Successful strategies for mitigation of a preclinical signal for phototoxicity in a dgat1 inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.9b00117

Diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor Pradigastat (1) was shown to be effective at decreasing postprandial triglyceride levels in a patient population with familial chylomicronemia syndrome (FCS). Although pradigastat does not cause photosensitization in humans at the high clinical dose of 40 mg, a positive signal was observed in preclinical models of phototoxicity. Herein, we describe a preclinical phototoxicity mitigation strategy for diarylamine containing molecules utilizing the introduction of an amide or suitable heterocyclic function. This strategy led to the development of two second-generation compounds with low risk of phototoxicity, disparate exposure profiles, and comparable efficacy to 1 in a rodent lipid bolus model for post-prandial plasma triglycerides.

Full text of DOI:10.1021/acsmedchemlett.9b00117

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Letter
Successful strategies for mitigation of a preclinical
signal for photo-toxicity in a DGAT1 inhibitor
Tyler J. Harrison, Daniel Bauer, Alina Berdichevsky, Xin Chen, Rohit Duvadie, Benjamin
Hoogheem, Panos Hatsis, Qian Liu, Justin Mao, Vasumathy Miduturu, Erik Rocheford,
Frederic Zecri, Richard Zessis, Rui Zheng, Qingming Zhu, Ryan Streeper, and Sejal J. Patel
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00117 • Publication Date (Web): 20 Jun 2019
Downloaded from http://pubs.acs.org on June 20, 2019
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Successful strategies for mitigation of a preclinical signal for
phototoxicity in a DGAT1 inhibitor
Tyler J. Harrison,*,a Daniel Bauer,d Alina Berdichevsky,b Xin Chen,a Rohit Duvadie,a Benjamin
Hoogheem,b Panos Hatsis,c Qian Liu,a Justin Mao,a Vasumathy Miduturu,a Erik Rocheford,b Frederic
Zecri,a Richard Zessis,b Rui Zheng,a Qingming Zhu,a Ryan Streeper,b Sejal J. Patela
b
c
aGlobal Discovery Chemistry, Cardiovascular and Metabolism, PK Sciences, Novartis Institutes for Biomedical
d
Research, 22 Windsor Street, Cambridge, MA 02139, USA; Preclinical Safety, Novartis Institutes for Biomedical
Research, 4002 Basel, Switzerland
ABSTRACT: Diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor Pradigastat (1) was shown to be effective at decreasing
postprandial triglyceride levels in a patient population with familial chylomicronemia syndrome (FCS). Although pradigastat does
not cause photosensitization in humans at the high clinical dose of 40 mg, a positive signal was observed in preclinical models of
phototoxicity. Herein we describe a preclinical phototoxicity mitigation strategy for diarylamine containing molecules utilizing the
introduction of an amide or suitable heterocyclic function. This strategy led to the development of two second-generation
compounds with low risk of phototoxicity, disparate exposure profiles and comparable efficacy to 1 in a rodent lipid bolus model
for post prandial plasma triglycerides.
KEYWORDS: DGAT1, diacylglycerol acyltransferase inhibitor, phototoxicity, pradigastat, diaryl amines, triglycerides
The accumulation of abnormal levels of triglyceride (TG) in
circulation and in tissues is associated with common diseases
like obesity and cardiovascular disease as well as rare diseases
such as familial chylomicronemia syndrome (FCS).1,2,3
DGAT1 plays a critical role in postprandial absorption of
dietary fat catalyzing the terminal and committed step in TG
synthesis in enterocyctes of the gut wall.4,5,6 Mice lacking
DGAT1 are long-lived7 and protected from diet-induced
obesity8, diabetes9 and atherosclerosis10 which is largely
believed to be a consequence of delayed lipid absorption in the
intestine11. Pradigastat (1) was developed as a novel DGAT1
inhibitor for the treatment of cardiometabolic diseases and FCS
and has been shown in this patient population to decrease
fasting and postprandial TG levels by reducing the rate of
chylomicron-TG secretion.12
extinction coefficient, MEC, exceeds 1000 Lmol-1cm-1).17 For
regulatory purposes, the in vitro 3T3-Neutral Red Uptake
(NRU) Phototoxicity Test is typically used.18 Compounds that
demonstrate phototoxicity in this in vitro assay (Photo-Irritation
Factor, PIF, above 5)18 may be further studied in vivo using the
murine photo-Local Lymph Node Assay (photo-LLNA).19 If
necessary, photosensitivity risk may be further assessed in
clinic.
Pradigastat is highly exposed in plasma and has a long t1/2 in
humans (125 h)20,21. As a result of the UV/vis absorption
(maxima at 310 nm and 330 nm; MEC, for both: ~30,000 Lmol-
1cm-1), pradigastat was profiled in both in vitro and in vivo
preclinical models and demonstrated the potential to induce
phototoxicity. Pradigastat did not, however, induce any
clinically relevant photosensitization at the high clinical dose of
40 mg per day in a dedicated clinical photosensitivity study and
it can be used without the need for sun-protective measures. 22
Drug induced phototoxicity is an acute, usually cutaneous,
adverse reaction which potentially limits therapeutic use.13,14
This does not only apply to topically applied chemicals
absorbing ultraviolet (UV) and/or visible (vis) light, but also to
those that reach light-exposed tissues such as skin (or in some
cases, eye) following systemic exposure. Compounds that
absorb sufficiently within sunlight range (290-700 nm) have the
potential to induce a phototoxic response15,16 and often require
further evaluation (usually, if the molar absorptivity or molar
Due to the potential concern over the observed preclinical
signal for phototoxicity, follow-on efforts were initiated prior
to the read-out of the clinical photosensitization study with
pradigastat. The proposed compound would be free from an in
vitro signal for phototoxicity in the 3T3-NRU assay (PIF below
5), would maintain an efficacy profile comparable to
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pradigastat and would offer diversification to guard against
unforeseen issues in late stage development.
Although the benzoxazole offered a moderate improvement to
the signal for phototoxicity and lent support to our hypothesis,
a fused bicyclic aromatic system also introduces additional
conjugation to the molecule and could carry an inherent
phototoxic risk. Existing SAR suggested that the extended
aromaticity at the A-ring was not necessary and the
benzoxazole ring was subsequently deconstructed.
The strong UV/vis absorption of 1 was hypothesized to be
derived from an efficient chromophore defined by the extended
conjugation of the A, B, C-aryl ring system. Initial efforts
focused on breaking up that conjugation (Figure 1). Saturation
of the central B-ring pyridine successfully abrogated the
phototoxic signal in the 3T3-NRU assay, but also completely
eliminated activity for DGAT1 inhibition.
Substituted oxadiazoles were investigated as potential
benzoxazole replacements where it was found that 1,3,4-
oxadiazoles offered superior metabolic stability over 1,2,4-
oxadiazoles in liver microsomes. A series of alkyl-substituted
1,3,4-oxadiazoles were prepared in order to probe the impact on
phototoxicity in the 3T3-NRU assay (Table 1).
9
10
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OH
D
O
C
F3C
pradigastat (1)
B
A
N
N
N
Table 1. In vitro data for compounds 1 and 3-6
N
H
OH
OH
O
O
F3C
N
N
H
R
Et
Me
N
Figure 1. Initial strategy for abolishing phototoxic potential from
pradigastat scaffold
O
4
S
6
O
O
3
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
As a subsequent strategy, a new hypothesis was developed
5
which
centered
around
blocking
the
known
Compound DGAT1 IC50 (nM)a Cell EC50 (nM)b
PIFc
56
photodegredation/photocyclization of diarylamines23,24, which
has been utilized in the photochemical synthesis of complex
carbazoles.25 To the extent that pradigastat could undergo a
photo-decomposition via a similar pathway (Figure 2), we
hypothesized that masking the available ortho-CH units in a
suitable heterocycle may be an effective means to block the
putative photocyclization event. We proposed that an oxazole
or oxadiazole could work in that regard.
1
3
4
5
6
55±50 (n=377)
540 (n=1)
71 ±55 (n=17)
206 (n=1)
NT
NT
1
150±79 (n=7)
41 ±20 (n=6)
9±7 (n=2)
27 ±15 (n=3)
12 ±6 (n=3)
8 (n=1)
22
a) DGAT1 membrane preparation assessing conversion of
F3C
R
F3C
R
diolein to triolein by LC/MS/MS; b) DGAT1 inhibitor effect on
cellular triglyceride production with intact C2C12 cells (see
supporting info); c) PIF from 3T3-NRU assay = IC50 -irradiation/IC50
+irradiation; chlorpromazine-HCl used as positive control in 3T3-NRU
assay (with PIF>6); NT=not tested; n=number of experiments
N
N
N
N
N
N
LIGHT
H
H
Figure 2. Proposed photodecomposition of predigastat via diaryl
amine photocyclization
Alkyl substitution on the oxadiazole ring appears to be well
tolerated whereby it was found that alkyl groups larger than
methyl are generally required for optimal potency. Larger
substituents such as cyclobutyl or tertiary-butyl (not shown)
offered good potency and did not show signs of phototoxicity
in the 3T3-NRU assay (PIF=1), further supporting the original
hypothesis. Thiadiazaole analogue, 6, was also prepared and
while it maintained good DGAT1 potency in vitro, a risk of
phototoxicity was observed in the 3T3-NRU assay (PIF=22).
Pradigastat analogue 2 was available from our internal archive
and the benzoxazole ring provided an initial test of the
hypothesis. We were excited to see that although this
modification to the scaffold did not abolish the in vitro phototox
signal, it did provide an improvement in observed phototoxic
potential in the 3T3-NRU assay relative to pradigastat (Figure
3).
OH
Our previously reported benzimidazole series of DGAT1
inhibitors26,27 demonstrated an amide function as a suitable
linker between the A and B rings and we hypothesized that an
amide linker could also be sufficient to prevent the putative
photocyclization event.
O
Pradigastat (1)
3T3-NRU assay:
PIF = 56
F3C
N
N
N
H
OH
Introduction of an oxazole amide A-ring in 7 (Figure 4) proved
effective at maintaining DGAT1 potency while removing the
phototoxicity signal in the 3T3-NRU assay.
O
2
3T3-NRU assay:
PIF = 17
MeO
N
N
O
N
H
Figure 3. Benzoxazole 2 improves phototoxic potential in 3T3-
NRU assay.
2
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OH
an accessible means to test the hypothesis of reducing the PSA
of 9 to below this perceived threshold. Removal of the pyridine
nitrogen to generate biphenyl analogue 10 (Table 3) resulted in
PSA = 97 Å2, logD7.4 = 2.4 and restored cell-based activity
(EC50 = 12 nM).
O
O
Me
N
7
N
N
H
Cell EC50 = 8 nM (n=1)
PIF = 1
O
In line with this discovery, we were pleased to find that the
oxabicyclooctane ring system was tolerated with the amide
linker as well, resulting in 11 (Table 3). The corresponding
biphenyl modification afforded 12 with minimal change to
DGAT1 potency. Importantly, both 10 and 12 remained free of
phototoxic potential in the 3T3-NRU assay (PIF=1).
Et
Figure 4. Amide function of Compound 7 is tolerated and non-
9
phototoxic in vitro
10
11
12
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With the identification of A-ring modifications that could lead
to elimination of the in vitro phototoxicity signal, as in 5 and 7,
we wanted to confirm that this strategy was consistent across
alternative C- and D-ring combinations in order to guard against
unexpected issues that could emerge in late stage development.
Internal SAR suggested that the C ring was less amenable to
modification and in the interest of diversifying away from the
pradigastat C,D-ring system, we instead focused efforts on
heteroatom insertion into the D-ring. Saturated nitrogen
heterocycles at the D-ring position, as in piperidine analogue,
8, resulted in substantial loss of activity in most cases. Oxygen-
containing heterocycles were considered as suitable alternatives
and an oxabicyclooctane ring system (9, Table 2) was
eventually identified.28
Table 3. In vitro data for oxabicyclooctanes 10-12
OH
OH
O
O
O
O
O
O
Me
N
O
X
N
N
N
N
H
11
12
H
(X=N)
10
(X=CH)
Et
Compound DGAT1 IC50 (nM)a Cell EC50 (nM)b PSA (Å2) PIFc
10
11
12
83 ±41 (n=5)
12 ±14 (n=5)
98 ±17 (n=2)
56 ±49 (n=2)
97
1
390 ±230 (n=3)
540 ±170 (n=10)
115
102
NT
1
Table 2. In vitro data comparison for compounds 1, 5, 8, 9
a) DGAT1 membrane preparation assessing conversion of
diolein to triolein by LC/MS/MS; b) DGAT1 inhibitor effect on
cellular triglyceride production with intact C2C12 cells (see
supporting info); c) PIF from 3T3-NRU assay = IC50 -irradiation/IC50
+irradiation; chlorpromazine-HCl used as positive control in 3T3-NRU
assay (with PIF>6); NT=not tested; n=number of experiments
OH
O
N
8
O
N
N
N
N
H
N
OH
In order to access oxabicyclooctane containing analogues such
as 10 and 12, the synthetic strategy utilized key intermediate 13
which was built up from methyl 2-(4-bromophenyl) acetate. A
O
O
O
9
double
Michael-Dieckmann
cyclization-decarboxylation
N
N
H
N
sequence (Scheme 1) was used to construct the corresponding
cyclohexanone.29 Overall reduction of the resulting methyl ester
to the neopentyl alcohol followed by Horner-Wadsworth-
Emmons olefination afforded the desired substrate for the
intramolecular oxa-Michael addition, which proceeded
smoothly with base to give key bromide intermediate 13.28 The
structure of the oxabicyclooctane ring system was confirmed in
the solid state by X-ray crystallographic analysis (ORTEP of 13
shown in Scheme 1).30
DGAT1a
Cellb
PSA
PAMPA
logPE pH6.8
Compound
logD7.4
3.6
pKa
4.1
IC50 (nM) EC50 (nM) (Å2)
55 ±50
71 ±55
(n=17)
1
5
8
9
75
-5
(n=377)
41 ±20
(n=6)
12 ±6
(n=3)
101 2.8
104 0.7
110 1.5
-5.8
< -6.7
-7.1
NT
3.8
12600±5900 2300
(n=2)
(n=1)
Scheme 1. Assembly of oxabicyclooctane intermediate 13
120 ±28
(n=2)
413 ±120
(n=2)
3.9
O
OMe
a) DGAT1 membrane preparation assessing conversion of diolein
to triolein by LC/MS/MS; b) DGAT1 inhibitor effect on cellular
triglyceride production with intact C2C12 cells (see supporting
info); NT=not tested; n=number of experiments
MeO2C
O
a
O
+
OMe
Br
b,c,d
Br
Br
2x
OH
OH
O
O
Compound 9 showed minimal loss of potency in the
biochemical assay (IC50 = 120 nM), but potency in the cell
based assay was more significantly impacted (>30-fold less
potent than 5). The decrease in cell-based potency was
attributed to reduced permeability and although changes to pKa
were modest, it coincided with a reduction in logD7.4 by 2 log
units and an increase in polar surface area (PSA). Upon closer
inspection of our diarylamine SAR, we realized that potent
DGAT1 inhibitors in this series tended toward PSA values less
than ~100 Å2. To that end, we targeted the pyridine B-ring as
e
OMe
Br
Br
f
O
O
OMe
13
13
ORTEP of
3
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Reagents and conditions: (a) i) KOtBu, THF ii) H2O, 85 °C, 21
h, 59%; (b) toluene, ethylene glycol, toluenesulfonic acid, 80 °C, 2
h; (c) DIBAl-H, dichloromethane, -78 °C, 30 min; (d) acetone,
Tmax (h)
F (%)
0.7
83
1
3
>100
>100
aVehicle (1): 5% NMP, 5%Captisol, 50mM Tris buffer;bVehicle
(10): 4% 1N NaOH, 20% PEG300, 50% of 20%Cremaphore EL,
50 mM Tris buffer pH 7.4; Vehicle (12): 2% 1N NaOH, 20%
PEG300, 50% of 20%Cremaphore EL, 50 mM Tris buffer pH 7.4
and 2N HCl for pH adjustment
H2O, toluenesulfonic acid, 75 °C,
1
h; (e) trimethyl
phosphonoacetate, NaH, MeOH,rt, 18 h 86% over 4 steps; (f) NaH,
1,4-dioxane, 100 °C, 30 min, 78 %.
c
Intermediate 13 was coupled to either arylaminophenyl boronic
ester 14 or to amidophenylboronic ester 15 to afford final
compounds 10 or 12 after methyl ester saponification,
respectively (Scheme 2).
9
In order to understand the efficacy profile of 10 and 12 in vivo,
the compounds were studied in rats subjected to an acute oral
lipid challenge to observe effects on postprandial triglycerides.
When administered at 5 mg/kg, 4 hours prior to lipid challenge,
both compounds (10 and 12) provided significant blunting of
the corresponding plasma triglyceride excursion (Figure 5),
demonstrating efficacy comparable to pradigastat from a related
study (while not compared head-to-head with compounds 10
and 12, in separate rat studies pradigastat similarly reduces TG
excursions after a lipid challenge by ~50-70%, consistent with
what is observed in human20).
10
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12
13
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Scheme 2. Elaboration of oxabicyclooctane intermediate 13
into 10 and 12
O
O
OH
O
a
N
14
N
N
10
H
O
O
O
O
OH
OMe
b
O
Br
13
15
O
N
N
Et
12
H
Me
O
B
O
B
O
O
O
O
O
N
N
Et
H
N
N
N
Me
H
15
14
Reagents and conditions: (a) i) 14, Pd(amphos)Cl2, CsF, 1,4-
dioxane-H2O, 90 °C, 15 h, 88%; ii) LiOH, THF-methanol-H2O, 40
°C, 5 h, 73% (b) 15, Pd(amphos)Cl2, CsF, 1,4-dioxane-H2O, 90
°C, 18 h, 58%; ii) LiOH, THF-methanol-H2O, rt, 18 h, 87%.
Figure 5: Rat lipid bolus data for 10 and 12; 5 mg/kg PO
administration (suspension, oral gavage) 4 h prior to lipid challenge
Consistent with rodent PK studies, exposure of 12 over the
course of the study was high in comparison to the moderate
exposure observed for 10 (Figure 6). Despite the differences in
exposure, comparable blunting of the triglyceride excursion
was observed in the rodent efficacy model, which is consistent
with efficacy driven by gut exposure. Adipose tissue specific
deletion of DGAT1 decreases fat mass and increases energy
expenditure suggesting that high systemic exposure of DGAT1
inhibitors like amide 12 may be beneficial.31, 32 However, in
these same studies, loss of DGAT1 in adipose tissue was
reported to lead to lipotoxic stress suggesting that lower
systemic exposure as observed with oxadiazole 10 may be
desired to minimize lipotoxicity.
In general, both the oxadiazole and amide head groups offered
solutions to the issue of in vitro phototoxicity. The amide head
group resulted in high permeability, low in vivo clearance in rat
and high exposures, consistent with the pradigastat profile.
The oxadiazole 10, by comparison, afforded lower exposures in
a rat pharmacokinetic (PK) study (Table 4).
Table 4. Pharmacokinetic data for 1, 10 and 12 in rat
Compound
1
10
12
Solubility pH6.8 (µM)
Caco-2: A-B (x 10-6 cm/s)
Caco-2: B-A (x 10-6 cm/s)
B-A/A-B ratio
<5
<5
13
8.1
2.3
0.28
99.7
5.9
11.8
2
18.3
7.7
0.4
>99
Plasma protein binding (Rat, %)
>99
IV dose, n = 2 (mg/kg)
AUC0-24h (nM*h)
CL (mL/min/kg)
Vd (L/kg)
1a
0.5b
5160
3.5
0.5c
51400
0.2
46000
1
0.1
5.1
0.7
0.3
T1/2 (h)
3.2
20
PO dose, n = 3 (mg/kg)
AUC0-24h (nM*h)
Cmax (nM)
5a
1.5b
1.5c
190000
42000
25800
6096
180000
28038
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Figure 6: Compound 10 and 12 plasma exposures in rat lipid bolus
study
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Neither 10 nor 12 showed a worsened GI tolerability profile in
preclinical models when compared to pradigastat.33 DGAT1
inhibitors as a class, however, have struggled with GI
tolerability and all DGAT1 inhibitors tested in clinic to date
show GI tolerability limitations.34 This is believed to be an on-
target effect and is consistent with human genetics.35 Until a
mitigation strategy for the GI side effects is known, DGAT1
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cardiometabolic disease.
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In summary, two second-generation compounds have been
identified from the pradigastat scaffold. Candidate molecules
10 and 12 have no observable preclinical signal for
phototoxicity, have a comparable GI tolerability profile to
pradigastat and retain full efficacy in a rodent lipid bolus model
for postprandial plasma triglycerides.36 In addition, the two
compounds identified offer disparate PK profiles with respect
to exposure and half-life as well as some structural
diversification relative to pradigastat. Importantly, it was
found that masking of the A-ring
ortho-hydrogens of the
diaryl amine with a suitable heterocycle or amide function
successfully removed the phototox liability and provides
potential for a general solution to phototoxicity arising from the
diarylamine substructure.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Synthetic Procedures, analytical data and assay protocols (PDF)
AUTHOR INFORMATION
(11) Lee, B.; Fast, A.M.; Zhu, J.; Cheng, J.-X.; Buhman, K.K.
Intestine-specific
Corresponding Author
expression
of
acyl
CoA:diacylglycerol
*Phone: 617-871-7199. Email: tyler.harrison@novartis.com
acyltransferase 1 reverses resistance to diet-induced hepatic steatosis
and obesity in Dgat1-/- mice. J. Lipid Res. 2010, 51, 1770-1780.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Jason Elliott for his helpful suggestions in
preparation of this manuscript, Thomas Gilmore for contributions
to scale-up of lead compounds, Todd Stawicki for bioanalysis of
triglyceride content, Ina Dix and Birger Dittrich for generating the
solid-state structure of intermediate 13 and Lan Wang for
contributions to the DGAT1 biochemical assay.
ABBREVIATIONS
CsF, caesium fluoride; DGAT1, Diacylglycerol O-acyltransferase
1; DIBAl-H, diisobutyl aluminum hydride; FCS, familial
chylomicronemia syndrome; GI, gastrointestinal; h, hours; HCl,
hydrochloric acid; KOtBu, potassium tert-butoxide; LiOH, lithium
hydroxide; LLNA, local lymph node assay; MEC, molar extinction
coefficient; NaH, sodium hydride; NaOH, sodium hydroxide;
NRU, neutral red uptake; NT, not tested; PAMPA, parallel artificial
membrane permeability assay; PEG, polyethylene glycol; PK,
pharmacokinetics; PIF, photo-irritation factor; PSA, polar surface
area; SAR, structure activity relationship; TG, triglyceride; UV,
ultraviolet; vis, visible.
(19) Schümann, J.; Boudon, S.; Ulrich, P.; Loll, N.; Garcia, D.;
Schaffner, R.; Streich, J.; Kittel, B.; Bauer, D. Integrated preclinical
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(33) Description of preclinical models for GI tolerability will be
published in due course.
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R.; Coppola, G. M.; Daniels, T.; Forster, C. J.; Gilmore, T. A.; Gong,
Y.; Jain, M.; Kanter, A.; Kwak, Y.; Li, J.; Meyers, C. D.; Neubert, A.
D.; Szklennik, P.; Tedesco, V.; Thompson, J.; Truong, D.; Yang, Q.;
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(36) Compounds 10 and 12 are currently parked prior to IND-
enabling studies.
Insert Table of Contents artwork here
O
O
OH
O
OH
O
O
OH
O
N
N
N
H
F3C
O
10
N
N
O
N
N
Et
Pradigastat (1)
H
H
N
Me
12
6
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