Strategies for the modulation of phase II metabolism in a series of PKCε inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.082

Extensive phase II metabolism of an advanced PKCε inhibitor resulted in sub-optimal pharmacokinetics in rat marked by elevated clearance. Synthesis of the O-glucuronide metabolite as a standard was followed by three distinct strategies to specifically temper phase II metabolic degradation of the parent molecule. In this study, it was determined that the introduction of proximal polarity to the primary alcohol generally curbed O-glucuronidation and improved PK and physical chemical properties while maintaining potency against the target. Utilization of a Jacobsen hydrolytic kinetic resolution to obtain optically enriched final compounds is also discussed.

Full text of DOI:10.1016/j.bmcl.2014.05.082

Accepted Manuscript
Strategies for the modulation of phase II metabolism in a series of PKCε inhib-
itors
Jeremy J. Clemens, Timothy Coon, Brett B. Busch, Juliana L. Asgian, Sarah
Hudson, Andreas Termin, Tina B. Flores, Dao Tran, Peggy Chiang, Sam Sperry,
Ray Gross, Jeffrey Abt, Roger Heim, Sandra Lechner, Heather Twin, John
Studley, Guy Brenchley, Philip N. Collier, Francoise Pierard, Andrew Miller,
Chau Mak, Vadims Dvornikovs, Juan-Miguel Jimenez, Dean Stamos
PII:
S0960-894X(14)00591-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.082
BMCL 21696
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 April 2014
20 May 2014
22 May 2014
Please cite this article as: Clemens, J.J., Coon, T., Busch, B.B., Asgian, J.L., Hudson, S., Termin, A., Flores, T.B.,
Tran, D., Chiang, P., Sperry, S., Gross, R., Abt, J., Heim, R., Lechner, S., Twin, H., Studley, J., Brenchley, G.,
Collier, P.N., Pierard, F., Miller, A., Mak, C., Dvornikovs, V., Jimenez, J-M., Stamos, D., Strategies for the
modulation of phase II metabolism in a series of PKCε inhibitors, Bioorganic & Medicinal Chemistry Letters (2014),
doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.082
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Bioorganic & Medicinal Chemistry Letters
jo u rn al h ome p ag e : w ww .e lse vie r .c om
Strategies for the modulation of phase II metabolism in a series of PKCε
inhibitors.
Jeremy J. Clemensa,*, Timothy Coona, Brett B. Buscha, Juliana L. Asgiana, Sarah Hudsona,†, Andreas
Termina, Tina B. Floresb, Dao Tranb, Peggy Chiangb, Sam Sperryb,‡, Ray Grossa, Jeffrey Abta, Roger
Heimc,§ , Sandra Lechnerc, Heather Twind, John Studleyd, Guy Brenchleyd, Philip N. Colliere,, Francoise
Pierardd, Andrew Millerd, Chau Makd, Vadims Dvornikovse, Juan-Miguel Jimenezd and Dean Stamosa
aDepartment of Chemistry and Drug Innovation, Vertex Pharmaceuticals, 11010 Torreyana Road, San Diego, California 92121, United States
bDepartment of Drug Metabolism and Pharmacokinetics, Vertex Pharmaceuticals, 11010 Torreyana Road, San Diego, California 92121, United States
cDepartment of Biology, Vertex Pharmaceuticals, 11010 Torreyana Road, San Diego, California 92121, United States
dDepartment of Chemistry, Vertex Pharmaceuticals (Europe) Ltd, 86-88 Jubilee Avenue, Milton Park, Abingdon, Oxfordshire OX14 4RW, United Kingdom
eDepartment of Medicinal Chemistry, Vertex Pharmaceuticals, 50 Northern Avenue, Boston, MA 02210, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Extensive phase II metabolism of an advanced PKCε inhibitor resulted in sub-optimal
pharmacokinetics in rat marked by elevated clearance. Synthesis of the O-glucuronide
metabolite as a standard was followed by three distinct strategies to specifically temper Phase II
metabolic degradation of the parent molecule. In this study, it was determined that the
introduction of proximal polarity to the primary alcohol generally curbed O-glucuronidation and
improved PK and physical chemical properties while maintaining potency against the target.
Utilization of a Jacobsen hydrolytic kinetic resolution to obtain optically enriched final
compounds is also discussed.
Revised
Accepted
Available online
Keywords:
PKCε
Phase II
Metabolism
Glucuronidation
Polarity
2009 Elsevier Ltd. All rights reserved.
In the discovery and development of novel drug candidates,
drug metabolism is an area of intense scrutiny as undesirable
metabolic instability can alter the pharmacokinetics and
pharmacodynamics of a compound while potentially producing
metabolites leading to toxicity by various mechanisms.1-2
Conjugative phase II metabolism specifically renders typically
lipophilic xenobiotics much more polar, increasing their
clearance potential and suppressing exposure of the parent
compound.3 Although often observed as a post-phase I oxidation
event, O-glucuronidation can occur directly on compounds
containing alkyl alcohols, phenols and carboxylic acids. This
type of metabolic pathway is a significant contributor to the
clearance of drugs such as propofol, morphine and various
NSAIDS.4-5 When significantly observed, the attenuation of this
metabolic clearance pathway is therefore pursued in the
development of drug candidates.
nervous and inflammatory systems leading to investigation of the
enzyme as a therapeutic target for diseases such as chronic pain
and cancer.6-12
Although diversely expressed, PKCε is
abundantly present in neuronal cells and has been implicated
specifically in primary afferent nociceptor sensitization as well as
mechanical hyperalgesia.13 As part of a program targeting
therapeutics for neuropathic pain, we identified a series of PKCε
inhibitors represented by compound 1 which demonstrated potent
activity (PKCε Ki = 25nM, Figure 1). In an effort to attenuate
observed O-glucuronidation of 1 resulting in metabolite 2, a
rationally designed set of diverse functionalities was introduced
in place of the metabolically susceptible ethyl alcohol sidechain.
The synthesis of 1 as well as related analogues relied on key
intermediates 6 and 7, the syntheses of which are shown in
Scheme 1.
Commercially available 2-fluoro-4-iodonicotinaldehyde is
treated with in situ generated trifluoromethyl anion giving aldol
product 3. Manganese oxide oxidation followed by cyclization
with hydrazine affords pyrazolopyridine compound 5. Protection
Protein kinase C epsilon (PKCε) is a serine/threonine kinase
belonging to the novel class of the greater family of PKC
enzymes. PKCε signaling has been implicated in immunologic,
———
∗ Corresponding author. Tel.: + 1-858-404-6701; e-mail: jeremy_clemens@vrtx.com
† Present Address: ACTUS Biotechnologies, EnduRX Pharmaceuticals, Inc., San Diego, California 92121, United States
‡ Present Address: eFFECTOR Therapeutics, 11180 Roselle Street, San Diego, California 92121, United States
§ Present Address: Aspyrian Therapeutics, Inc., 11189 Sorrento Valley Road, Suite 104, San Diego, California 92121, United States

the two enantiomers using chiral supercritical fluid
chromatography gave the chiral alcohols 12 and 13 for which
absolute stereochemical configuration was not determined.
Bulkier secondary alcohols 14 – 16 were prepared from 11 by a
sequence of ozonolysis then addition of the appropriate
commercial Grignard reagent followed by coupling to 7 and trityl
deprotection. The secondary trifluoromethyl alcohol 17 was
prepared from the ozonolysis product (aldehyde) of 11 by the
carbene catalyzed trifluoromethylation procedure reported by
Song et al followed by coupling to 7 during which the OTMS
group is cleaved and finally trityl deprotection.16
Dihydroxylation of 11 using OsO4 followed by protection of the
diol as the dimethyl ketal and subsequent coupling to 7 and
trityl/ketal deprotection furnished the diol analogue 18. The
individual enantiomers of 18 were separated using chiral
supercritical fluid chromatography affording 19 and 20.
Stereochemical configuration of both 19 and 20 was proven by a
chiral synthesis of 20 which commenced with epoxidation of 11.
A Jacobsen hydrolytic kinetic resolution of the resulting epoxide
using acetic acid, water and catalytic (S,S)-(+)-N,N′-bis(3,5-di-
tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt(II)
Figure 1. Structures of compound 1 and metabolite 2.
produced the S-epoxide and R-diol in yields of 94% (98% ee) and
94% (91% ee), respectively (yield based on expected ½ molar
equivalent of products).17 The chiral diol was then protected as
the dimethyl ketal followed by coupling to 7 and trityl/ketal
deprotection giving 20. Methoxyalcohol 21 was prepared from
11 by the sequence of epoxidation, coupling to 7, epoxide ring
opening by methanol under basic conditions and finally trityl
deprotection. Ozonolysis of 11 and treatment of the resulting
aldehyde with DAST followed by coupling to 7 and trityl
deprotection gave the gem-difluoro analogue 22.
Scheme 1. Reagents and conditions: (i) TMS-CF3, TBAF, THF, -10°C to
0°C, 30 min then HCl, 77%; (ii) MnO2, PhCH3, reflux, 3h, 89%; (iii)
hydrazine monohydrate, 1,4-dioxane, 50°C, 15 min, 96%; (iv) NaH, DMF,
0°C, 20 min then trityl chloride, rt, 2h, 99%; (v) B(pin)2, Pd(dppf)Cl2, KOAc,
DMF, 105°C, 14h, 82%.
of the pyrazolopyridine was achieved regiospecifically at the N1
position by use of the trityl group giving 6. Conversion of 6 from
the aryl iodide to the aryl boronic acid pinacol ester gives the key
intermediate compound 7.
An alternative synthetic approach provided an ethyl acetate
handle which allowed for the construction of numerous
additional functional groups to replace the metabolically
susceptible alcohol (Scheme 4). Conversion of the bis-bromide
10 to the mono-boronic acid pinacol ester followed by coupling
to 6 gave an intermediate bromide which was subjected to Heck
coupling with vinyl acetate followed by hydrogenation of the
olefin to give compound 24 along with a minor amount of 23.
Simple trityl deprotection of 24 afforded the O-acetylated
compound 25. Deacetylated compound 23 was subjected to trityl
deprotection to give 32. Compound 1 can be produced from 24
by saponification with sodium hydroxide followed by trityl
deprotection. Treatment of 1 with sodium hypochlorite/sodium
bromide and TEMPO produced carboxylic acid 27 directly. The
obtained alcohol en route to 1 prior to trityl deprotection could be
methylated with methyl iodide and sodium hydride or converted
to the fluoride using DAST producing 26 and 28 respectively
after trityl deprotections. Additionally, the aforementioned
alcohol was subjected to Mitsunobu reaction with
diphenylphosphoryl azide followed by Staudinger reduction and
trityl deprotection to give amine 29. The intermediate amine en
route to 29 prior to trityl deprotection was acylated with acetic
anhydride as well as sulfonylated using methanesulfonyl chloride
to give 30 and 31 respectively after trityl deprotections.
Compound 10 is another key intermediate in the syntheses of
1 and related analogues. The synthesis of compound 10
commences with
a SNAr displacement of fluoride from
commercially available 1,3-dibromo-5-fluorobenzene by ethyl-2-
cyanoacetate (Scheme 2). Subsequent Krapcho decarboxylation
followed by cyclopropanation gives the key dibromide 10.
Scheme 2. Reagents and conditions: (i) ethyl-2-cyanoacetate, NaH, NMP,
120°C, 24h, 98%; (ii) LiCl, DMSO, H2O, 165°C, 3h, quant.; (iii) 1,2-
dibromoethane, tetrabutylammonium bromide, PhCH3, NaOH, H2O, rt, 16h,
94%.
Although compound 1 demonstrated good in vitro Phase I
metabolic stability in rat liver microsomes (+NADPH/-UDPGA)
with 84% remaining after 30 minutes, the compound
demonstrated high clearance (CL = 87.1 mL/min/kg) in a single
low dose PK study (Sprague Dawley rat, 0.5mpk iv).18 Direct
phase II metabolism was implicated as a significant contributor
to clearance as incubation of 1 with rat liver S9 fraction (-
NADPH/+UDPGA) resulted in 17% remaining parent compound
after 1h. Mass spectrometric analysis of in vitro (rat liver S9)
Treatment of 10 with the allyl Grignard reagent prepared
under Knochel’s conditions provided mono-allylated bromide
11.14 From compound 11 a wide variety of analogues could be
prepared using the olefin as a functional handle (Scheme 3).
Secondary alcohols 12 and 13 were prepared by
oxymercuration/demercuration of 11 followed by Suzuki
coupling under Fu’s conditions to aryl boronic acid 7.15 Acidic
deprotection of the trityl functionality and finally separation of

.
i
Scheme 3. Reagents and conditions: (i) CuCN, PrMgCl⋅LiCl, 3-bromo-1-propene, THF, 0°C to rt, 1h, 92%; (ii) HgOAc2, NaBH4, NaOH, THF, H2O, rt, 2h,
69%; (iii) 7, K3PO4, Pd2(dba)3, P(tBu)3HBF4, THF, H2O, rt, 2.5h, 38−97%; (iv) TES, TFA, DCM, 0°C, 2h, 45−85%; (v) chiral supercritical fluid chromatography;
(vi) O3, DCM, MeOH, -78°C, 1h then dimethylsulfide, -78°C to rt, 16h, quant.; (vii) alkyl magnesium bromide, THF, -78°C to rt, 16h, 16-46%; (viii) 1,3-bis-(1-
adamantyl)imidazol-2-ylidene (cat.), TMSCF3, DMF, rt, 16h, 46%; (ix) OsO4, NMO, acetone, H2O, rt, 16h, quant.; (x) 2,2-dimethoxypropane, p-TsOH, acetone,
H2O, rt, 16h, 73-84%; (xi) mcpba, DCM, rt, 20h, 96%; (xii) H2O, HOAc, catalytic (S,S)-(+)-N,N′-bis(3,5-di-tert-butylsalicylidene)-1,2-
cyclohexanediaminocobalt(II), THF, 0°C to rt, 72h, 94% (98% ee) for S-epoxide, 94% (91% ee) for R-diol; (xiii) HCl, DCM, rt, 3h, 70%; (xiv) LiOH, MeOH,
THF, rt, 16h, quant.; (xv) DAST, DCM, -20°C, 2h, 86%. *Absolute stereochemistry unknown.
Scheme 4. Reagents and conditions: (i) iPrMgCl⋅LiCl, THF, -20°C, 1.5h then 2-methoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, -20°C to rt, 16h, 84%; (ii) 6,
Pd(OAc)2, dppb, tBuNH2, butyronitrile, H2O, 120°C, 16h, 69%; (iii) vinyl acetate, Pd(P(tBu)3)2, DIEA, 1,4-dioxane, microwave irradiation, 130°C, 30min, 73%;
(iv) H2 (1 atm), 10% Pd/C, MeOH, EtOAc, rt, 5h, 94%; (v) TES, TFA, DCM, 0°C, 2h, 40−89%; (vi) NaOH, THF, MeOH, 0°C, 1h, quant.; (vii) MeI, NaH, DMF,
rt, 3h, 94%; (viii) NaOCl, TEMPO, NaBr, acetone, H2O, 0°C, 20 min, 57%; (ix) DAST, DCM, 0°C to rt, 1h, 50%; (x) DPPA, PPh3, DIAD, 0°C to rt, 16h, 59%;
(xi) H2 (1 atm), 10% Pd/C, MeOH, EtOAc, rt, 1h, 72%; (xii) acetyl chloride, TEA, DCM, rt, 2h, 97%; (xiii) MsCl, TEA, DCM, rt, 2h, 73%.
and in vivo rat plasma samples indicated glucuronidation of the
parent compound was a major metabolite. Postulating that
glucuronidation was occurring on the primary hydroxyl group of
1, we set out to synthesize this potential metabolite as proof
(Scheme 5). The synthesis of 2 was carried out using
commercially available tribenzoylglucuronic acid methyl ester
trichloroacetimidate which reacted with trityl protected
compound 33 to give the protected glucuronide 34 in 52%
yield.19 Removal of the trityl group by treatment with TFA/TES
followed by saponification using sodium carbonate afforded 2
which demonstrated identical chromatographic properties to our
major metabolite thereby providing strong evidence that the

Scheme 5. Reagents and conditions: (i) 2,3,4-tri-O-benzoyl-α-D-glucuronic acid methyl ester trichloroacetimidate, BF3⋅OEt2, DCM, -10°C, 75 min then TEA, -
10°C to rt, 5 min, 52%; (ii) TES, TFA, DCM, 0°C, 2h, 42%; (iii) Na2CO3, MeOH, H2O, rt, 4 days, 55%.
Table 1. Primary alcohol replacements
Compound
R
PKCε Ki (nM)20
Rat Liver S9 stability
(-NADPH/+UDPGA,
% remaining)21
Strategy
1
17
25 18
78 12
32
28
78
67
Remove Hydroxyl/Replace with Isostere
90
2
22
194 43
145 48
88
26
25
90
5
119
8
29
30
31
12
13
16
183 23
91
95
98
42
37
0
32
50
9
6
34 12
51 28
Introduce Proximal Steric Bulk
30
2
14
15
49
6
17
19
215
3
7
17
267
71
27
18
446 33
26 12
95
59
Introduce Proximal Polarity

19
20
21
37 17
41 19
77 18
67
65
51
glucuronidation of compound 1 occurred at the primary hydroxyl
group.
potency along with greatly improved rat liver S9 stability and
improved physical chemical properties over 1, including a clean
CYP inhibitory profile (CYP3A4, CYP2C9 and CYP2D6 IC50’s
all > 20µM). Conversion of the primary alcohol of diol 18 to a
methoxy group to give 21 improved rat liver S9 stability
similarly to the diol, but with a larger loss in potency. Based on
the in vitro profile of 20, the compound was selected for a single
low dose PK study (Table 2) (Sprague Dawley rat, 0.5mpk iv).
As directly compared to 1, compound 20 demonstrated
improvements in both clearance and half-life which encouraged
us to examine the PK in a higher species. Compound 20 was
next advanced to low dose PK analysis in dog where oral
administration gave a 30-fold improvement in AUC and CL/F as
compared to compound 1 at the same dose with iv clearance of
20 measured at approximately half hepatic blood flow.
With evidence of metabolic fate chiefly consisting of primary
alcohol glucuronidation of parent to give 2 we next set out to
alter this metabolic pathway in an attempt to improve plasma
clearance and CNS exposure. To meet this task, we envisioned a
three-pronged approach to attenuate recognition of the primary
alcohol functionality of 1 by glucuronyl transferases while still
maintaining potency against PKCε and otherwise not diminishing
the leadlike properties of the compound. Specifically our
approach to modulate glucuronidation consisted of:
Removing or replacing the hydroxyl group with an
isosteric/isoelectronic group; 2.) Introducing steric bulk
1.)
proximal to the alcohol or 3.) Introducing polarity in the vicinity
of the alcohol (Table 1).22
As shown in Table 1, removal of the labile hydroxyl group
giving 32 resulted in greatly improved rat liver S9 metabolic
stability accompanied by a 3 fold drop in potency. In fact,
removal of the hydrogen bond donating character of the hydroxyl
group by replacement with mono- and di-fluoro groups or
capping with a methyl or acetate group led to unacceptable drops
in potency (90-194 nM). Conversion to the primary, basic amine
29 greatly improved rat liver S9 stability, however the functional
group was not tolerated as a hydroxyl replacement in terms of
potency. Retaining the hydrogen bond donating capacity of the
amine but utilizing an acetate or methanesulfonamide to
eliminate basicity provided us our first potent compounds with
greatly improved rat liver S9 stability. Both 30 and 31, however
demonstrated moderate human cytochrome P450 isoform
inhibition (30: CYP2D6 IC50 = 15 µM, 31: CYP3A4 IC50 = 7
µM).23
Table 2. Pharmocokinetic results for compounds 1 and 20.
Cmpnd
1
Rat
87
1
Dog
n.d.
20
Rat
55
20
Dog
16.9
Species
CL*(mL/min/kg)
T1/2*(h)
0.23
n.d.
0.51
1.7
CL/F** (mL/min/kg)
1500
0.13
6.7
56
4.4
6.1
AUC** (µg⋅h/mL)
T1/2**(h)
*Compounds dosed at 0.5 mg/kg iv bolus into Sprague Dawley rats (Rat,
3 subjects) or 1 mg/kg iv bolus into Beagle dogs (Dog, 3 subjects).
**Compounds dosed at 10 mg/kg by oral gavage into Beagle dogs (Dog, 3
subjects). n.d. = no data.
The introduction of alkyl groups adjacent to the labile
hydroxyl group was envisioned as a method to sterically
encumber the formation of the glucuronide. As is demonstrated
in Table 1, however, rat liver S9 stability for these compounds
was poor although potency could be retained with some analogs
(12 and 16). Compound 17 with an adjacent CF3 group
significantly enhanced rat liver S9 stability, however introduction
of this lipophilic electron-withdrawing group greatly decreased
potency.
In conclusion we have demonstrated a strategy for the
mitigation of direct O-glucuronidation of a lead molecule
resulting in improved drug-like properties and pharmacokinetics.
Specifically we have shown that the introduction of proximal,
polar functionality (when allowed from a potency standpoint) can
be a superior method to the introduction of bulky, lipophilic
groups intended to sterically impede this metabolic pathway.
The improvement of rat liver S9 stability for compound 20
compared to compound 1 was shown to give an improved
pharmacokinetic profile that translated well to dog. Compound
20 has been selected for further in vivo profiling and details
surrounding those studies will be reported in due course.
With the hypothesis at hand that clearance potential of the
molecules could actually be enhanced by the introduction of
sterically bulky groups, thereby increasing the overall
lipophilicity of the molecules, we set out to introduce polar
functionality in the vicinity of the labile hydroxyl group. In fact,
converting the hydroxyl directly to the polar carboxylic acid 27
resulted in a vast improvement in rat liver S9 stability, albeit at a
large potency cost. Conversion to the racemic diol 18 constituted
a significant step in the program as the potency of 1 was retained
along with a 3.5-fold increase in rat liver S9 stability. Both
individual diol enantiomers, 19 and 20 demonstrated acceptable
Acknowledgements. We thank Professor Michael E. Jung for
useful discussions surrounding the Jacobsen hydrolytic kinetic
resolution. We also thank Caroline Decker, William Kargo,
Rohan Gandhi and Dennis Fiorino for PK and pharmacology

assay (coupling substrate phosphorylation-induced ATP
consumption to NADH consumption) using full-length
recombinant PKCε enzyme (Invitrogen, Madison, WI). Assays
were carried out in 1536- or 3456-well plates containing 100mM
HEPES (pH 7.5), 10mM MgCl2, 25mM NaCl, 0.1mM EDTA,
0.01% Brij, 2mM DTT, 0.002% Triton X-100, 200µg/ml
support. Thanks also to Sabine Hadida and Weichao Chen for
useful discussions.
References and Notes.
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York, 2009; Vol. 186, Chapter 1.
phosphatidylserine,
20µg/ml
diacylglycerol,
500µg/ml
phosphoenolpyruvate, 65µg/ml pyruvate kinase, 20µg/ml lactate
dehydrogenase, 325µM NADH, 150µM substrate peptide
(ERMRPRKRQGSVRRRV), 18nM PKCε, and 60µM ATP (ATP
Km = 47/41µM, 1536/3456) in a final volume of 4.8/1.6µl,
1536/3456. Serial dilutions of inhibitors were prepared in DMSO
and the reaction was initiated by the addition of ATP. Initial rate
data were determined from the rate of change of absorbance at
340nM (corresponding to stoichiometric consumption of NADH)
2. Iyer, R.; Zhang, D. In: Drug Metabolism in Drug Design and
Development; Zhang, D., Zhu, M., Humphreys, W. G., Eds.;
Wiley – Interscience: Hoboken, 2008; Vol. 1, Chapter 9.
3. Kerns, E. H.; Di, L. Drug-like Properties: Concepts, Structure
Design and Methods; Elsevier: Oxford, 2008; Vol. 1, Chapter 11.
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over 20 min at room temperature, using
a Topography
5. Kuehl, G. E.; Lampe, J. W.; Potter, J. D.; Bigler, J. Drug
Metabolism and Disposition, 2005, 33, 1027.
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CA) or an EnVision Multilabel Plate Reader (PerkinElmer,
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protocol (Genedata, San Francisco, CA). Protocol adapted from:
Jimenez, J-M.; Boyall, D.; Brenchley, G.; Collier, P. N.; Davis, C.
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21. Rat Liver S9 Stability Assay: Compounds were tested at 10µM
(0.1M Tris buffer (pH
= 7.4)) in triplicate with a final
concentration of 0.1% DMSO. Rat Liver S9 (Xenotech, LLC.)
were added to a final protein concentration of 5mg/mL and
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a
11. Gonelli, A.; Mischiati, C.; Guerrini, R.; Voltan, R.; Salvadori, S.;
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10ng/mL of an internal standard as a quality control check.
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LCMS. Percent parent remaining was calculated by dividing the
average of 60 min area ratio by the average of 0 min area ratio.
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23. CYP Inhibition Assays: Compounds were tested in three high-
throughput assays (384-format) to assess for inhibition of human
CYP3A4, CYP2C9 and CYP2D6. 10-point compound titrations
were prepared in DMSO ranging from 0.06µM to 30µM with a
final assay concentration of 0.3% DMSO. A solution containing
0.1mg/mL of pooled HLM (BD Ultra Pool 150) and
corresponding substrate at Km (Testosterone (CYP3A4),
Diclofenac (CYP2C9), and Dextromethorphan (CYP2D6) at
50µM, 12µM and 3µM, respectively) was added to compound
plates. Reaction was initialized by adding 1mM NADPH (Sigma).
All reagents were prepared in 0.1mM phosphate and 3mM MgCl2,
pH 7.4 (Sigma). Compound plates were incubated for 10min at
37°C, 5% CO2. Reaction was quenched with 2 parts of acetonitrile
with a corresponding isotopic internal standard. Samples were
centrifuged and supernatant was put in a SPE-MS (RapidFire®-
MS) system for analysis. Analyte was product formed. The area
ratios of analyte peak area to IS peak area were converted to
percent inhibition from blank control and inhibited control. Dose
response curves were generated and IC50 values determined using
Genedata®.
17. Schaus, S. E.; Brandes, B. D.; Larrow, J. F.; Tokunaga, M.;
Hansen, K. B.; Gould, A. E.; Furrow, M. E.; Jacobsen, E. N. J.
Am. Chem. Soc. 2002, 124, 1307.
18. Rat Liver Microsome Stability Assay: Compounds were incubated
at 37 ºC and shaken for 30 minutes in a phosphate buffered
solution with either rat or human liver microsomes and NADPH as
cofactor. A time zero control was similarly prepared, however
with NADPH excluded. The final incubation concentrations were
1 µM compound (0.2% DMSO), 0.5 mg/mL liver microsome, 2
mM NADPH, and 0.1 M phosphate. Reactions were quenched
and proteins precipitated by the addition of 2 volume equivalents
of ice cold acetonitrile containing an internal standard. Following
a centrifugation step, aliquots from the quenched incubations were
further diluted with 4 volume equivalents of a 50% aqueous
methanol solution and then subjected to LC/MS/MS analysis for
quantitation of parent compound. Microsome stability values
were calculated as the percent of substrate remaining after 30
minutes referenced against the time zero control.
19. Lucas, R.; Alcantara, D.; Morales, J. C. Carbohydr. Res., 2009,
344, 1340.
20. PKCepsilon biochemical assay experimental: Compounds were
screened for their ability to inhibit PKCε in a coupled-enzyme

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Strategies for the modulation of phase II
metabolism in a series of PKCε inhibitors.
Jeremy J. Clemens, Timothy Coon, Brett B. Busch, Juliana L. Asgian, Sarah Hudson, Andreas Termin, Tina
Flores, Dao Tran, Peggy Chiang, Sam Sperry, Ray Gross, Jeffrey Abt, Roger Heim, Sandra Lechner, Heather
Twin, John Studley, Guy Brenchley, Philip N. Collier, Francoise Pierard, Andrew Miller, Chau Mak, Vadims
Dvornikovs, Juan-Miguel Jimenez and Dean Stamos