Structure-activity relationships for lipoprotein lipase agonists that lower plasma triglycerides in vivo

Article abstract of DOI:10.1016/j.ejmech.2015.08.058

The risk of cardiovascular events increases in individuals with elevated plasma triglyceride (TG) levels, therefore advocating the need for efficient TG-lowering drugs. In the blood circulation, TG levels are regulated by lipoprotein lipase (LPL), an unstable enzyme that is only active as a non-covalently associated homodimer. We recently reported on a N-phenylphthalimide derivative (1) that stabilizes LPL in vitro, and moderately lowers triglycerides in vivo (Biochem. Biophys. Res. Commun. 2014, 450, 1063). Herein, we establish structure-activity relationships of 51 N-phenylphthalimide analogs of the screening hit 1. In vitro evaluation highlighted that modifications on the phthalimide moiety were not tolerated and that lipophilic substituents on the central phenyl ring were functionally essential. The substitution pattern on the central phenyl ring also proved important to stabilize LPL. However, in vitro testing demonstrated rapid degradation of the phthalimide fragment in plasma which was addressed by replacing the phthalimide scaffold with other heterocyclic fragments. The in vitro potency was retained or improved and substance 80 proved stable in plasma and efficiently lowered plasma TGs in vivo.

Full text of DOI:10.1016/j.ejmech.2015.08.058

Accepted Manuscript
Structure-activity relationships for lipoprotein lipase agonists that lower plasma
triglycerides in vivo
Rémi Caraballo, Mikael Larsson, Stefan K. Nilsson, Madelene Ericsson, Weixing
Qian, Nam Phuong Nguyen Tran, Tomas Kindahl, Richard Svensson, Valeria Saar,
Per Artursson, Gunilla Olivecrona, Per-Anders Enquist, Mikael Elofsson
PII:
S0223-5234(15)30241-5
10.1016/j.ejmech.2015.08.058
EJMECH 8094
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 February 2015
Revised Date: 25 August 2015
Accepted Date: 28 August 2015
Please cite this article as: R. Caraballo, M. Larsson, S.K. Nilsson, M. Ericsson, W. Qian, N.P. Nguyen
Tran, T. Kindahl, R. Svensson, V. Saar, P. Artursson, G. Olivecrona, P.-A. Enquist, M. Elofsson,
Structure-activity relationships for lipoprotein lipase agonists that lower plasma triglycerides in vivo,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.08.058.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-activity relationships for lipoprotein lipase
agonists that lower plasma triglycerides in vivo
a
b
b
b
c
Rémi Caraballo, Mikael Larsson, Stefan K. Nilsson, Madelene Ericsson, Weixing Qian,
a
a
d
b
Nam Phuong Nguyen Tran, Tomas Kindahl, Richard Svensson, Valeria Saar, Per
d
b
c
a, c, *
Artursson, Gunilla Olivecrona, Per-Anders Enquist and Mikael Elofsson
a
Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden
b
Department of Medical Biosciences, Umeå University, SE-901 87 Umeå, Sweden
Laboratories for Chemical Biology Umeå, Chemical Biology Consortium Sweden, Umeå
c
University, SE-901 87 Umeå, Sweden
d
Department of Pharmacy, Uppsala University, Drug Optimization and Pharmaceutical
Profiling Platform, Chemical Biology Consortium Sweden, Science for Life Laboratory
Uppsala, SE-751 23 Uppsala, Sweden.
*
Corresponding author; Email: mikael.elofsson@umu.se; Phone: +46-90-7869328; Fax:
+46-90-7867655
Abstract
The risk of cardiovascular events increases in individuals with elevated plasma triglyceride
(TG) levels, therefore advocating the need for efficient TG-lowering drugs. In the blood
circulation, TG levels are regulated by lipoprotein lipase (LPL), an unstable enzyme that is
only active as a non-covalently associated homodimer. We recently reported on a N-
phenylphthalimide derivative (1) that stabilizes LPL in vitro, and moderately lowers
triglycerides in vivo (Biochem. Biophys. Res. Commun. 2014, 450, 1063). Herein, we
establish structure-activity relationships of 51 N-phenylphthalimide analogues of the
1

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screening hit 1. In vitro evaluation highlighted that modifications on the phthalimide moiety
were not tolerated and that lipophilic substituents on the central phenyl ring were functionally
essential. The substitution pattern on the central phenyl ring also proved important to
stabilize LPL. However, in vitro testing demonstrated rapid degradation of the phthalimide
fragment in plasma which was addressed by replacing the phthalimide scaffold with other
heterocyclic fragments. The in vitro potency was retained or improved and substance 80
proved stable in plasma and efficiently lowered plasma TGs in vivo.
Keywords: Lipoprotein Lipase, LPL, triglyceride, structure-activity relationship, agonist
1
. Introduction
Coronary Artery Disease (CAD) is a medical term encompassing a set of conditions
responsible for the narrowing and hardening of blood vessels. CAD is classified as the
world’s leading cause of death and, therefore, the prevention, control and treatment of its
associated risk factors remain essential.[1, 2] For instance, the probability of developing
CAD increase significantly with age,[3] family history,[4] obesity,[5] diabetes,[6] high blood
pressure and/or high blood cholesterol levels.[7] Likewise and even though it has long been
debated, hypertriglyceridemia is an independent risk factor of CAD.[8, 9] Abnormally
elevated plasma triacylglycerol (TG) levels is a frequently encountered lipid disorder closely
associated with other CAD risk factors.[10, 11]
Plasma TGs originate from exogenous and endogenous sources. Dietary TGs are packed
into large lipoprotein particles (chylomicrons) in the intestine whereas endogenous TGs are
assembled into very low-density lipoproteins (VLDLs) in the liver. After entering the blood
circulation these TG enriched lipoproteins are processed by lipoprotein lipase (LPL), a TG
specific hydrolase enzyme.[12-14] The released free fatty acids and monoglycerides are
stored or directly utilized for energy production in adipose or muscle tissues, respectively.
2

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The remnant lipoproteins are absorbed by the liver or, in the case of VLDL remnants, they
are further processed into low-density lipoproteins (LDL).
LPL plays a central role in the regulation of plasma TG levels.[12, 15] The enzyme, which
is only active as an unstable non-covalently associated homodimer, is produced and secreted
from parenchymal cells. LPL is then transported and anchored to its site of action at the
luminal surface of endothelial cells in capillaries.[14] Several endogenous factors such as
angiopoietin-like protein (ANGPTL) 3 and 4 regulate LPL activity.[15, 16] ANGPTL3 is
reported to inhibit LPL[17] whereas ANGPTL4 directly promotes the dissociation of the
active homodimer into inactive monomeric species[18].
Causes for abnormally elevated plasma TG levels are gathered into two categories: familial
and acquired hypertriglyceridemia.[10] Familial hypertriglyceridemia is believed to be a
consequence of an accumulation of inherited deleterious alleles in genes regulating
lipoprotein catabolism, including LPL.[19] The condition is said acquired when it is induced
by environmental factors such as obesity, type 2 diabetes mellitus or high alcohol
consumption. Lifestyle changes, e.g. low fat diet, physical activity and alcohol restriction
usually lower plasma TG levels in hypertriglyceridemic patients; however, for patients for
whom lifestyle changes have been ineffective, medication with fibrates, niacin and/or n-3
fatty acids may be necessary.[20]
Herein, we report on the design, synthesis and evaluation of small molecules that stabilize
LPL in the presence of ANGPTL4 and, therefore, prevent the dissociation and inactivation of
homodimeric LPL. As a result, a significant increase in LPL-mediated hydrolysis was seen in
vitro with a number of compounds. Structure-activity relationships (SAR) based on the
screening hit were established and the optimized compounds were found to be efficacious in
vivo.
2
. Results and discussion
3

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High throughput screening of 17340 compounds provided the foundation for this medicinal
chemistry program.[21] The screening hit 1 (Figure 1a), a substituted N-phenylphthalimide,
was selected as starting point on the basis of its positive effects on LPL stability. Compound
1
preserved LPL activity at 37 °C or when incubated at room temperature with the enzyme in
the presence of ANGPTL4. Chemical diversity around the N-phenylphthalimide scaffold
Figure 1b) can be achieved by starting from readily available building blocks. All
(
compounds in the present study were investigated for effects on LPL activity and stability, in
the presence of ANGPTL4, according to previously described protocols.[21]
The synthesis of 1 was straightforward and in good yields (Scheme 1). Compound 2 was
converted to the corresponding acid chloride upon treatment with oxalyl chloride. Thereafter,
coupling with p-toluidine provided 3 in 90% yield. Direct amination of 3 with piperidine
under basic conditions yielded 4 (94%).[22] Subsequent reduction of the nitro group using tin
dichloride in refluxing ethanol[23] or sodium borohydride and palladium on charcoal in
methanol[24] afforded 5 in nearly quantitative yields. Finally, the condensation reaction
between aniline 5 and a substituted phthalic anhydride (6) in refluxing glacial acetic acid
gave 1 in 89% yields.[25]
2.1. Relevance of the carboxylic acid
Compound 7, which only differs from the selected hit (1) by the absence of the carboxylic
acid functional group, was also evaluated during the primary screen (Table 1).[21]
Interestingly, it could not preserve LPL activity which indicates that a substituent on the
1
phthalimide moiety (R in Figure 1b) is essential. To probe the role of the carboxylic acid, a
set of compounds in which the carboxylic acid present in 1 was moved or replaced with other
functional groups was first investigated (Table 1).
The synthesis of the N-phenylphthalimide derivatives followed the synthetic route to 1
(Scheme 1), using the appropriate phthalic anhydride derivatives in the last step (83-97%
4

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yields).[25] Some of the target compounds necessitated an additional transformation. The
methyl ester 12 was conveniently obtained in high yield (97%) by reacting 1 with
trimethylsilyldiazomethane.[26] The carboxamide derivative 11 was synthesized by treatment
of 1 with oxalyl chloride to form the corresponding acid chloride and the activated carboxylic
acid was then reacted with hexamethyldisilazane and provided 11 in 11% yield after
recrystallization. Finally, microwave-assisted cyanation of 8 using copper cyanide as the
nitrile source gave 10 in 24% yield.
The substances were subsequently evaluated for stabilization of LPL activity.[21] In brief,
ANGPTL4 and LPL were added to a phosphate buffer solution containing the compounds
previously dissolved in DMSO and a detergent. The mixtures were incubated for 10 min at
2
5 °C and the remaining LPL activity was determined by addition of a substrate mix
®
containing Intralipid . LPL-mediated hydrolysis was terminated after 45 min and the released
free fatty acids were quantified using a titration kit (NEFA HR2 from Wako Chemicals). All
enzymatic activity measurements were normalized to DMSO controls where 100% represent
LPL incubated without ANGPTL4.
The effect of the compounds on LPL activity is summarized in Table 1. Solely 1
contributed to a significantly greater enzymatic activity. Indeed, a replacement of the
carboxylic acid by another small functional group at the position 5 of the phthalimide
(
compounds 7-12) resulted in the limited or absence of LPL-mediated hydrolysis. The low
activity of 11 at 25 µM underlined the importance of having an acidic functional group
carboxylic acid in 1 vs. carboxamide in 11). It also demonstrated that electron-withdrawing
(
substituents have no direct effect on the compound potency. As expected from the initial
screen, 7 was confirmed inactive. Similarly, moving the carboxylic acid to position 4 of the
phthalimide (13) completely suppressed the effect observed for 1. These results are
conspicuous and evidence the need of having a carboxylic acid group at position 5 of the
5

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phthalimide. Consequently, it was decided that newly designed compounds all should include
a carboxylic acid functional group at this position.
2.2. Substituent scrambling
Efforts to optimize the screening hit then focused on the substituents on the central phenyl
2
3
ring (R and R in Figure 1b). A series of seven analogues that present a scrambling of the
two substituents around the central phenyl ring were synthesized (Table 2). In addition, three
other substances where the 1-piperidinyl group in compound 23 was replaced by a chloride
(14), a phenyl- or tolylamino derivative (15 and 16, respectively) were also analyzed.
Compounds 17-22 were prepared using the same synthetic route as in Scheme 1 with use of
the appropriate starting materials. A different synthetic strategy was however adopted for the
preparation of compounds 15, 16 and 23, using 3-bromo-5-aminobenozoic acid (24) as
starting building block. During the amide coupling and the direct amination reactions
(Scheme 2, steps a and b/c respectively), the unprotected amino group present in 24 would
compete with the reacting amines and therefore a non-negligible amount of homo-coupling
products would form. To circumvent this problem 24 was conveniently converted into the
corresponding formamidine product upon reaction with the Vilsmeier reagent.[27] A
subsequent amide coupling with p-toluidine formed 25 (60%), in a one-pot procedure.
Compound 25 was subsequently reacted with aromatic or aliphatic amines under Buchwald
coupling conditions[28, 29] and after formamidine cleavage in refluxing
ethylenediamine,[27] compounds 28, 29 and 30 were isolated in 53-73% yields. The
condensation reaction between the resulting amines and 6 yielded the final products (50-
6
2%).
Modification of the substitution pattern of 1 generally proved detrimental, or at best non-
beneficial, for LPL stabilization (Tables 1 and 2). Thus, displacing the p-tolylcarbamoyl
substituent to position 2 of the central phenyl ring (19) did not improve the hit potency.
6

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Moving the 1-piperidinyl group to position 5 (23) or 6 (20) of the ring considerably or
slightly reduced LPL activity, respectively. Interestingly, reversing the substituents (21)
resulted in a compound with low activity. Sterically congested compounds 18 and 22 were
also demonstrated poorly active. Finally, 17 showed a reduced potency compared to the
screening hit. As a result, the substitution pattern of 1 was concluded as the pattern of choice
for LPL stabilization.
The set of modified derivatives of 23 (14, 15 and 16) also provided valuable information.
While 14 proved inactive, the replacement of 1-piperidinyl by a phenylamino group (15)
improved the potency of the compound in comparison to 23. However, elongation of the
phenylamino substituent of 15 with a methyl group (16) caused a dramatic loss of activity,
thus suggesting that larger groups are not tolerated.
2.3. Substituent variation.
A set of 33 compounds with the 1,3,4 pattern on the central phenyl ring (cf. Figure 1) was
designed and synthesized (Table 3). N-phenylphthalimide (31), despite the absence of a
carboxylic acid functional group, was added to this series on the basis of its previously
reported hypolipidemic activity in mice.[30] Challenging 31 in our in vitro assay would
indicate a similar or different mode of action than our analogues. For comparison 32, the
carboxylated version of 31, was also evaluated. The target compounds were synthesized as
described in Scheme 1 using of the appropriate building blocks. For compound 54, O-
alkylation was achieved in 75% yield by treating 3 with the alcohol derivative under basic
conditions in DMSO.[31]
Table 3 presents the effect on LPL activity of one or a combination of two modifications at
the central phenyl ring in respect to 1 (Table 1). Importantly, the previously reported 31 and
3
2 proved completely inactive under our experimental conditions, thus suggesting that 1 and
the other active analogues might preserve LPL activity via a different mechanism.
7

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The introduction of a more polar substituent (51-54 and 63) resulted in a complete loss of
LPL activity. Similarly, 55, 56 and 64 that have an electron donating or electron withdrawing
group on the central fragment were not active. It was not surprising to find that the
combination of two unfavorable modifications (45 and 46) resulted in inactive analogues.
Also, the replacement of the methyl group on the carbamoyl substituent with a methyl-ether
(34) decreased the compound potency.
Continuing the analysis on the carbamoyl substituent, the addition of a chloride (48) caused
a significant increase in potency. However, the corresponding fluoride analogue 50 did not
exhibit any enhanced potency in comparison to the screening hit. Likewise, trifluoromethyl
group in 35 and 49 did not contribute to LPL stabilization. Benzylic (44) and aliphatic (47)
substituents were found inactive and less potent than 1, respectively. Removal of the methyl
group (33) significantly reduced LPL-mediated hydrolysis whereas its shift (36) decreased
LPL activity. Interestingly, increasing the bulkiness of the substituent (37, 39-40 and 43)
proved favorable at 6.25 µM; however, the efficacy of the compounds remained relatively
low (≤ 71% of control) at higher concentrations regardless to the size of the substituent.
Substitution of the 1-piperidinyl moiety with a phenylamino group (57) was confirmed as a
beneficial transformation for LPL activity. Compounds 38, 41-42 and 58 showed similar
potencies as 57 and, therefore cumulative effects resulting from two favorable modifications
were not observed. Finally, the aliphatic derivatives (59-62) provided valuable information.
Short aliphatic chains (59 and 62) showed a detrimental effect on LPL activity whereas a
longer chain (60) enhanced the compound’s potency to stabilize LPL. However, further
elongation of the tail (61) almost turned the compound inactive, thus, probing the importance
of the length of the aliphatic chain.
8

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Several compounds e.g. 41, 42, 58, reach a plateau at the two or three highest
concentrations tested. This could possibly be due to low solubility or that these compounds
act as partial agonists.
2.4. Evaluation of the in vitro plasma stability.
Screening hit 1 was selected as model compound for the absorption, distribution,
metabolism and excretion (ADME) profiling of the N-phenylphthalimide derivatives.
Unfortunately, 1 rapidly degraded during the initial plasma protein binding study (2%
remaining after 4 h; see experimental section for details). To verify the poor stability,
compound 1 was incubated and sampled over 24 hours in human plasma which showed a
short half-life (t1/2 = 1.5 h). The degradation mechanism was investigated by LC-MS/MS and
the first and second metabolites were identified as the partially (65 and 66) and fully (5)
hydrolyzed N-phenylphthalimide derivatives (Table 4). Thus, 65 was formed predominantly
(51% after 24 h) while 66 and 5 were produced in lower quantities (24% and 23% after 24 h,
respectively).
In order to establish whether the metabolites could stabilize LPL, they were synthesized
and evaluated in our enzymatic assay (Table 4). Regioisomers 65 and 66 were obtained by
addition of 5 to anhydride 6 in acetic acid. After completion of the reaction, the two
regioisomers were separated using HPLC. Despite a mild effect on LPL activity at 25 µM,
compounds 65 and 66 remained less potent than 1. The second metabolite (5) was found
completely inactive in our in vitro assay. Although these results highlight the beneficial effect
of the rigid bicyclic scaffold over the ring-opened structures, investigations on the N-
phenylphthalimide compounds were discontinued as a consequence of their poor plasma
stability. The previously reported plasma TG lowering-effect of compound 1 in mice should
probably be reconsidered as the effect of a cocktail of substances (1 + metabolites) rather
9

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than the effect of 1 alone.[21] Consequently, the intrinsic potency of compound 1 was
probably underestimated in vivo.
2.5. Design and synthesis of compounds stable in plasma
A set of compounds (Table 5) where the hydrolysis-prone phthalimide fragment was
replaced by other bicyclic scaffolds was designed and synthesized. The selected heterocyclic
rings included benzimidazole 71, benzofuran 77, indole 78 and indazole 80-82 derivatives.
Compound 71 was synthesized in four steps starting from intermediate 5 (Scheme 3). The
aniline derivative was converted into aryl bromide 67 (90%) via a Sandmeyer reaction.[32]
Halogen exchange using a copper-catalyzed procedure provided aryl iodide 68 in excellent
yields.[33] Benzimidazole 70 was prepared in moderate yields by direct arylation of 69 with
6
8.[34] Subsequent methyl ester hydrolysis under basic conditions yielded the target
compound (71, 90%).
Benzofuran and indole derivatives were prepared using the synthetic route displayed in
Scheme 4. Cross-coupling reaction between aryl bromide 67 and trimethylsilylacetylene gave
7
2 in 60% yields after cleavage of the trimethylsilyl group.[35] A cascade reaction involving
a Sonogashira coupling between aryl iodide 73 or 74 and acetylene 72 followed by an
intramolecular ring-closure yielded benzofuran (75, 67%) and indole (76, 51%) derivatives,
respectively.[36] In this reaction the trifluoroacetyl leaving group in 74 is needed for the ring-
closure to occur during the coupling. Subsequent methyl ester hydrolysis yielded the targeted
benzofuran 77 (80%) and indole 78 (77%).
2
H-Indazole derivatives 80-82 were synthesized according to a previously reported one-pot
three-component procedure (Scheme 5).[37] With our substrates, higher yields were however
obtained when the imine intermediate was synthesized separately. The imine was prepared by
refluxing 5 and 79[38] in ethanol.
10

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The newly synthesized compounds were tested for stabilization of LPL (Table 5).
Compound 71 was evaluated as the weakest LPL stabilizer of the series with a moderate
effect on LPL activity at 25 µM. At low concentrations, the benzofuran scaffold in 77 proved
beneficial over the phthalimide fragment in 1 and the indole derivative 78 appeared potent.
However, a drop in LPL activity was observed for both compounds at 25 µM. The underlying
reason is most likely precipitation of the substances due to poor aqueous solubility. Indazole
isomers 80 and 81 were found to have similar potency as 1. Interestingly, the shift of the
carboxylic acid from position 5 (81) to position 6 (80) did not affect the compound ability to
stabilize LPL. In order to verify the importance of the carboxylic acid group in this
compound class, 82 was investigated and found inactive in agreement with the previous SAR.
The results depicted in Table 5 clearly demonstrate that heterocyclic fragments can
efficiently replace the phthalimide scaffold. In order to validate our optimization strategy, the
drug-likeness of compounds 77 and 80 was subjected to ADME profiling in vitro (Table 6).
Unlike 1, the substances proved stable in human plasma (> 93% stability). Both compounds
strongly bound to plasma proteins with < 0.01% of estimated unbound fraction (see
experimental section for details). The high protein binding indicates a limited tissue
exposure, which could be advantageous since LPL is operating in plasma. The kinetic
solubility of compounds 77 and 80 was tested in phosphate buffered saline solution at neutral
and acidic pH (Table 6). Because of the presence of lipophilic substituents on their core
structure, 77 and 80 were expected to be poorly soluble in aqueous solution. The
experimental measurements confirmed this hypothesis. Ionization of the acid function at pH
7
.4 (< 2 µM for both substances) or the apparent protonation of the different basic functions
at pH 1.2 (7 µM for 77; < 2 µM for 80) did not significantly improve the solubility.
In vitro permeability was measured from transport rates across Caco-2 cell monolayers as
-6
previously reported.[39] Compounds 77 (Papp value = 1.2 × 10 cm/s) and 80 (P value = 23
app
11

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-6
×
10 cm/s) had a moderate and high intestinal permeability in the absorptive, apical to
basolateral direction (a-b), respectively. Comparable permeability coefficients were obtained
in the secretory, basolateral to apical (b-a) direction. Calculation of the efflux ratios (E = b-a
Papp / a-b P ) indicated that no apparent active efflux interfered with the transport of either
app
7
7 (E = 1.5) or 80 (E = 1.4). Incubation in human liver microsomes showed very high
metabolic stability for 77 (t1/2 > 80 h, CL < 17 µL/min/mg) and 80 (t > 80 h, CL < 17
int
1/2
int
µL/min/mg) which thus indicates a very low risk for oxidative metabolism in the liver.[40,
1] Compounds 77 and 80 were also tested in freshly prepared human hepatocytes, where
4
additional drug metabolizing enzymes are present and where 77 (t1/2 = 40 min, CL = 35
int
6
µL/min/10 cells) showed a higher degree of metabolism. In contrast, 80 (t > 2 h, CL =
1
/2
int
6
1
2 µL/min/10 cells) proved stable also under the in vivo-like conditions in the human
hepatocytes. In summary, the lead optimization was successful. Further, the compounds are
drug-like with regards to the predicted oral drug space.[42]
Compound 80, its regiosiomer 81 and the screening hit 1 were then further investigated in
vitro by dose-response analysis and determination of EC50 values (see the Supporting
Information for dose-response curves). EC values were found to be 6.0 µM (80), 5.0 µM
5
0
(81) and 6.6 µM (1) showing that the compounds have similar potencies and reach full
efficacy.
2.6. Previously reported hypolipidemic agents.
Our approach to the discovery of new and potent hypotriglyceridemic agents offers the
advantage to assess the in vitro evaluation of the target compounds. LPL is unstable and
difficult to handle. Therefore, in vivo studies are often privileged. We wanted to investigate
whether previously known lipid lowering compounds could stabilize LPL in our enzymatic
assay. As discussed above, N-phenylphthalimide 31 and the carboxylated analogue 32 (Table
3
), which belongs to the same class of compounds as our screening hit, were inefficient in our
12

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assay thus suggesting that 1 and the other active analogues preserve LPL activity via a
different mechanism than 31. Other LPL activators (Figure 2), including Ibrolipim[43, 44],
also known as NO-1886, some 5-fluoro-1H-indole-2-carboxamides derivatives[45] and C10
that was recently reported as an LPL agonist preserving the enzyme from inhibition by
ANGPTL4[46] were tested in our system. None of these substances proved active at 25 µM
in our in vitro assay, confirming that the LPL-activating mechanisms are different compared
to the stabilizing effects of the compounds described in this study.
2.7. In vivo investigation.
In order to compare the in vivo efficacy with the in vitro findings on our compounds, 1, 71,
7, 78, 80, 81 and vehicle only were administered through intraperitoneal (i.p.) injections to
7
mice (n = 8/group), once daily for 4 days (10 mg/kg body weight per injection). Before the
last injection the mice were fasted overnight. On the next morning they were given a last
injection and after 60 min they were challenged with an oral olive oil gavage to monitor
postprandial lipid clearance. In the fasting state 80 was the only compound that caused
significantly lower TG levels than the vehicle control (Figure 3). However, 60 min after the
gavage all animals but those receiving compound 1 had significantly lower plasma TG levels
than the vehicle control. The low plasma stability of 1, described above, is most likely the
reason for the modest efficacy of this compound.[21] At 120 min, a similar pattern was seen
compared to vehicle control. At 180 min, the TG values for most compounds were
significantly lower than for compound 1. These data demonstrate that compounds 71, 77, 78,
8
0 and 81 are superior to the previously reported compound 1 for accelerating postprandial
lipid clearance. In early studies, a single i.p. injection was found to be less efficient than the
repeated injections for 4 days in lowering TG levels (data not shown).
Potentially the TG lowering effect could the result of transcriptional activation.[47] To
address this we profiled compound 80 for agonist activity against a set of nuclear receptors
13

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(
see Experimental section for details). For all receptors the EC values exceeded 100 µM.
5
0
These results supported our theory that the efficacy in vivo of the compounds is due to a
direct effect on LPL.
The most commonly used drugs for lowering TG are niacin, fibrates and fish oil. We have
not benchmarked these drugs against our compounds, mainly because their mode of action is
different. All three currently approved drugs affect TG levels by lowering VLDL synthesis to
some extent,[48, 49] while our compounds appear to increase the clearance of plasma TG. In
support of this we detected increased LPL activity in homogenates of subcutaneous white
adipose tissue for the most potent compounds at 3 h after the lipid gavage (preliminary data,
not shown).
3
. Conclusions
Compounds with greater potencies and stability than the screening hit (1) were designed,
synthesized and evaluated in vitro. The SAR analysis illustrated the necessity of having a
carboxylic acid functional group at the position 5 (or 6) of the bicyclic ring. On the central
phenyl ring, we confirmed the substitution pattern and evidenced the need of having
lipophilic substituents. Larger substituents at the carbamoyl group were probed as beneficial
for the potency (43). At position 4 of the central fragment, cyclic and more rigid substituents
were not obligatory, and thus, an n-hexylamino group (60) added a favorable effect.
However, the length of the aliphatic tail was demonstrated as highly important (61 and 62).
Unfortunately, additive effect of favorable modifications could not be evidenced. The
metabolic stability of the screening hit was also significantly improved by replacing the
hydrolysis-prone phthalimide scaffold by other heterocyclic fragments. In addition to the
SAR analysis, we challenged previously reported hypotriglyceridemic agents in our test
system and, consequently, we demonstrated that our compounds have a different mode of
action on LPL activity than those previously reported. Injections of the optimized lead
14

ACCEPTED MANUSCRIPT
compounds (71, 77, 78, 80 and 81) to mice demonstrated that all compounds lowered plasma
TG levels after a lipid gavage compared to mice treated with vehicle or with the previously
identified, but unstable, compound 1.
In conclusion, we have developed novel TG lowering compounds that preserve the activity
of LPL via a mechanism based upon stabilization of the dimeric enzyme from inactivation by
ANGPTL4. Preliminary experiments in mice revealed that these compounds can blunt a
postprandial elevation of plasma TG levels in vivo.
4
. Experimental section
.1. General Chemical Procedures
4
LC-MS was carried out with a Waters LC system equipped with an Xterra C18 column (50
×
19 mm, 5 ꢀm, 125 Å), eluted with a linear gradient of acetonitrile in water, both of which
contained formic acid (0.2 %). A flow rate of 1.5 mL/min was used and detection was
performed at 254 nm. Mass spectra were obtained on a Water micromass ZQ 2000 using
1
13
positive and negative electrospray ionization. H NMR and C NMR spectra were recorded
with a Bruker DRX-400 or DRX-360 spectrometer. NMR experiments were conducted at 298
K in DMSO-d (residual solvent peak = 2.50 ppm (δH) and 39.52 (δC)). Compounds 9, 13,
6
1
4, 27, 33, 34, 36, 37, 44, 51, 55, 56, 57, 59, 64 and C10 were purchased from ChemBridge
Corp. or Sunblochem HB, analyzed with LC-MS, and used without further purification. All
target compounds were ≥ 95% pure according to HPLC UV-traces, except the low-active
compounds 9, 13 and 65 that were ≥ 90% pure.
4.2. Synthetic Procedures
Procedure A: Synthesis of an acyl chloride and coupling to an aniline derivative
(Exemplified by 3)
15

ACCEPTED MANUSCRIPT
A catalytic amount of DMF was added to a stirred solution of 2-chloro-5-nitrobenzoic acid
(2) (4.00 g, 19.85 mmol) and oxalyl chloride (8.65 mL, 99.19 mmol) in DCM (50 mL) at 0
°
C under nitrogen atmosphere. After 20 min, the reaction was allowed to warm to room
temperature and to proceed for a further 2 h. The reaction mixture was concentrated, re-
dissolved in DCM and concentrated again. Triethylamine (13.83 mL, 99.22 mmol) and p-
toluidine (2.13 g, 19.85 mmol) were added to the crude product dissolved in DCM (50 mL).
The reaction was stirred for 5 h at room temperature under nitrogen atmosphere. After
completion, the reaction mixture was diluted with DCM and successively washed with HCl
(
aq., 1M), NaHCO (aq., sat.), water and brine. The organic phase was dried over MgSO ,
3
4
filtered, and concentrated to dryness. The crude amide was purified by column
chromatography (SiO , EtOAc/n-heptane, 7:3 -> 9:1) to give 3 in 90% yield.
2
Procedure B: Synthesis of an aromatic amide prepared from 3-aminobenzoic acid
derivatives (Exemplified by 25)
Preparation of the Vilsmeier reagent: oxalyl chloride (404 µL, 4.63 mmol) was added
dropwise to a stirred solution of DMF (358 µL, 4.63 mmol) in DCM (5 mL) at 0 °C under
nitrogen atmosphere (note: a white foam is forming). The reaction was allowed to warm to
room temperature and to proceed for 30 min. 3-Amino-5-bromobenzoic acid (500 mg, 2.31
mmol) in DCM (5 mL) was added to the Vilsmeier reagent at 0 °C and the reaction was
stirred for 1 h. Pyridine (561 µL, 6.95 mmol) was added to the mixture at 0 °C and the
reaction was allowed to warm to room temperature. After 30 min, p-toluidine (248 mg, 2.31
mmol) was added and the reaction was stirred for a further 2 h. The mixture was then diluted
with DCM and successively washed with NaHCO , water and brine. The organic phase
3
was dried over MgSO and concentrated to dryness. The crude amide was purified by column
4
chromatography (SiO , DCM/methanol, 5:1) to give 25 in 60% yield.
2
Procedure C: Direct amination of aryl chlorides with amino compounds (Exemplified by 4)
16

ACCEPTED MANUSCRIPT
Piperidine (665 µL, 6.72 mmol) and triethylamine (937 µL, 6.72 mmol) were
simultaneously added to a stirred solution of 3 (1.70 g, 5.85 mmol) in ethanol (15 mL). The
mixture was heated at reflux temperature for 5 h. If the reaction was not completed,
additional amine (e.g. 2 eq.) was added and the reaction was allowed to proceed overnight.
After completion, the mixture was slowly cooled to room temperature. The product was
filtered off, washed with cold ethanol and dried under vacuum to give 4 as a yellow
crystalline powder in 94% yield.
Procedure D: Direct amination of aryl bromides with aniline derivatives (Exemplified by
2
7)
An oven-dried microwave tube was charged with Pd(OAc) (1 mol%, 0.6 mg) and xPhos (3
2
mol%, 4.0 mg). The vessel was evacuated and backfilled with nitrogen (this process was
repeated 3 times). Degassed 1,4-dioxane (0.5 mL) and degassed H O (4 mol%, 0.2 µL) were
2
successively added and the solution was heated at 80 °C for 1.5 min (dark green color). A
second oven-dried microwave tube was charged with NaOt-Bu (37.4 mg, 0.39 mmol), 25
(100 mg, 0.28 mmol) and p-toluidine (35.7 mg, 0.33 mmol). The vessel was evacuated and
backfilled with nitrogen (this process was repeated 3 times). The activated catalyst solution
was transferred from the first microwave tube into the second one using a degassed syringe.
Additional 1,4-dioxane (0.5 mL) was then added in order to ease the stirring. The solution
was heated at 80-90 °C for 5 h. After completion, the reaction mixture was cooled down to
room temperature, diluted with EtOAc, washed with water and brine, dried over MgSO and
4
concentrated to dryness. The crude product was purified by column chromatography (SiO2,
DCM/methanol, 95:5) to give 27 in 79% yield.
Procedure E: Direct amination of aryl bromides with piperidine (Exemplified by 28)
25 (113 mg, 0.31 mmol), Copper (I) iodide (5 mol%, 3.18 mg), (R)-BINOL (20 mol%, 18
mg) and tripotassium phosphate (133.5 mg, 0.63 mmol) were charged in an oven-dried
17

ACCEPTED MANUSCRIPT
microwave tube. The vessel was evacuated and backfilled with nitrogen (this process was
repeated 3 times) and piperidine (311 µL, 3.15 mmol) was added via a degassed syringe. The
reaction was heated, solvent-free, at 110 °C for 1 day. The crude product was directly used in
the next step (formamidine cleavage) without further purification.
Procedure F: Formamidine cleavage (Exemplified by 30)
27 (81 mg, 0.21 mmol) was dissolved in ethanol (5 mL) and ethylenediamine (63 µL, 0.94
mmol) was added to the mixture. The reaction was heated at reflux temperature for 6 h before
being concentrated. The crude product was purified by column chromatography (SiO2,
EtOAc/n-heptane, 1:1) to give 30 in 93% yield.
Procedure G: O-Alkylation of aryl chlorides and alcohol (Exemplified by the synthesis of
5
4)
3
(300 mg, 1.03 mmol) and potassium hydroxide (85.3 mg, 1.55 mmol) were dissolved in
DMSO (3 mL) and the mixture was stirred at room temperature under nitrogen atmosphere.
-(2-Methoxyethoxy)ethanol (147 µL, 1.24 mmol) was then added and the reaction was
2
heated at 60 °C for 1 day. After cooling to room temperature, the reaction mixture was
acidified with HCl (aq., 5%). The precipitate was filtered off, washed with water and dried
under vacuum to give pure product as a yellow powder in 75% yield.
Procedure H: Reduction of nitro compounds, method 1 (Exemplified by 5)
4
(800 mg, 2.36 mmol) and Pd/C (31 mg) was dissolved/suspended in methanol (20 mL)
and stirred vigorously. NaBH (222.8 mg, 5.89 mmol) was added portion wise over a period
4
of 30 min and the reaction was allowed to perform until hydrogen gas release ended. If the
reaction was not completed, additional NaBH was added. The solution was filtered through a
4
®
pad of Celite and concentrated. The crude product was purified by column chromatography
(
SiO , EtOAc/n-heptane, 1:9 -> 1:1) to give 5 in 90% yield.
2
18

ACCEPTED MANUSCRIPT
Procedure I: Reduction of nitro compounds, method 2 (Exemplified by 5)
SnCl (18.07 g, 80.09 mmol) was added to a solution of 4 (5.44 g, 16.05 mmol) in ethanol
2
(100 mL) and the mixture was heated for 3 h at reflux temperature. After completion, the
reaction mixture was allowed to cool down to room temperature. The mixture was then
®
diluted with water and, after removal of the white solid using a Celite pad, the product was
extracted with ethyl acetate. Pure product was obtained by crystallization.
Procedure J: Phthalamido compound synthesis by cyclocondensation of phthalic anhydride
derivatives and amino compounds (Exemplified by 1)
5
(500 mg, 1.62 mmol) and 1,2,4-benzenetricarboxylic anhydride (6, 311 mg, 1.62 mmol)
were refluxed in glacial acetic acid (2.5 mL). After 5 h, the reaction mixture was allowed to
cool down to rt. The product was filtered off, washed abundantly with n-heptane and dried
under vacuum to give 1 as a yellow powder in 89% yield.
Procedure K: Methyl ester synthesis (Exemplified by 12)
1
(50 mg, 0.10 mmol) was dissolved in a DCM/methanol mixture (1.6:0.2 mL) and the
mixture was stirred at room temperature and under nitrogen atmosphere.
Trimethylsilyldiazomethane (2M in hexane; 62 µL, 0.12 mmol) was added dropwise over a
period of 10 min. After the nitrogen gas release ended, the reaction mixture was diluted in
DCM. The organic phase was successively washed with NaHCO , water and brine, dried
3
over MgSO and concentrated to dryness to give 12 as a white powder in 97% yield.
4
Procedure L: Carboxamide synthesis (Exemplified by 11)
A catalytic amount of DMF was added to a stirred solution of 1 (50 mg, 0.10 mmol) and
oxalyl chloride (45 µL, 0.52 mmol) in THF (1 mL) at 0 °C and under nitrogen atmosphere.
After 30 min, the reaction was allowed to warm to room temperature and to perform for a
further 4 h. Then, the reaction mixture was concentrated, dissolved in DCM and concentrated
19

ACCEPTED MANUSCRIPT
again. The crude acyl chloride was dissolved in DCM (1 mL) and the mixture was stirred at 0
°
C and under nitrogen atmosphere. Hexamethyldisilazane (65 µL, 0.31 mmol) in cold DCM
(0 °C, 0.5 mL) was rapidly added to the acyl chloride solution and the reaction was allowed
to warm to room temperature overnight. After removal of the solvent, the crude product was
dissolved in acetone. The remaining solids were filtered off and the filtrate was concentrated.
Recrystallization in DCM gave 11 in 11% yield.
Procedure M: Cyanation of aryl bromides (Exemplified by 10)
8
(100 mg, 0.19 mmol) and Copper (I) cyanide (25.9 mg, 0.29 mmol) were charged in a
microwave tube and NMP (0.2 mL) was added. The vessel was sealed and the reaction, under
nitrogen atmosphere, was heated at 175 °C for 20 h. The reaction was allowed to cool down
to room temperature and the mixture was diluted in ethyl acetate before being filtered
®
through a Celite pad and concentrated. The product was purified on column chromatography
(
SiO , EtOAc/n-heptane, 1:5 -> 1:0) to give 10 as a white solid in 29% yield.
2
Procedure N: Synthesis of amides using phthalic anhydride derivatives and amino
compounds (Exemplified by 65 and 66)
(1.1 equiv.) and 1,2,4-benzenetricarboxylic anhydride (6, 29 mg, 0.15 mmol) were stirred
5
in glacial acetic acid (2 mL) for 20 h at room temperature. After removal of the solvent, 65
and 66 were separated by reverse phase HPLC (Acetonitrile/Water: 4:6 -> 1:1, with 0.005%
of formic acid). The fractions containing pure compound 65 or 66 were combined and
lyophilized to give the regioisomers in 7% and 9% yields, respectively.
Procedure O: Synthesis of aryl bromides via Sandmeyer reaction (Exemplified by 67)
A concentrated HBr solution (0.5 mL, 8 equiv.) was added dropwise at 5 °C to a suspension
of 5 (350 mg, 1.13 mmol) in acetonitrile (3.5 mL); a pink pale precipitate formed. Then, a
cold aqueous solution of sodium nitrite (1 equiv., 0.35 M) was added dropwise while the
20

ACCEPTED MANUSCRIPT
temperature was kept below 5 °C. A cold solution of copper (I) bromide (340.6 mg, 2.1
equiv.) in HBr (0.5 mL, 8 equiv.) was added dropwise to the formed diazonium salt. The
reaction was stirred at room temperature for 1 h and then heated to reflux temperature for an
additional hour. The reaction mixture was diluted with DCM and washed with brine. The
organic phase was dried over MgSO , filtered, and concentrated to dryness. The crude was
4
purified by column chromatography (SiO , gradient EtOAc/n-heptane) to give 67 in 90%
2
yield.
Procedure P: Halogen exchange in aryl halides (Exemplified by 68)
6
7 (600 mg, 1.6 mmol), copper (I) iodide (30.6 mg, 0.1 equiv.) and sodium iodide (482 mg, 2
equiv.) were charged in an oven-dried microwave tube. The sealed vessel was evacuated and
backfilled with nitrogen (this process was repeated 3 times) and N,N'-
dimethylethylenediamine (34 µL, 0.2 equiv.) followed by degassed 1,4-dioxane (2.1 mL)
were added via a degassed syringe. The reaction was heated at 110 °C for 1 day. After
cooling down to room temperature, the reaction mixture was diluted with DCM and
successively washed with water and brine. The organic phase was dried over MgSO , filtered
4
and concentrated to dryness. The crude was purified by column chromatography (SiO2,
gradient EtOAc/n-heptane) to give 68 in 94% yield.
Procedure Q: Direct arylation of methyl 1H-benzimidazole-5-carboxylate (Exemplified by
7
0)
Methyl 1H-benzimidazole-5-carboxylate (69, 30 mg, 0.17 mmol, 1 equiv.), 68 (143.2 mg, 2
equiv.), copper (I) iodide (65 mg, 2 equiv.), and palladium (II) acetate (1.9 mg, 0.05 equiv.)
were charged in an oven-dried microwave vial. The vessel was evacuated and backfilled with
nitrogen (this process was repeated 3 times) and dry DMF (0.8 mL) was added via a degassed
syringe. The reaction was heated at 140 °C for 4 days. After cooling down to room
temperature, the reaction mixture was diluted with ethyl acetate and successively washed
21

ACCEPTED MANUSCRIPT
with water and brine. The organic phase was dried over MgSO , filtered, and concentrated to
4
dryness. The crude was purified by column chromatography (SiO , DCM/methanol, 95:5) to
2
give 70 in 56% yield.
Procedure R: Methyl ester hydrolysis
An aqueous solution of potassium hydroxide (37 mM) was added to an ethanolic solution
of methyl ester derivative (25 mM). The reaction mixture was heated to reflux temperature
for 1 day. After cooling down to room temperature and the pH of the solution was set to 2-3
using HCl (aq., 1 M). The precipitate was filtered off, washed with cold water and dried
under vacuum.
Procedure S: Sonogashira coupling reaction of alkyne derivatives with aryl bromides
(Exemplified by 72)
6
7 (0.11 mmol, 1 equiv.), copper (I) iodide (0.1 equiv.) and bis(triphenylphosphine)
palladium(II) chloride (0.05 equiv.) were charged in an oven-dried microwave tube. The
vessel was evacuated and backfilled with nitrogen (this process was repeated 3 times) and dry
DMF (1.2 mL) followed by triethylamine (2.0 equiv.) were added via a degassed syringe.
The reaction was allowed to perform for 5 min at room temperature and
trimethylsilylacetylene (3.0 equiv.) was added. The reaction was heated at 60 °C for 2 days.
After cooling down to room temperature, the reaction mixture was diluted with ethyl acetate
and washed with brine. The organic phase was dried over MgSO , filtered, and concentrated
4
to dryness. The crude was dissolved in a methanol/THF mixture (1:4) and potassium
carbonate (2 equiv.) was added. After overnight stirring, the reaction mixture was diluted
with ethyl acetate and washed with brine. The organic phase was dried over MgSO , filtered,
4
and concentrated to dryness. The crude was purified by column chromatography to yield 72
in 60% yield.
22

ACCEPTED MANUSCRIPT
Procedure T: Synthesis of Benzofuran and Indole derivatives via Sonogashira coupling
reaction of alkyne with aryl iodides (Exemplified by 72)
73 (0.11 mmol, 1 equiv.), 72 (2 equiv.), copper (I) iodide (0.1 equiv.) and
bis(triphenylphosphine) palladium(II) chloride (0.05 equiv.) were charged in an oven-dried
microwave tube. The vessel was evacuated and backfilled with nitrogen (this process was
repeated 3 times) and dry DMF (1.2 mL) followed by triethylamine (2.7 equiv.) were added
via a degassed syringe. The reaction was heated under microwave irradiation conditions at
1
10 °C for 20 min. After cooling to room temperature, the reaction mixture was diluted with
ethyl acetate and washed with brine. The organic phase was dried over MgSO , filtered, and
4
concentrated to dryness. The crude was purified by column chromatography to give 75 in
6
7% yield.
Procedure U: Synthesis of 2H-Indazole derivatives (Exemplified by 80)
5
(3 mmol, 1 equiv.) and 79 (1 equiv.) were dissolved in ethanol (70 mL) and the mixture
was refluxed for 20 min. After cooling down to room temperature, the formed precipitate was
filtered off, washed carefully with cold ethanol and dried under vacuum. The preformed
imine (1.77 mmol. 1 equiv.) and cesium carbonate (1.0 equiv.) were dissolved in dry,
degassed DMSO (80 mL) and the mixture was heated at 100 °C. A freshly prepared DMSO
solution (4 mL) of copper iodide (0.2 equiv.) and TMEDA (0.2 equiv.) was added to the
reaction vessel. Sodium azide (2.0 equiv.) was then added in small portions and the reaction
was stirred at 120 °C for 24 h. The brown mixture was allowed to reach room temperature,
diluted in ethyl acetate and successively washed with HCl (1 M), brine, dried over Na SO ,
2
4
filtered and concentrated to give a brown solid. The crude solid was triturated with ethanol
and then ethanol was pipetted off. The solids were dissolved in 50 ml of hot ethanol and the
product was precipitated with 200 ml of heptane. After cooling to ca. 10 °C, the solids were
vacuum filtered to give 80 in 56% yield. (Note: The reaction proved more efficient when the
23

ACCEPTED MANUSCRIPT
imine was preformed. A one-pot procedure where all the reactants were added together also
provided the desire product, albeit a lower reaction efficiency.)
4.3. Tabulated compounds
1,3-Dioxo-2-(4-(piperidin-1-yl)-3-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic acid
(1). 1 was synthesized by procedures A, C, H (or I) and J.
1
H-NMR (400 MHz, DMSO-d ): δ 13.80 (bs, 1H), 11.43 (s, 1H), 8.41 (dd, J = 1.5, 7.7 Hz,
6
1H), 8.30 (d, J = 1.4 Hz, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.88 (d, J = 2.6 Hz, 1H), 7.66 (d, J =
8.3 Hz, 2H), 7.56 (dd, J = 2.6, 8.5 Hz, 1H), 7.43 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 8.6 Hz, 2H),
2.95-3.05 (m, 4H), 2.28 (s, 3H), 1.62-1.73 (m, 4H), 1.48-1.58 (m, 2H).
1
3
C-NMR (100.5 MHz, DMSO-d ): δ 166.36, 166.35, 165.84, 163.92, 151.41, 136.40,
6
1
36.36, 135.41, 135.03, 132.49, 132.16, 130.57, 129.38, 129.07, 126.81, 123.77, 123.33,
20.80, 119.32, 53.70, 25.95, 23.29, 20.50.
1
+
ESI-MS m/z calcd for C H N O (M+H) 484.18; found: 484.21.
2
8
25
3
5
5-(1,3-dioxoisoindolin-2-yl)-2-(piperidin-1-yl)-N-p-tolylbenzamide (7). 7 was synthesized
by procedures A, C, H and J.
1
H-NMR (400 MHz, DMSO-d ): δ 11.46 (s, 1H), 7.94-8.00 (m, 2H), 7.88-7.94 (m, 2H),
6
7.86 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.55 (dd, J = 2.6, 8.5 Hz, 1H), 7.43 (d, J =
8.6 Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 2.95-3.07 (m, 4H), 2.28 (s, 3H), 1.62-1.74 (m, 4H),
1.48-1.59 (m, 2H).
+
ESI-MS m/z calcd for C H N O (M+H) 440.19; found: 439.97.
2
7
25
3
3
5-(5-bromo-1,3-dioxoisoindolin-2-yl)-2-(piperidin-1-yl)-N-p-tolylbenzamide (8). 8 was
synthesized by procedures A, C, H and J.
1
H-NMR (360 MHz, DMSO-d ): δ 11.43 (s, 1H), 8.17 (d, J = 1.8 Hz, 1H), 8.1 (dd, J = 1.8,
6
7
.9 Hz, 1H), 7.9 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.54
24

ACCEPTED MANUSCRIPT
(dd, J = 2.7, 8.4 Hz, 1H), 7.43 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 3.01 (t, J = 4.8
Hz, 4H), 2.28 (s, 3H), 1.61-1.73 (m, 4H), 1.48-1.58 (m, 2H).
+
ESI-MS m/z calcd for C H BrN O (M+H) 518.10; found: 518.11.
2
7
24
3
3
5-(5-amino-1,3-dioxoisoindolin-2-yl)-2-(piperidin-1-yl)-N-p-tolylbenzamide (9).
1
H-NMR (400 MHz, DMSO-d ): δ 11.53 (s, 1H), 7.80 (d, J = 2.6 Hz, 1H), 7.65 (d, J = 8.4
6
Hz, 2H), 7.58 (d, J = 8.3 Hz, 1H), 7.50 (dd, J = 2.6, 8.5 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H),
7
.18 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 2.0 Hz, 1H), 6.86 (dd, J = 2.0, 8.3 Hz, 1H), 6.56 (bs,
2
H), 2.95-3.03 (m, 4H), 2.28 (s, 3H), 1.62-1.74 (m, 4H), 1.49-1.58 (m, 2H).
+
ESI-MS m/z calcd for C H N O (M+H) 455.20; found: 455.02.
2
7
26
4
3
5-(5-cyano-1,3-dioxoisoindolin-2-yl)-2-(piperidin-1-yl)-N-p-tolylbenzamide (10). 10 was
synthesized by procedure M.
1
H-NMR (400 MHz, DMSO-d ): δ 11.41 (s, 1H), 8.51 (s, 1H), 8.37 (dd, J = 1.4, 7.7 Hz,
6
1
H), 8.14 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 2.6 Hz, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.55 (dd, J =
.6, 8.5 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.18 (d, J = 8.1 Hz, 2H), 2.98-3.05 (m, 4H), 2.28
2
(s, 3H), 1.63-1.72 (m, 4H), 1.50-1.58 (m, 2H).
+
ESI-MS m/z calcd for C H N O (M+H) 465.18; found: 465.38.
2
8
24
4
3
1,3-dioxo-2-(4-(piperidin-1-yl)-3-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxamide
(11). 11 was synthesized by procedure L.
1
H-NMR (360 MHz, DMSO-d ): δ 11.44 (s, 1H), 8.33-8.43 (m, 3H), 8.05 (d, J = 7.8 Hz,
6
1
H), 7.87 (d, J = 2.6 Hz, 1H), 7.79 (bs, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.56 (dd, J = 2.7, 8.6
Hz, 1H), 7.43 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 8.7 Hz, 2H), 2.98-3.04 (m, 4H), 2.28 (s, 3H),
.64-1.72 (m, 4H), 1.50-1.58 (m, 2H).
ESI-MS m/z calcd for C H N O (M+H) 483.20; found: 483.27.
1
+
2
8
26
4
4
25

ACCEPTED MANUSCRIPT
Methyl-1,3-dioxo-2-(4-(piperidin-1-yl)-3-(p-tolylcarbamoyl)phenyl)isoindoline-5-
carboxylate (12). 12 was synthesized by procedure K.
1
H-NMR (360 MHz, DMSO-d ): δ 11.42 (bs, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.32 (s, 1H),
6
8
.33 (s, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.56
(dd, J = 2.5, 8.5 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.18 (d, J = 8.1 Hz, 2H), 3.95 (s, 3H),
2
.97-3,05 (m, 4H), 2.28 (s, 3H), 1.62-1,72 (m, 4H), 1.49-1.58 (m, 2H).
+
ESI-MS m/z calcd for C H N O (M+H) 498.20; found: 498.07.
2
9
27
3
5
1,3-dioxo-2-(4-(piperidin-1-yl)-3-(p-tolylcarbamoyl)phenyl)isoindoline-4-carboxylic acid
(13).
1
H-NMR (400 MHz, DMSO-d ): δ 13.69 (bs, 1H), 11.43 (s, 1H), 7.90-8.10 (m, 3H), 7.85
6
(d, J = 2.6 Hz, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.55 (dd, J = 2.6, 8.6 Hz, 1H), 7.42 (d, J = 8.6
Hz, 1H), 7.18 (d, J = 8.6 Hz, 2H), 2.98-3.06 (m, 4H), 2.28 (s, 3H), 1.63-1.72 (m, 4H), 1.49-
1
.59 (m, 2H).
ESI-MS m/z calcd for C H N O (M+H) 484.18; found: 483.97.
+
2
8
25
3
5
2-(3-chloro-5-(p-tolylcarbamoyl)phenyl)-1,3-dioxoisoindoline-5-carboxylic acid (14).
1
H-NMR (400 MHz, DMSO-d ): δ 13.80 (bs, 1H), 10.37 (s, 1H), 8.44 (dd, J = 1.4, 7.8 Hz,
6
1
H), 8.34 (s, 1H), 8.15 (t, J = 2.22 Hz, 1H), 8.13 (d, J = 7.7 Hz, 1H), 8.02 (t, J = 1.7 Hz, 1H),
.82 (t, J = 1.9 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 2.29 (s, 3H).
7
+
ESI-MS m/z calcd for C H ClN O (M+H) 435.07; found: 434.90.
2
3
15
2
5
1,3-dioxo-2-(3-(phenylamino)-5-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic acid
(15). 15 was synthesized by procedures B, D, F and J.
1
H-NMR (360 MHz, DMSO-d ): δ 13.76 (bs, 1H), 10.20 (s, 1H), 8.64 (s, 1H), 8.42 (dd, J =
6
1
.1, 7.4 Hz, 1H), 8.32 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.63 (m, 3H), 7.42 (t, J = 1.5 Hz, 1H),
26

ACCEPTED MANUSCRIPT
7
.37 (t, J = 1.5 Hz, 1H), 7.29 (t, J = 7.48 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.3
Hz, 2H), 6.91 (t, J = 7.3 Hz, 1H), 2.28 (s, 3H).
+
ESI-MS m/z calcd for C H N O (M+H) 492.15; found: 492.28.
2
9
21
3
5
1,3-dioxo-2-(3-(p-tolylamino)-5-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic
acid
(16). 16 was synthesized by procedures B, D, F and J.
1
H-NMR (360 MHz, DMSO-d ): δ 13.02 (bs, 1H), 10.18 (s, 1H), 8.49 (s, 1H), 8.42 (d, J =
6
7
.8 Hz, 1H), 8.32 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.55 (t, J = 1.3
Hz, 1H), 7.36 (s, 1H), 7.28 (s, 1H), 7.06-7.17 (m, 6H), 2.27 (s, 3H), 2.24 (s, 3H).
+
ESI-MS m/z calcd for C H N O (M+H) 506.16; found: 505.82.
3
0
23
3
5
1,3-dioxo-2-(5-(piperidin-1-yl)-2-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic acid
(17). 17 was synthesized by procedures A, C, I and J.
1
H-NMR (400 MHz, DMSO-d ): δ 13.72 (bs, 1H), 10.33 (s, 1H), 8.39 (dd, J = 1.5, 7.7 Hz,
6
1
H), 8.26 (s, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.8 Hz, 1H),
.25 (d, J = 2.7 Hz, 1H), 7.19 (dd, J = 2.5, 8.4 Hz, 1H), 7.06 (d, J = 8.0 Hz, 2H), 3.28-3.38
7
(m, 4H), 2.22 (s, 3H), 1.54-1.71 (m, 6H).
+
ESI-MS m/z calcd for C H N O (M+H) 484.18; found: 484.04.
2
8
25
3
5
1,3-dioxo-2-(2-(piperidin-1-yl)-6-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic acid
(18). 18 was synthesized by procedures A, C, I and J.
1
H-NMR (400 MHz, DMSO-d ): δ 13.71 (bs, 1H), 10.38 (s, 1H), 8.41 (d, J = 7.6 Hz, 1H),
6
8
.30 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.60 (t, J = 7.9 Hz, 1H), 7.40-7.51 (m, 4H), 7.06 (d, J =
8
.0 Hz, 2H), 2.69-2.78 (m, 4H), 2.22 (s, 3H), 1.24-1.41 (m, 6H).
+
ESI-MS m/z calcd for C H N O (M+H) 484.18; found: 483.93.
2
8
25
3
5
1,3-dioxo-2-(4-(piperidin-1-yl)-2-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic acid
(19).
27

ACCEPTED MANUSCRIPT
1
H-NMR (400 MHz, DMSO-d ): δ 13.65 (bs, 1H), 10.33 (s, 1H), 8.38 (dd, J = 1.0, 7.7 Hz,
6
1
H), 8.25 (s, 1H), 8.01 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.9 Hz, 1H),
.25 (d, J = 2.7 Hz, 1H), 7.19 (dd, J = 2.7, 8.8 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 3.30-3.40
7
(m, 4H), 2.22 (s, 3H), 1.55-1.73 (m, 6H).
+
ESI-MS m/z calcd for C H N O (M+H) 484.18; found: 483.97.
2
8
25
3
5
1,3-dioxo-2-(2-(piperidin-1-yl)-5-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic acid
(20). 20 was synthesized by procedures A, C, I and J.
1
H-NMR (400 MHz, DMSO-d ): δ 13.79 (bs, 1H), 11.21 (s, 1H), 8.43 (dd, J = 1.5, 7.8 Hz,
6
1
H), 8.32 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H),
.38 (d, J = 2.0 Hz, 1H), 7.26 (dd, J = 1.8, 8.4 Hz, 1H), 7.18 (d, J = 8.5 Hz, 2H), 2.93-3.03
7
(m, 4H), 2.29 (s, 3H), 1.60-1.71 (m, 4H), 1.45-1.55 (m, 2H).
+
ESI-MS m/z calcd for C H N O (M+H) 484.18; found: 483.97.
2
8
25
3
5
1,3-dioxo-2-(3-(piperidin-1-yl)-4-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic acid
(21). 21 was synthesized by procedures A, C, I and J.
1
H-NMR (400 MHz, DMSO-d ): δ 13.79 (bs, 1H), 10.10 (s, 1H), 8.45 (dd, J = 1.4, 7.7 Hz,
6
1
H), 8.36 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.06 (dd, J = 2.3, 8.5 Hz, 1H), 7.94 (d, J = 2.2 Hz,
H), 7.63 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 8.6 Hz, 2H), 2.75-2.88
1
(m, 4H), 2.27 (s, 3H), 1.26-1.43 (m, 6H).
+
ESI-MS m/z calcd for C H N O (M+H) 484.18; found: 483.97.
2
8
25
3
5
1,3-dioxo-2-(2-(piperidin-1-yl)-3-(p-tolylcarbamoyl)phenyl)isoindoline-5-carboxylic acid
(22). 22 was synthesized by procedures A, C, I and J.
1
H-NMR (400 MHz, DMSO-d ): δ 13.79 (bs, 1H), 10.45 (s, 1H), 8.46 (dd, J = 1.5, 7.7 Hz,
6
1
H), 8.36 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.46-7.54 (m, 2H), 7.31
28