A carboxylic acid isostere screen of the DHODH inhibitor Brequinar

Article abstract of DOI:10.1016/j.bmcl.2020.127589

Dihydroorotate dehydrogenase (DHODH) enzymatic activity impacts many aspects critical to cell proliferation and survival. Recently, DHODH has been identified as a target for acute myeloid differentiation therapy. In preclinical models of AML, the DHODH inhibitor Brequinar (BRQ) demonstrated potent anti-leukemic activity. Herein we describe a carboxylic acid isostere study of Brequinar which revealed a more potent non-carboxylic acid derivative with improved cellular potency and good pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2020.127589

Journal Pre-proofs
A Carboxylic Acid Isostere Screen of the DHODH Inhibitor Brequinar
Lindsey G. DeRatt, E. Christine Pietsch, Alexandra Tanner, Paul Shaffer,
Edgar Jacoby, Weixue Wang, Faraz Kazmi, Xiaochun Zhang, Ricardo M.
Attar, James P. Edwards, Scott D. Kuduk
PII:
S0960-894X(20)30700-9
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127589
BMCL 127589
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
5 June 2020
22 September 2020
24 September 2020
Please cite this article as: DeRatt, L.G., Christine Pietsch, E., Tanner, A., Shaffer, P., Jacoby, E., Wang, W.,
Kazmi, F., Zhang, X., Attar, R.M., Edwards, J.P., Kuduk, S.D., A Carboxylic Acid Isostere Screen of the
DHODH Inhibitor Brequinar, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/
j.bmcl.2020.127589
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A Carboxylic Acid Isostere Screen of the DHODH Inhibitor Brequinar
Lindsey G. DeRatt,a E. Christine Pietsch,b Alexandra Tanner,c Paul Shaffer,d Edgar Jacoby,e Weixue Wang,c
Faraz Kazmi,f Xiaochun Zhang,b Ricardo M. Attar,b James P. Edwards,a Scott D. Kuduka
a Discovery Chemistry, Janssen Pharmaceutical Research & Development, 1400 McKean Rd, Spring
House, PA 19477.
b Oncology Discovery, Janssen Pharmaceutical Research & Development, 1400 McKean Rd, Spring
House, PA 19477.
c Discovery Technology and Molecular Pharmacology, Janssen Pharmaceutical Research & Development,
Spring House, PA 19477
d Structural and Protein Sciences, Janssen Pharmaceutical Research & Development, 1400 McKean Rd,
Spring House, PA 19477.
e Discovery Chemistry, Janssen Pharmaceutical Research & Development, Turnhoutseweg 30, 2340
Beerse, Belgium.
f Drug Metabolism and Pharmacokinetics, Janssen Pharmaceutical Research & Development, 1400
McKean Rd, Spring House, PA 19477.
Dihydroorotate dehydrogenase (DHODH) enzymatic activity impacts many aspects critical to cell
proliferation and survival. Recently, DHODH has been identified as a target for acute myeloid
differentiation therapy. In preclinical models of AML, the DHODH inhibitor Brequinar (BRQ)
demonstrated potent anti-leukemic activity. Herein we describe a carboxylic acid isostere study of
Brequinar which revealed a more potent non-carboxylic acid derivative with improved cellular potency
and good pharmacokinetic properties.
Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow resulting from mutations that
occur in normal blood stem cells.1 In AML, leukemic cells lose the ability to differentiate into adult white
blood cells. This leads to accumulation of abnormal myeloid progenitor cells (myeloblasts) in the bone
marrow, which are characterized by excessive proliferation and disrupt the production of normal blood
cells.2
The chemotherapeutic standard of care for AML has undergone little change over the last four decades.3
Although a rare disease, AML is the most common type of acute leukemia in adults with >20,000 new
cases reported per year in the US.4 The rapid progression of untreated AML results in mortality within
weeks to months and, along with the poor survivorship of treated patients (5 year survival rate <30%),
highlights an unmet medical need for improved therapeutics.
With the success of differentiation therapy in acute promyelocytic leukemia (APL), which represents a
small subset (10-15%) of all AML patients, this approach represents a powerful treatment strategy for
the remaining 90% of the patient population.5 In 2016, reports by Sykes et. al.6 demonstrated the central
role that dihydroorotate dehydrogenase (DHODH) plays in AML by regulating myeloid differentiation in
both in vitro and in vivo models across a range of AML subtypes independent of oncogenic mutations.
DHODH are flavin mononucleotide (FMN)-binding flavoproteins that catalyze the conversion of L-

dihydroorotate (DHO) to orotate (ORO), which represents the rate limiting step in the de novo
pyrimidine biosynthesis pathway.7 As such, inhibition of DHODH in AML represents a metabolic
vulnerability that leads to differentiation and/or apoptosis.8
With the discovery of the role of DHODH in AML, a renewed energy has been focused on identifying
novel DHODH inhibitors as therapeutic agents for cancer treatment.9 Brequinar (BRQ), a biphenyl
quinoline carboxylic acid with sub-nanomolar hDHODH enzyme inhibition, was the first DHODH inhibitor
to enter clinical trials for oncology indications, albeit for solid tumors and not heme malignancies.10 The
results of several Phase II studies revealed no objective responses in the majority of patients and the
drug had a narrow therapeutic window halting further clinical development.
Previous SAR studies surrounding BRQ focused on modification of the bulky biaryl subunit and quinoline
core that exemplified the importance of the carboxylic acid moiety at C4 (Figure 1).11 This carboxylate
forms a key salt bridge interaction with Arg 136 (vide infra) in the hDHODH protein leading to a high
affinity of BRQ for hDHODH. However, an exhaustive isostere study focused on replacement of the
carboxylic acid functionality has not been reported.12 It is well known that the presence of a carboxylic
acid in a drug or drug candidate can be responsible for limited permeability, metabolic stability, and
toxicities.13
it
subun
enyl
F
biph
quinoline
N
4
F
Me
isostere replacement
HO
O
Brequinar (1)
Figure 1. Brequinar (BRQ), a potent DHODH inhibitor
To date, carboxylic acid and salts thereof have been the only acceptable substituents at the C4 position
of BRQ and its structural derivatives due to the aforementioned interactions with Arg136. Other
functionalities, such as the ethyl ester or primary amide, drastically reduce or completely abolish the
inhibitory activity.14 Herein, structure activity relationship (SAR) studies on the replacement of the
carboxylic acid with isosteres on the BRQ core structure are disclosed.
In our attempt to identify acceptable replacements for the carboxylic acid functionality, a number of
novel and previously reported14 DHODH inhibitors were prepared and evaluated in both an enzymatic
and cellular assay. In our hDHODH enzymatic assay, BRQ 1 was confirmed to have exceptional inhibitor
activity with an IC50 = 0.48 nM (Table 1). However, in a cellular proliferation assay with MOLM-13 AML
cells, a large 125-fold shift was observed, with a measured IC50 of 60 nM. The large difference can likely
be attributed to the low permeability due to the lipophilic carboxylic acid nature of BRQ. Consistent with
findings in an earlier report,14 the hydroxymethyl compound 2 led to a large (>300-fold) loss in
biochemical activity, but only a 11-fold loss in cellular activity. Additionally, derivatives with fluorinated
substituents at the C4 position (compounds 3-5) were not well-tolerated and diminished activity was
observed. A characteristic replacement of the carboxylic acid group in the form of a tetrazole (6)
showed reduced enzymatic activity (IC50 = 223 nM) and complete loss of activity in the cellular assay.
Similarly, the oxadiazolone 7 was largely inactive in both assays. Interestingly, the incorporation of a

primary amide 8 provided a compound with an IC50 of 47 nM in the enzymatic assay (100-fold loss vs
BRQ), but only a 2-fold loss of activity was observed in the cellular assay (IC50 = 128 nM) showing a
substantial reduction in shift between the two assays.14
Efforts to modulate the pKa of the amide nitrogen to engage key hydrogen bonding interactions were
then pursued. Both the cyanoacetamide 9 and the acetylcarbamate 10 did not provide an improvement
in the inhibitory activity. The sulfonamide 11 was similarly potent in the biochemical assay, yet the
cellular potency was reduced ~2-fold as compared to the primary amide 8. Surprisingly, the N-
hydroxyacetamide 12 demonstrated excellent potency with an IC50 of 1.4 nM in the enzymatic assay,
and only a 16-fold shift in the cellular potency (IC50 = 23 nM). This derivative is more potent in MOLM-13
than BRQ and marks a substantial improvement in the shift between biochemical and cellular assays as
well. Moreover, the N-methoxyacetamide 13 further improved the biochemical activity and cellular
potency to IC50 = 0.12 nM and IC50 = 9.0 nM, respectively. This represents ~6-fold potency enhancement
in the MOLM-13 cellular assay compared to BRQ. Further SAR showed that activity was reduced
modestly when elongating the alkyl substituent on the hydroxyamide to N-ethoxyacetamide 14, but still
an improvement compared to BRQ. Lastly, the N-hydroxy-N-methylacetamide 15 was prepared to study
the effects of the disruption of potential hydrogen bonding interactions. Interestingly, 15 maintained
activity in the biochemical assay though this result may be attributed to the observed hydrolytic
instability of 15.15 Additionally, BRQ and 13 were tested using a THP-1 cell line and shown to have a
measured proliferation IC50 = 300 nM and 50 nM, respectively. This result confirms the MOLM-13
observation and demonstrates that the trend in cellular activity is not restricted to one cell type.
Table 1. Effects of Carboxylic Acid Replacement on Enzymatic and Cellular Potencya
F
N
F
Me
R
DHODH Enzymatic MOLM-13 Cellular
Compound
R =
Assay IC50 (nM)
Assay IC50 (nM)
O
1 (BRQ)
0.48
166
160
60
700
OH
OH
2
3
F
>3000
F
OH
CF3
4
5
808
74
640
O
>3000
CF3
N
N
N
H
N
6
223
>3000
O
N
O
7
8
9
1560
47
>3000
128
N
H
O
NH2
O
CN
116
>3000
N
H

O
O
O
Me
10
11
12
13
14
1250
32
403
300
23
N
H
O
O
O
S
N
Me
H
O
OH
OMe
OEt
1.4
N
H
O
0.12
4.2
9
N
H
O
O
33
N
H
OH
15
7.7
5100
N
Me
aValues represent the average of n > 2 experiments. Inter-assay variability <30%. DCIP absorbance assay has a LLOQ of 0.5 nM.
The smaller shift in biochemical versus cellular activity is worth noting for the amide class of compounds
which can be attributed to the reduced lipophilicity leading to better permeability. For reference, the
LogP of BRQ was calculated to be 6.39. The lower cLogP for amide 8 (5.36) results in a more permeable
analog with a corresponding reduced shift between the biochemical and cellular assays. For compounds
12 and 13, the LogP was calculated to be 5.08 and 5.43, respectively. As shown in Table 2, the
permeability of 13 was greatly improved, resulting in less of a shift between assays. Additionally, good
human microsomal metabolic stability was observed, although the mouse showed higher clearance for
all carboxamides relative to BRQ.
Table 2. Physicochemical Properties of BRQ Derivatives.
MOLM-13
A→B (+Pgp
Human/Mouse
LM t1/2 (min)
Compound
R =
Cellular Assay
cLogP
inh.)b
IC50 (nM) a
O
1 (BRQ)
8
60
8.99
19.7
6.39
5.36
>180/>180
135/65
OH
O
128
NH2
O
OH
12
13
14
23
9
8.43
22.8
11.1
5.08
5.43
5.96
>180/36
>180/57
>180/18
N
H
O
OMe
OEt
N
H
O
33
N
H
aValues represent the average of n > 2 experiments. Inter-assay variability <30%. bMDCK cells.
With the improved cellular activity observed for the N-methoxyamide 13, the pharmacokinetic profile
was obtained in mouse for comparison with BRQ. Compound 13 demonstrated high oral bioavailability,
a larger Vdss compared to BRQ, and a t1/2 of 2.35 h. The clearance was still low, which was notable
considering the stability in mouse liver microsomes was considerably lower (t1/2 = 57 min) compared to
BRQ (t1/2 > 180 min). Additionally, the plasma protein binding (PPB) was high for both compounds.

Overall, this shows that good pharmacokinetics and improved potency can be achieved with derivatives
that lack the carboxylic acid functionality found in BRQ.
Table 3. Mouse pharmacokinetics of BRQ and 13.a
Compound
1 (BRQ)
13
F (%)
72
108
t1/2 (h)
-*
2.35
Cl (ml/min/kg) Vdss (L/kg)
PPB h/m (% free)
0.39/0.73
0.2
0.3
5.09
1.15
0.55/0.77
a C57 Mice (n = 6). Oral dose 10 mg/kg, PEG400/water (70:30) vehicle; IV dose = 2 mg/kg, PEG400/water (70:30) vehicle. *not determined
due to high extrapolated AUC.
To further understand the improvement in activity of 13, a crystal structure with the DHODH protein
was obtained through a co-crystallization experiment (pdb 7K2U) (Figure 2a). The electron density for
most of the inhibitor was excellent except for the methoxy group which was much weaker, perhaps
indicating some mobility of this group in the binding site. The fluorophenyl is modeled in two
conformations that were supported by the electron density. Comparing the structure of DHODH bound
to 13 with the published structure of hDHODH bound to des-fluoro-BRQ analog (pdb 1D3G) shows 13
adopts a very similar binding mode with only slight rearrangement of the sidechain of Arg136 in the
binding site (Figure 2b). The carbonyl of the amide of 13 made two hydrogen bonds with the sidechain
of Arg136, similar to what is observed with BRQ. The nitrogen of the amide donated a hydrogen bond to
the backbone carbonyl of Thr360, an interaction not observed with BRQ. Additionally, the oxygen of the
methoxy made a hydrogen bond with the sidechain of Arg136. This slight change in binding pattern can
be rationalized in part by the difference in pKa of the N-methoxyamide (measured: 7.31 ± 0.01;
calculated: 8.6) compared to the carboxylic acid (measured: 4.15 ± 0.05; calculated: 2.4).16 Analogous to
BRQ, numerous hydrophobic interactions with many regions of the protein were also observed.
Figure 2. a) Crystal structure of 13 (7K2U) b) Overlay of 13 (7K2U) and des-fluoro-BRQ analog (1D3G).
DHODH/compound 13 are shown in wheat/green, respectively. 1D3G overlay shown in cyan. Dashed
lines represent hydrogen bonds between 13 and DHODH.
a)
b)
The synthesis of the target compounds 2-5 is described in Scheme 1. Compound 2 was prepared from
the requisite BRQ methyl ester via reduction with DIBAL. Oxidation using DMP afforded the aldehyde
intermediate, which was treated with DAST and TMSCF3 to provide 3 and 4, respectively. Further
oxidation of 4 with DMP afforded ketone 5.

Ar
c
F
F
F
3
Ar
Ar
Ar
a
b
N
MeO
Int I
O
OH
H
O
F
2
Int II
Me
= Ar
Ar
Ar
e
F
F
d
F
O
OH
F
F
F
4
5
Scheme 1. Synthesis of compounds 2-5. Reagents and conditions: a) DIBAL, toluene, DCM, -78 °C to r.t.,
99%. b) DMP, DCM, r.t., 87%. c) DAST, DCM, -78 °C to r.t., 80%. d) TMSCF3, K2CO3, DMF, r.t. then 1N HCl,
89%. e) DMP, DCM, r.t., 76%.
Amides 8, 9, 12, and 13 were synthesized using PyBOP as a coupling agent with the appropriate amine
(Scheme 2). Compounds 14 and 15 were prepared through conversion of BRQ to the acid chloride then
treatment with the corresponding amine. The primary amide 8 was then treated with NaH and MsCl to
generate compound 10. Compound 11 was formed in an analogous fashion using NaH, LiOtBu and
CO(OMe)2. Additionally, compound 8 was treated with TFA in pyridine to form the nitrile intermediate
which was further converted to tetrazole 6. Lastly, oxadiazolone 7 was obtained through a two-step
process from the nitrile intermediate.
8: R1 = R2 = H
9: R1 = H, R2 = CN
Ar
Ar
12: R1 = H, R2 = OH
13: R1 = H, R2 = OMe
14: R1 = H, R2 = OEt
15: R1 = Me, R2 = OH
R1
a or b
N
R2
O
HO
BRQ (
O
1
)
F
Ar
N
Ar
Ar
Ar
c or d
e
f
N
N
HN
R
O
F
Me
= Ar
H2N
O
N NH
N
Int 3
8
6
10: R = CO2Me
11: R = SO2Me
g, h
Ar
HN
O
N
O
7
Scheme 2. Synthesis of compounds 6-15. Reagents and conditions: a) (COCl)2, DMF, DCM then either
NH2OEt or NHMeOH, 73% and 28% over two steps, respectively. b) PyBOP, Et3N, DMF and either NH4Cl,
NH2CN, NH2OH•HCl, or NH2OMe, 18%, 34%, 55%, and 38%, respectively. c) NaH, MsCl, THF 0 °C to r.t.,
44%. d) NaH, LiOtBu, CO(OMe2), THF, 0 °C to r.t. ,37%. e) TFA, pyridine, 1,4-dioxane, 0 °C to r.t., 73%. f)
NaN3, NH4Cl, DMF, 130 °C, 21%. g) NH2OH (50% aqueous), EtOH, r.t. to 80 °C. h) CDI, DBU in 1,4-dioxane,
100 °C, 27% over two steps.
In summary, an SAR study on the carboxylic acid moiety of Brequinar revealed a subset of isosteres that
led to improved activity in both biochemical and cellular assays. Additionally, the enhanced permeability
of these compounds corresponded to a lower shift between the two assays. In particular, N-
methoxyamide 13 exhibited good pharmacokinetic properties in mouse and represents a compelling
non-carboxylic acid starting point for further modifications of the aromatic regions of the compound to
further reduce lipophilicity.

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