Overcoming Time-Dependent Inhibition (TDI) of Cytochrome P450 3A4 (CYP3A4) Resulting from Bioactivation of a Fluoropyrimidine Moiety

Article abstract of DOI:10.1021/acs.jmedchem.8b01326

Herein we describe structure-activity relationship (SAR) and metabolite identification (Met-ID) studies that provided insight into the origin of time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4) by compound 1. Collectively, these efforts revealed that bioactivation of the fluoropyrimidine moiety of 1 led to reactive metabolite formation via oxidative defluorination and was responsible for the observed TDI. We discovered that substitution at both the 4- and 6-positions of the 5-fluoropyrimidine of 1 was necessary to ameliorate this TDI as exemplified by compound 19.

Full text of DOI:10.1021/acs.jmedchem.8b01326

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Article
Overcoming Time Dependent Inhibition (TDI) of Cytochrome P450 3A4
(CYP3A4) Resulting from Bioactivation of a Fluoropyrimidine Moiety
Mihir Baran Mandal, Kaushik Mitra, Diane Grotz, Xinjie Lin, Jairam Palamanda, Pramila Kumari, Alexei
V. Buevich, John P. Caldwell, Xia Chen, Kathleen Cox, Leonard Favreau, Lynn A. Hyde, Matthew
E. Kennedy, Reshma Kuvelkar, Xiaoxang Liu, Robert Mazzola, Eric M. Parker, Diane Rindgen,
Edward C. Sherer, Hongwu Wang, Zhaoning Zhu, Andrew W Stamford, and Jared N Cumming
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01326 • Publication Date (Web): 02 Nov 2018
Downloaded from http://pubs.acs.org on November 3, 2018
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Overcoming Time Dependent Inhibition (TDI) of
Cytochrome P450 3A4 (CYP3A4) Resulting from
Bioactivation of a Fluoropyrimidine Moiety
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Mihirbaran Mandal,*,‡ Kaushik Mitra,# Diane Grotz,# Xinjie Lin,# Jairam Palamanda,# Pramila
Kumari,# Alexei Buevich,† John P. Caldwell,‡ Xia Chen,§ Kathleen Cox,# Leonard Favreau,#
Lynn Hyde,§ Matthew E. Kennedy,§ Reshma Kuvelkar,§ Xiaoxiang Liu,‡ Robert D. Mazzola,‡
Eric Parker,§ Diane Rindgen,# Edward Sherer, Hongwu Wang, Zhaoning Zhu,‡, Andrew W.
Stamford,‡, and Jared N. Cumming‡
‡Department of Medicinal Chemistry, #Department of Pharmacokinetics, Pharmacodynamics,
and Drug Metabolism, †Department of NMR Structure Elucidation, §Department of
Neuroscience, and Department of Modeling and Informatics
Merck & Co., Inc., 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
ABSTRACT
Herein we describe structure activity relationship (SAR) and metabolite identification
(Met-ID) studies that provided insight into the origin of time dependent inhibition (TDI) of
cytochrome P450 3A4 (CYP3A4) by compound 1. Collectively, these efforts revealed that
bioactivation of the fluoropyrimidine moiety of 1 led to reactive metabolite formation via
oxidative defluorination and was responsible for the observed TDI. We discovered that
substitution at both the 4- and 6-positions of the 5-fluoropyrimidine of 1 was necessary to
ameliorate this TDI as exemplified by compound 19.
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INTRODUCTION
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More than half of all drugs are metabolized by cytochrome P450 3A4 (CYP3A4), and
thus inhibition of this enzyme by any new therapeutic is highly undesirable due to the potential
for drug-drug interactions (DDI) in patients.1 Time dependent inhibition (TDI) of CYP3A4 is of
particular concern because of its potential etiology in reactive metabolite formation and resulting
potential for hepatotoxicity.2 As a consequence, drug discovery efforts generally include in vitro
assays early in the screening funnel for measurement of both direct and time dependent CYP3A4
inhibition. Many functional groups capable of causing TDI upon bioactivation and reactive
metabolite formation have been identified, and related mitigation strategies have been devised.2
Among those reports, CYP3A4 TDI due to bioactivation of a pyrimidine moiety is less
prevalent.3
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From our efforts on design and development of BACE1 inhibitors for the potential
treatment of Alzheimer’s disease,4 we identified compound 1 as a lead in our fused pyrrolidine
series that demonstrated robust lowering of amyloid beta (Aβ40) in the cerebral spinal fluid (CSF)
of rats upon oral administration.5 Unfortunately, among other off-target liabilities, compound 1
demonstrated CYP3A4 TDI. As a result, we initiated extensive SAR studies to understand and
subsequently overcome this effect while preserving BACE1 potency and pre-clinical in vivo
activity. In this manuscript, we present evidence that bioactivation of the pyrimidine moiety and
subsequent reactive metabolite formation is responsible for the observed TDI with compound 1.
We also detail the accompanying SAR and ultimate remediation of the TDI while identifying
analog 19 that maintained the overall in vitro and in vivo profile of the original lead compound in
that chemical series.
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RESULTS AND DISCUSSION
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Discovery of compound 1 was an important milestone for our BACE1 inhibitor program
as it represented a distinctly different arrangement of substituents in the enzyme active site than
had been previously reported in the literature.5 The fused pyrrolidine ring enabled facile
exploration of the S2'' subsite through derivatization of the nitrogen of said ring. After early
SAR exploration, we identified N-pyrimidine-substituted derivatives as particularly interesting
given their overall balance of in vitro and cellular potency and in vivo activity, albeit with a
significant TDI liability in the series. The profile of compound 1 – no direct inhibition of
CYP3A4 (co-incubation IC50 > 30 µM) with significant TDI (pre-incubation IC50 = 1.2 µM) –
suggested that one or more metabolites derived upon pre-incubation likely inhibited CYP3A4.
Given the interest in this general structure, we began a concerted effort to engineer this liability
out of the series.
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Figure 1. Most likely sites of potential metabolism of compound 1 by CYP 3A4 as predicted by
MetaSite. The sites are represented by the red circles on the structure, and the likelihood of
metabolism at those sites is expressed by the percentage of relative score.
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Analysis of compound 1 by MetaSite6 suggested that the most likely routes of liver-based
metabolism were oxidation of several sites on the cyanophenyl ring and N-demethylation.
Additional, potential reactive sites were flagged on the pyrrolidine, thiophene, and, to a lesser
extent, pyrimidine rings (Figure 1). From this analysis, of particular interest to us was the
potential for oxidation on the cyanophenyl ring to generate reactive intermediates. Given that we
had previously demonstrated tolerance for modification of this ring,5,7 in contrast to known
limited tolerance for modification of the N-methyl moiety,8 we investigated the impact of
substitution and ultimately removal of the cyanophenyl ring. As shown in Table 1, blocking of
the potential metabolic sites of the cyanophenyl ring with halogens, which should have also
served to deactivate other sites on the ring, or with a methoxy group failed to significantly alter
the TDI profile (modest attenuation with α-halo analogues 2 and 3, no change with β-substituted
analogues 4 and 5). Removal of the cyano moiety entirely from the phenyl ring also did not
change the TDI liability (analogues 6 and 7). Interestingly, even the complete absence of the
cyanophenyl ring did not alleviate the TDI issue in analog 8, suggesting that moiety was not
responsible for the observed TDI in this series.
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Table 1. CYP 3A4 TDI SAR around the cyanophenyl ring
HN
N
O
HN
N
R1
S
N
N
F
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CYP 3A4
BACE1
Cpd
1
R1
pre-incubation
Ki (nM)a
IC50 (μM)b
NC
NC
4
1.2
5.7
5.5
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2
4
F
NC
3
10
Cl
NC
4
5
6
2
1
1.6
1.6
1.0
F
NC
MeO
F
10
F
Cl
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8
1
1.2
0.9
Br
100
aProtocols for determination of Ki values against purified human BACE1 and of BACE1 IC50 values in HEK293
cells have been previously described.4d bPre-incubation CYP3A4 IC50 was obtained following 30 minute pre-
incubation in human liver microsomes supplemented with NADPH.
Concurrent with the above efforts, we examined SAR around the changes to the N-
heteroaryl substituent on the pyrrolidine ring (Table 2). Replacement of the fluoropyrimidine
moiety with a fluoropyridine in analog 95 led to a modest attenuation of the TDI, and most
strikingly, both fluorophenyl analog 105 and unsubstituted pyrimidine analog 115 did not inhibit
CYP3A4 after pre-incubation (IC50 > 30 µM). While replacement of a phenyl with a pyrimidine
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and fluorination of aryl or heteroaryl rings are techniques often used to increase metabolic
stability,9 these data suggested that combination of these modifications in the 5-fluoropyrimidine
moiety of compound 1 was responsible for the observed TDI.
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Table 2. CYP3A4 TDI SAR around the pyrrolidine N-heteroaryl substituent
HN
N
O
HN
NC
N
S
R1
CYP 3A4
BACE1
Cpda
R1
pre-incubation
Ki (nM)b
IC50 (μM)c
N
F
N
N
1
4
1.2
9
6
6.4
>30
>30
F
10
26
11
F
N
11
N
aCompounds were reported previously and used here to build hypothesis.5 bProtocols for determination of Ki values
against purified human BACE1, and BACE1 IC50 values, have been previously described.4d cPre-incubation
CYP3A4 IC50 was obtained following 30 minute pre-incubation in human liver microsomes supplemented with
NADPH.
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The above observation presented a significant potential challenge for this series in our BACE
inhibitor program since, as previously disclosed,5 5-fluoropyrimidine analog 1 provided the best
overall balance of potency, selectivity over cathepsin-D, and good pharmacokinetics in rat. We
therefore examined SAR at the 4- and 6-positions of the 5-fluoropyrimidine moiety with the
intent to identify modifications that attenuated the TDI liability while maintaining the overall
profile of compound 1 (Table 3). To that end, mono substitution on the fluoropyrimidine with
either alkyl (analogues 12 and 13) or alkoxy moieties (analogues 14,10 15, and 16) did not
mitigate the CYP3A4 TDI issues. In contrast, mono substitution with amino moieties such as
pyrrolidine or morpholine (analogues 17 and 18) or disubstitution on the fluoropyrimidine ring
(19 and 20) led to significant improvement in the TDI liability. While all of the above
modifications were generally well-tolerated in terms of BACE1 affinity, among the selected
analogs with an improved TDI profile that were evaluated in vivo in rat, only compound 19
exhibited a similar reduction in levels of CSF Aβ40 to that of compound 1 (54% vs. 52% for
compounds 19 and 1, respectively) following a 10 mg/kg oral dose. In addition, compound 19
displayed an acceptable PK profile when dosed orally (10 mg/kg) in Wistar Han rats, having
bioavailability and a Cmax of 54% and 5.8 µM respectively (Table 4).
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Table 3. CYP3A4 TDI SAR around substitution on the N-fluoropyrimidine moiety
HN
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NC
N
S
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N
R2
R1
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Cpd
R1
R2
Ki (nM)
CYP 3A4
Rat CSF Aβ40
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BACE1a pre-incubation % Reduction
IC50 (μM)b
(dose, mpk)
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9
1
H
Me
Et
H
H
4
0.7
1
1.2
52 (10)
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18
19
20
0.6
0.9
1.3
0.7
1.2
>30
>30
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38 (10)
49 (10)
45 (30)
20 (30)
12 (30)
2 (10)
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H
OMe
OEt
H
1
H
1
O-nPr
H
1
N
H
3
N
O
H
1
17 (10)
54 (10)
ND
Me
Me
Me
OMe
1
0.7
11
aProtocols for determination of Ki values against purified human BACE1, and BACE1 IC50 values, have been
previously described.4d bPre-incubation CYP3A4 IC50 was obtained following 30 minute pre-incubation in human
liver microsomes supplemented with NADPH.
Table 4. Rat pharmacokinetic parameters of compound 19
AUC(0-∞)
(µM*hr)a
Cmax
PPB
Cl
Vd,ss
t1/2 (h)b
2.1
F%
54
(µM)a
(% bound) (mL/min/kg)b
(L/kg)b
34
5.8
99.7 5.6
1.0
a data from 10 mg/kg oral dose. data from 2 mg/kg intravenous dose.
b
To understand the molecular basis for CYP3A4 TDI in this series, with a particular
interest in identification of reactive metabolites derived from fluoropyrimidine moiety, we
conducted metabolite identification studies on compound 1 in two different systems – human
liver microsomes and recombinant human CYP3A4. Both systems produced N-demethylation11
and a variety of oxidized products. In addition, in the recombinant CYP3A4 system, a minor
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metabolite (0.26%) with a molecular weight corresponding to a net oxidation and defluorination
with general structure A was observed by LCMS (Figure 2). Analogous N-demethylated
oxidative defluorination metabolism was also observed at much lower levels (0.07%). To trap
the reactive intermediate formed in this oxidative defluorination process, the incubation with
recombinant CYP3A4 was repeated in presence glutathione (GSH), and an LCMS signal was
observed corresponding to a GSH adduct of general formula B (Figure 2). Prompted by these
results, reinvestigation of the incubation of compound 1 in human liver microsomes showed that
an oxidative defluorination metabolite was indeed formed in that system as well at very low
levels (0.01%).
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HN
HN
HN
HN
N
N
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O
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N
N
S
S
(+OH-F+GSH-2H)
(+OH-F)
N
F
N
F
N
N
B
A
Figure 2. Key metabolites observed following incubation with rCYP3A4 in absence or presence
of glutathione (metabolite A or adduct B, respectively).
In thinking about the mechanism and products for the observed oxidative defluorination,
we considered that a net ipso process was most likely, and thus the incubation products in the
absence or presence of glutathione would be compounds 21 and 22, respectively (Figure 3). En
route to those observed species, we proposed that either epoxide 23, likely formed by direct
oxidation of the pyrimidine ring,12 or the resulting quinone iminium intermediate 24 were the
possible reactive intermediates that underwent GSH addition to form adduct 22. Oxidative
defluorination product 21 was likely formed by two electron reduction of quinone iminium
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intermediate 24 by NADPH mediated by P450 reductase.13 While the specific reasons for the
TDI SAR for pyrimidine substitution in Table 3 are unclear, we speculate that substitution that
decreases the ability to form epoxide 23 and quinone iminium intermediate 24, either through
steric or electronic changes, would resolve the TDI.
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HN
N
O
NC
HN
N
N
N
S
N
N
N
F
N
N
N
GS
OH
OH
1
21
22
NADPH /
P450 reductase
[O]
N
N
N
N
F
N
N
GSH
GSH
O
O
24
23
Figure 3. Possible mechanisms for formation of reactive intermediates and resulting oxidative
defluorination metabolite 21 and glutathione adduct 22.
To provide additional evidence that the CYP3A4 TDI for compound 1 was due to
formation of reactive intermediates and that compound 19 addressed that issue, we determined
the rate constant (kobs) for 6β-hydroxylation of testosterone in human liver microsomes in the
presence of each compound at two different concentrations (Figure 4). For compound 1,
inhibition of the CYP3A4-mediated hydroxylation was observed at both 2 µM and 10 µM, and
the magnitude was dependent on both the concentration of 1 and time (Figure 4A), key indicators
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of the involvement of metabolically derived reactive intermediates. In fact, the kobs for
compound 1 at 10 µM was nearly identical to that of mifepristone (0.69 min-1 and 0.66 min-1,
respectively), a known time- and metabolism-based inhibitor of CYP3A4.14 Gratifying and in
concert with its more moderate TDI, the profile of compound 19 represented a significant
improvement over that of compound 1 (Figure 4B), specifically, there was no inhibition of
testosterone 6β-hydroxylation observed at 2 μM, and the kobs at 10 μM was reduced relative to 1
(of 0.022 min-1 versus 0.069 min-1, respectively).
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A
5
6
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100
Rate Constant
( min-1)
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0.006
Methanol (Blank)
10
0.043
0.069
0.066
0.062
Compound 1 (2 µM)
Compound 1 (10 µM)
Mifepristone (10 µM)
Mifepristone (50 µM)
0
5
10
15
20
25
30
35
Preincubation Time (min)
B
100
Rate Constant
( min-1)
0.006
0.009
0.022
0.066
0.062
Methanol (Blank)
Compound 19 (2 µM)
Compound 19 (10 µM)
Mifepristone (10 µM)
Mifepristone (50 µM)
10
0
5
10
15
20
25
30
35
Preincubation Time (min)
Figure 4. Time-dependent inhibition of testosterone 6-hydroxylation by compound 1 (Panel A)
and 19 (Panel B) in human liver microsomes. The kobs for each compound was measured at
concentrations of 2 μM (red circle) and 10 μM (green square) of compound. Mifepristone at 10
µM and 50 µM (black diamond and triangle, respectively) is included as a positive control.
Methanol (blue diamond) is included as a blank negative control. The number of replicates from
each experiment is given as “n”. Panel A (Compound 1): n=3 for each data point except at 30
min at 10 µM concentration of 1 (n=2). CV < 20 % for all data points. Panel B (Compound 19):
n=3 for all data points. CV < 20 % for all data points.
CONCLUSIONS
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Manifestation of CYP TDI as a result of bioactivation of drug molecules is a complex process,
and identification of structural elements in the molecules responsible for the TDI remains a very
difficult task. Once identified, SAR studies to mitigate this off-target liability without major
deterioration of potency, pharmacokinetics, and related in vivo activity is a major challenge in
drug discovery. Over the years, many functional groups prone to manifest TDI upon
bioactivation have been identified and mitigation strategies developed. However, TDI
originating from pyrimidine moieties are rare, and thus related mitigation strategies are less well
characterized. Our SAR analysis and investigations coupled with Met-ID studies supported that
reactive metabolite generation through bioactivation of the fluoropyrimidine moiety of
compound 1 was responsible for its observed CYP3A4 TDI15 and ultimately that substitution of
the fluoropyrimidine could mitigate this TDI liability while maintaining a desirable overall
molecular profile in analog 19.
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CHEMISTRY
Intermediates 25 and 26, which we previously reported,5 served as key intermediates for
synthesis of all of the novel analogues described in this article (Scheme 2). Synthesis of
analogues 2 – 7 began with Suzuki cross-coupling of compound 25 with the analogous aryl
boronic acids followed by fluoride-mediated deprotection of the Teoc group, N-arylation of the
corresponding free pyrrolidine with 2-chloro-5-fluoropyrimidine, and TFA-mediated
deprotection of the Boc group to afford the desired final compounds. Analog 8 was made via
fluoride-mediated deprotection of the Teoc group from intermediate 25 directly followed by N-
arylation of the corresponding free pyrrolidine with 2-chloro-5-fluoropyrimidine and TFA-
mediated deprotection of the Boc group. Analogues 12, 13, and 15 - 20 were prepared from
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intermediate 26 via N-arylation with the corresponding substituted 2-chloro-5-fluoro pyrimidines
followed by TFA-mediated deprotection of the Boc group to afford the desired final compounds.
The protocols for the synthesis of substituted 2-chloro pyrimidines used for the preparation of
analogues in Table 3 are described in the supporting information. Syntheses of compounds 1, 9,
10, 11 and 14 were reported previously.5
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Scheme 2. Synthesis of compounds 2 - 8 and 12, 13, and 15 - 20.
HN
N
O
BocN
N
HN
O
a, b, c, e
HN
N
R1
S
N
F
N
Teoc
Br
S
N
2 - 7
25
HN
HN
N
O
c, e
N
Br
S
N
F
N
8
HN
HN
N
O
BocN
HN
N
O
NC
d, e
N
S
NC
N
F
N
H
N
S
R2
R1
12, 13, 15 - 20
26
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Reagents and conditions: (a) R1-Boronic acid, dichloro[1,1’-bis(diphenylphosphino)ferrocene]-
palladium(II) dichloromethane, tert-butanol, K2CO3, 60 °C; (b) TBAF, THF, 0 °C; (c) 2-chloro-
5-fluoropyrimidine, Pd2(dba)3, (2-biphenylyl)di-tert-butylphosphine (JohnPhos) and Na-OtBu,
50 °C; (d) 2-Chloro-4-R1-5-fluoro-6-R2-pyrimidine, Pd2(dba)3, (2-biphenylyl)di-tert-butyl-
phosphine (JohnPhos), and Na-OtBu, 60 °C; (e) TFA in dichloromethane, rt.
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EXPERIMENTAL SECTION
General. CYP3A4 TDI was assessed by measuring inhibition of 6β-hydroxylation of
testosterone by rCYP3A4 after 30 minutes of pre-incubation of the analogues in the presence of
NADPH to allow for metabolite formation and is reported here as the pre-incubation IC50. Direct
inhibition of CYP3A4 was measured without NADPH pre-incubation and is reported here as the
co-incubation IC50. Protocols for more detailed measurement of kobs for compounds 1 and 19 are
in the supplemental material. Measurement of BACE1 Ki and IC50 values and determination of
rat CSF Aβ40 lowering in vivo were carried out following our previously described protocols.4d
Synthesis. All the reagents and solvents obtained from commercial sources were used without
further purification. Air sensitive chemistries were performed under an atmosphere of nitrogen
or argon. Purification of the final compounds to >95% purity were carried out either using a
prepacked silica gel cartridge (Analogix, Biotage, or ISCO) or a reverse phase C18 column
(mobile phase, A = 0.05% TFA in water and B = 0.05% TFA in acetonitrile, gradient = 10% to
95% B over 10 min). All NMR data unless otherwise specified were collected using either at
400 or 600 MHz on a Varian or Bruker instrument. Chemical shifts are reported in ppm relative
1
to the residual solvent peak in the indicated solvent, and for H NMR, multiplicities, coupling
constants in Hertz, and numbers of protons are indicated parenthetically. Microwave assisted
reactions were performed in a Smith synthesizer from Personal Chemistry. Purity and MS
information was obtained via LC-electrospray-mass spectroscopy with a C18 column using a
gradient of 5% to 95% MeCN in water with 0.05% TFA as the mobile phase. The purity of the
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samples was assessed using a UV detector at 254 nm. An additional analytical reverse phase
HPLC system was used to assess the purity of final compounds using an ELSD detector and a
UV detector monitoring both 220 nm and 254 nm.
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General Procedure A: Synthesis of analogues 2 – 7 from intermediate 25
To a solution of bromide 25 (1 equiv.) and appropriately substituted aryl boronic acid
(1.5 equiv.) in t-BuOH (4 mL) was added dichloro[1,1’-bis(diphenylphosphino)-
ferrocene]palladium(II) dichloromethane (0.15 equiv.) followed by aqueous 1N K2CO3 (1.5
equiv.). The resulting mixture was heated at 60 °C for 1 h, or until the reaction was determined
to be complete, before it was cooled, diluted with ethyl acetate and washed with water. The
organic layer was dried, concentrated and the residue was purified by silica gel chromatography
using 0 to 100% ethyl acetate in hexanes to provide the product. To this product was added 1M
TBAF (2.8 equiv.) in THF at 0 °C and the solution was stirred at room temperature for 4 h or
until the reaction was determined to be complete. The reaction mixture was poured into a
saturated aqueous solution of NaHCO3 and extracted with ethyl acetate. The organic layer was
concentrated and residue was purified by silica gel chromatography using 100% ethyl acetate to
provide a free pyrrolidine intermediate.
To a solution of the above pyrrolidine intermediate (1 equiv.) and 2-chloro-5-
fluoropyrimidine (1.2 equiv.) in toluene (5 mL/mmol of pyrrolidine) were added
tris(dibenzylidene-acetone)dipalladium(0) (0.05 equiv.), (2-biphenylyl)di-tert-butylphosphine
(JohnPhos, 0.13 equiv.) and Na-OtBu (2.7 equiv.). The resulting solution was degassed and
heated at 50 °C for 16 h or until the reaction was determined to be complete, then cooled to room
temperature, filtered through a pad of Celite, and the crude material was purified via silica gel
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chromatography using ethyl acetate in hexanes as the eluent to give an N-arylated product. The
Boc protecting group was removed through treatment of this material with 20% TFA/CH2Cl2
(2.5 mL/mmol). The deprotected compounds were purified by reverse phase HPLC (C18) using
0 to 90% water / acetonitrile with 0.05% TFA.
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2-Fluoro-5-(5-((4aR,7aR)-6-(5-fluoropyrimidin-2-yl)-2-imino-3-methyl-4-oxooctahydro-
7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (2)
Analog 2 was prepared from intermediate 25 and (3-cyano-4-fluorophenyl)boronic acid
1
according to General Procedure A. H NMR (600 MHz, CDCl3)  8.25 (s, 2H), 7.77 - 7.70 (m,
2H), 7.43 - 7.41 (m, 1H), 7.33 (s, 1H), 7.28 - 7.23 (m, 1H), 4.57 (d, J = 12.2 Hz, 1H), 4.36 (s,
1H), 3.98 (d, J = 12.5 Hz, 1H), 3.89 - 3.82 (m, 2H), 3.37 (s, 3H). m/z: 466.3.
2-Chloro-5-(5-((4aR,7aR)-6-(5-fluoropyrimidin-2-yl)-2-imino-3-methyl-4-oxooctahydro-
7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (3)
Analog 3 was prepared from intermediate 25 and (4-chloro-3-cyanophenyl)boronic acid
1
according to General Procedure A. H NMR (600 MHz, CDCl3)  8.26 (s, 2H), 7.80 (d, J = 1.8
Hz, 1H), 7.67 (dd, J = 2.1, 7.9 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.47 (s, 1H), 7.35 (s, 1H), 4.59
(d, J = 12.5 Hz, 1H), 4.36 (s, 1H), 3.99 (d, J = 12.2 Hz, 1H), 3.90 - 3.83 (m, 2H), 3.38 (s, 3H).
m/z: 482.2.
3-Fluoro-5-(5-((4aR,7aR)-6-(5-fluoropyrimidin-2-yl)-2-imino-3-methyl-4-oxooctahydro-
7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (4)
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Analog 4 was prepared from intermediate 25 and (3-cyano-5-fluorophenyl)boronic acid
5
6
1
according to General Procedure A (12% yield). H NMR (400 MHz, CDCl3)  10.55 (br, 1H),
7
8
9
8.25 (s, 2H), 7.59 (s, 1H), 7.50 (d, J = 1.5 Hz, 1H), 7.44 (ddd, J = 1.3, 2.5, 9.4 Hz, 1H), 7.30
(ddd, J = 1.2, 2.5, 7.7 Hz, 1H), 7.24 (d, J = 1.5 Hz, 1H), 4.31 - 4.21 (m, 2H), 4.13 (d, J = 12.4
Hz, 1H), 3.94 (dd, J = 8.6, 11.2 Hz, 1H), 3.70 (t, J = 8.8 Hz, 1H), 3.36 (s, 3H). m/z: 466.3.
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3-(5-((4aR,7aR)-6-(5-Fluoropyrimidin-2-yl)-2-imino-3-methyl-4-oxooctahydro-7aH-
pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)-5-methoxybenzonitrile (5)
Analog 5 was prepared from intermediate 25 and (3-cyano-5-methoxyphenyl)boronic
1
acid according to General procedure A (19% yield). H NMR (400 MHz, CD3OD)  8.43 (s,
2H), 7.89 (d, J = 0.7 Hz, 1H), 7.68 (s, 1H), 7.60 (s, 1H), 7.52 - 7.45 (m, 1H), 7.21 (d, J = 0.7 Hz,
1H), 4.47 (d, J = 12.1 Hz, 1H), 4.26 - 4.12 (m, 3H), 4.08 (dd, J = 7.3, 9.5 Hz, 1H), 3.89 (s, 3H),
3.35 (s, 3H). m/z: 478.3.
(4aR,7aR)-7a-(4-(3,5-Difluorophenyl)thiophen-2-yl)-6-(5-fluoropyrimidin-2-yl)-2-imino-3-
methyloctahydro-4H-pyrrolo[3,4-d]pyrimidin-4-one (6)
Analog 6 was prepared from intermediate 25 and (3,5-difluorophenyl)boronic acid
1
according to General Procedure A. H NMR (600 MHz ,CDCl3)  8.27 (s, 2 H), 7.45 (s, 1H),
7.32 (s, 1H), 7.02 (d, J = 6.4 Hz, 2H), 6.76 (t, J = 8.7 Hz, 1H), 4.58 (d, J = 12.2 Hz, 1H), 4.35 (s,
1H), 3.99 (d, J = 12.5 Hz, 1H), 3.91 - 3.82 (m, 2H), 3.37 (s, 3H). m/z: 459.3.
(4aR,7aR)-7a-(4-(3-Chlorophenyl)thiophen-2-yl)-6-(5-fluoropyrimidin-2-yl)-2-imino-3-
methyloctahydro-4H-pyrrolo[3,4-d]pyrimidin-4-one (7)
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Analog 7 was prepared from intermediate 25 and (3-chlorophenyl)boronic acid according
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to General Procedure A. H NMR (600 MHz, CDCl3)  8.22 (s, 2H), 7.50 (s, 1H), 7.40 (d, J =
7
8
9
7.6 Hz, 1H), 7.36 (s, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.27 - 7.24 (m, 1H), 4.31 (d, J = 11.3 Hz, 1H),
4.22 (t, J = 10.4 Hz, 1H), 3.96 (dd, J = 2.0, 12.2 Hz, 1H), 3.80 (t, J = 10.1 Hz, 1H), 3.65 (t, J =
9.2 Hz, 1H), 3.30 (s, 3H). m/z: 457.3.
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Preparation of (4aR,7aR)-7a-(4-bromothiophen-2-yl)-6-(5-fluoropyrimidin-2-yl)-2-
imino-3-methyloctahydro-4H-pyrrolo[3,4-d]pyrimidin-4-one (8)
Following removal of the Teoc group from intermediate 25 using a procedure analogous
to that described in the relevant section of General Procedure A, the resulting free pyrrolidine
(0.027 g, 0.063 mmol) was mixed with toluene (0.5 mL), sodium tert-butoxide (0.021 g, 0.219
mmol), Pd2(dba)3 (0.007 g, 7.64 µmol) and BINAP (0.01 g, 0.016 mmol). The resulting mixture
was degassed and back filled with nitrogen (x3). To this solution was added a solution of 2-
chloro-5-fluoropyrimidine (0.016 g, 0.121 mmol) in toluene (0.1 mL), and the mixture was
heated at 60 °C for 12 h. The reaction mixture was filtered through a pad of Celite, and the crude
material was purified via silica gel chromatography using 0 to 100% ethyl acetate in hexanes as
the eluent to give an N-arylated product. The Boc protecting group from the N-arylated product
was removed through treatment with 20% TFA/CH2Cl2 (1 mL). The deprotected compound was
purified by reverse phase HPLC (C18) using water/acetonitrile with 0.1% formic acid, and was
1
converted to its HCl salt to provide compound 8 (0.0013 g, yield 5%). H NMR (400 MHz,
CD3OD) δ 8.37 (d, J = 0.9 Hz, 2H), 7.54 (d, J = 1.4 Hz, 1H), 7.22 (d, J = 1.5 Hz, 1H), 4.38 (d, J
= 12.2 Hz, 1H), 4.21 – 3.99 (m, 4H), 3.33 (s, 3H). m/z: 427.2.
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General Procedure B: Synthesis of analogues 12, 13, 15 – 20 from intermediate 26.
To a solution of the pyrrolidine 26 (1 equiv.) and an appropriately substituted 2-chloro-5-
fluoro pyrimidine (1.2 equiv.) in toluene (5 mL/mmol of pyrrolidine) were added
tris(dibenzylidene-acetone)dipalladium(0) (0.1 equiv.), (2-biphenylyl)di-tert-butylphosphine
(JohnPhos, 0.15 equiv.), and Na-OtBu (2.2 equiv.). The resulting solution was degassed, and
heated at 60 °C for 2 h, or until the reaction was determined to be complete, and then cooled to
rt. The reaction mixture was filtered through a pad of Celite, and the crude material was purified
via silica gel chromatography using ethyl acetate in hexanes as the eluent to give an N-arylated
product. The Boc protecting group of the N-arylated product was removed through treatment
with 20% TFA/CH2Cl2 (2.5 mL/mmol). The deprotected compounds were purified by reverse
phase HPLC (C18) using 0−90% water/acetonitrile with 0.05% TFA.
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3-(5-((4aR,7aR)-6-(5-Fluoro-4-methylpyrimidin-2-yl)-2-imino-3-methyl-4-oxooctahydro-
7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (12)
Analog 12 was prepared from intermediate 26 and 2-chloro-5-fluoro-4-methylpyrimidine
1
following General Procedure B (0.035 g, 35% yield). H NMR (400 MHz, CD3OD)  8.27 (d, J
= 2.2 Hz, 1H), 8.05 (t, J = 1.5 Hz, 1H), 7.98 (td, J = 1.5, 8.1 Hz, 1H), 7.90 (d, J = 1.5 Hz, 1H),
7.70 (d, J = 1.1 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 4.49 (d, J = 12.4 Hz,
1H), 4.27 - 4.06 (m, 4H), 3.35 (s, 3H), 2.44 (d, J = 2.2 Hz, 3H). m/z: 462.3.
3-(5-((4aR,7aR)-6-(4-Ethyl-5-fluoropyrimidin-2-yl)-2-imino-3-methyl-4-oxooctahydro-7aH-
pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (13)
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Analog 13 was prepared from intermediate 26 and 2-chloro-4-ethyl-5-fluoropyrimidine
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1
following General Procedure B (0.063 g, 60% yield). H NMR (400 MHz, CD3OD)  8.24 (d, J
7
8
9
= 2.2 Hz, 1H), 8.04 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.90 (d, J = 1.5 Hz, 1H), 7.69 (d, J = 1.1
Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H), 4.49 (d, J = 12.1 Hz, 1H), 4.29 - 4.05
(m, 4H), 3.35 (s, 3H), 2.84 - 2.74 (m, 2H), 1.28 (t, J = 7.5 Hz, 3H). m/z: 476.3.
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3-(5-((4aR,7aR)-6-(4-Ethoxy-5-fluoropyrimidin-2-yl)-2-imino-3-methyl-4-oxooctahydro-
7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (15)
Analog 15 was prepared from intermediate 26 and 2-chloro-4-ethoxy-5-fluoropyrimidine
1
following General Procedure B. H NMR (400 MHz, CD3OD)  8.23 (d, J = 4.0 Hz, 1H), 8.06
(s, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 1.5 Hz, 1H), 7.73 (s, 1H), 7.67 (d, J = 7.7 Hz, 1H),
7.60 (t, J = 8.0 Hz, 1H), 4.58 - 4.51 (m, 3H), 4.24 (s, 4H), 3.35 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H).
m/z: 492.3.
3-(5-((4aR,7aR)-6-(5-Fluoro-4-propoxypyrimidin-2-yl)-2-imino-3-methyl-4-oxooctahydro-
7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (16)
Analog 16 was prepared from intermediate 26 and 2-chloro-5-fluoro-4-
1
propoxypyrimidine following General Procedure B. H NMR (400 MHz, CD3OD)  8.13 (d, J =
3.7 Hz, 1H), 8.05 (t, J = 1.5 Hz, 1H), 7.98 (td, J = 1.5, 8.1 Hz, 1H), 7.91 (d, J = 1.5 Hz, 1H), 7.71
(d, J = 1.1 Hz, 1H), 7.67 (td, J = 1.5, 7.7 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 4.54 - 4.49 (m, 1H),
4.45 (t, J = 6.6 Hz, 2H), 4.28 - 4.06 (m, 4H), 3.35 (s, 3H), 1.91 - 1.80 (m, 2H), 1.03 (t, J = 7.3
Hz, 3H). m/z: 506.3.
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3-(5-((4aR,7aR)-6-(5-Fluoro-4-(pyrrolidin-1-yl)pyrimidin-2-yl)-2-imino-3-methyl-4-
oxooctahydro-7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (17)
Analog 17 was prepared from intermediate 26 and 2-chloro-5-fluoro-4-(pyrrolidin-1-
yl)pyrimidine following General Procedure B (0.059 g, 51% yield). 1H NMR (400 MHz
,CD3OD)  8.06 (s, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 1.1 Hz, 1H), 7.91 (d, J = 7.0 Hz,
1H), 7.76 (d, J = 1.1 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.9 Hz, 1H), 4.36 - 4.12 (m,
5H), 3.98 - 3.87 (m, 2H), 3.85 - 3.72 (m, 2H), 3.35 (s, 3H), 2.18 - 2.05 (m, 2H), 2.04 - 1.92 (m,
2H). m/z: 517.3.
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3-(5-((4aR,7aR)-6-(5-Fluoro-4-morpholinopyrimidin-2-yl)-2-imino-3-methyl-4-
oxooctahydro-7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (18)
Analog 18 was prepared from intermediate 26 and 4-(2-chloro-5-fluoropyrimidin-4-
yl)morpholine following General Procedure B (0.021 g, 59% yield). 1H NMR (400 MHz,
CD3OD)  8.03 (s, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.91 (d, J = 1.1 Hz,
1H), 7.70 - 7.64 (m, 2H), 7.59 (t, J = 7.9 Hz, 1H), 4.29 - 4.06 (m, 5H), 3.99 - 3.91 (m, 4H), 3.83 -
3.75 (m, 4H), 3.33 (s, 3H). m/z: 533.3.
3-(5-((4aR,7aR)-6-(5-Fluoro-4,6-dimethylpyrimidin-2-yl)-2-imino-3-methyl-4-
oxooctahydro-7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (19)
Analog 19 was prepared from intermediate 26 and 2-chloro-5-fluoro-4,6-
1
dimethylpyrimidine following General Procedure B (0.040 g, 38% yield). H NMR (400 MHz,
CD3OD)  8.04 (t, J = 1.5 Hz, 1H), 7.96 (td, J = 1.5, 7.9 Hz, 1H), 7.89 (d, J = 1.5 Hz, 1H), 7.67
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(td, J = 1.5, 7.9 Hz, 1H), 7.65 (d, J = 1.5 Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H), 4.43 (d, J = 12.1 Hz,
1H), 4.21 - 4.06 (m, 3H), 4.01 (dd, J = 8.1, 10.3 Hz, 1H), 3.34 (s, 3H), 2.34 (d, J = 2.6 Hz, 6H).
m/z: 476.3.
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3-(5-((4aR,7aR)-6-(5-Fluoro-4-methoxy-6-methylpyrimidin-2-yl)-2-imino-3-methyl-
4-oxooctahydro-7aH-pyrrolo[3,4-d]pyrimidin-7a-yl)thiophen-3-yl)benzonitrile (20)
Analog 20 was prepared from intermediate 26 and 2-chloro-5-fluoro-4-methoxy-6-
methylpyrimidine following General Procedure B (0.10 g, 47% yield). 1H NMR (400 MHz,
CD3OD) δ 8.03 (t, J = 1.8 Hz, 1H), 7.96 (ddd, J = 7.9, 1.9, 1.2 Hz, 1H), 7.88 (d, J = 1.5 Hz, 1H),
7.66 (dt, J = 8.0, 1.4 Hz, 2H), 7.58 (t, J = 7.8 Hz, 1H), 4.45 (d, J = 12.3 Hz, 1H), 4.20 (dd, J =
10.3, 8.5 Hz, 1H), 4.15 – 4.01 (m, 3H), 3.99 (s, 3H), 3.34 (s, 3H), 2.29 (d, J = 2.9 Hz, 3H). m/z:
492.3.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Synthetic procedures for preparation of 2-chloro-5-fluoro-4-methylpyrimidine (27), 2-chloro-4-
ethyl-5-fluoropyrimidine (28), 2-chloro-5-fluoro-4-methoxypyrimidine (29), 2-chloro-4-ethoxy-
5-fluoropyrimidine (30), 2-chloro-5-fluoro-4-propoxypyrimidine (31), 2-chloro-5-fluoro-4-
(pyrrolidin-1-yl)pyrimidine (32), 4-(2-chloro-5-fluoropyrimidin-4-yl)morpholine (33), and 2-
chloro-5-fluoro-4,6-dimethylpyrimidine (34)
Incubation protocol for compound 1 with CYP3A4 in the presence and absence of glutathione
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Materials and methods for measurement of kobs for 1 and 19, HPLC traces for compounds 2 – 8.
12, 13, 15 – 20
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Molecular Formula Strings
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AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed:
MM. Telephone: +1 9087402369. E-mail: mihirbaran.mandal@merck.com
ACKNOWLEDGMENTS
We thank Junling Gao and Ross Yang for their help in LCMS analysis of the compounds, Tom
Bateman and Ian Bell for helpful discussions around metabolism and mechanism.
ABBREVIATIONS USED
Cat-D, cathepsin D; TDI, time dependent inhibition; CYP, cytochrome P450; NADPH, reduced
form of Nicotinamide adenine dinucleotide phosphate; rCYP3A4, recombinant cytochrome P450
3A4.
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inhibition, measurement of TDI should be included early in the screening funnel along with
direct inhibition studies.
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