2H, NH). 23a: (acetone-d6) d 3.94 (s, 3H, CH3), 7.28
(d, 1H, J = 8.9 Hz, Ar), 7.55 (dd, 1H, J = 8.8 Hz,
J = 2.7 Hz, Ar), 7.62 (d, 1H, J = 2.7 Hz, Ar), 8.54 (s, 2H,
Ar). 24a: (DMSO-d6) d 1.47 (s, 18H, CH3), 3.79 (s, 3H,
CH3), 7.18 (d, 1H, J = 8.9 Hz, Ar), 7.30 (d, 1H, J = 2.6 Hz,
Ar), 7.47–7.49 (m, 3H, Ar), 9.41 (s, 2H, NH).
28. LPAAT-a and LPAAT-b assays were conducted using
5,50-dithiobis(2-nitrobenzoic acid) (DTNB) as the colori-
metric reagent according to the method described in 16.
IC50 values were determined from experiments using 10
concentrations (0.001–40 lM) of each compound in
duplicate and fit to a sigmoidal curve using GraphPad
Prism (GraphPad Software, Inc., San Diego, CA). Dupli-
cate values typically varied by less than 5% and the R2
values for the goodness-of-fit were P0.95.