The kinetic deuterium isotope effect as applied to metabolic deactivation of imatinib to the des-methyl metabolite, CGP74588

Article abstract of DOI:10.1016/j.bmc.2013.03.038

There has recently been a burgeoning interest in impeding drug metabolism by replacing hydrogen atoms with deuterium to invoke a kinetic isotope effect. Imatinib, a front-line therapy for both chronic myeloid leukemia and of gastrointestinal stromal tumours, is often substantially metabolised via N-demethylation to the significantly less active CGP74588. Since deuterium-carbon bonds are stronger than hydrogen-carbon bonds, we hypothesised that the N-trideuteromethyl analogue of imatinib might be subject to a reduced metabolic turnover as compared to imatinib and lead to different pharmacokinetic properties, and hence improved efficacy, in vivo. Consequently, we investigated whether the N-trideuteromethyl analogue would maintain target inhibition and show a reduced propensity for N-demethylation in in vitro assays with liver microsomes and following oral administration to rats. The N-trideuteromethyl compound exhibited similar activity as a tyrosine kinase inhibitor as imatinib and similar efficacy as an antiproliferative in cellular assays. In comparison to imatinib, the trideuterated analogue also showed reduced N-demethylation upon incubation with both rat and human liver microsomes, consistent with a deuterium isotope effect. However, the reduced in vitro metabolism did not translate into increased exposure of the N-trideuteromethyl analogue following intravenous administration of the compound to rats and no significant difference was observed for the formation of the N-desmethyl metabolite from either parent drug.

Full text of DOI:10.1016/j.bmc.2013.03.038

Bioorganic & Medicinal Chemistry 21 (2013) 3231–3239
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The kinetic deuterium isotope effect as applied to metabolic
deactivation of imatinib to the des-methyl metabolite, CGP74588
⇑
Paul W. Manley , Francesca Blasco, Jürgen Mestan, Reiner Aichholz
Disease Area Oncology, Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
There has recently been a burgeoning interest in impeding drug metabolism by replacing hydrogen atoms
with deuterium to invoke a kinetic isotope effect. Imatinib, a front-line therapy for both chronic myeloid
leukemia and of gastrointestinal stromal tumours, is often substantially metabolised via N-demethylation
to the significantly less active CGP74588. Since deuterium–carbon bonds are stronger than hydrogen–
carbon bonds, we hypothesised that the N-trideuteromethyl analogue of imatinib might be subject to
a reduced metabolic turnover as compared to imatinib and lead to different pharmacokinetic properties,
and hence improved efficacy, in vivo. Consequently, we investigated whether the N-trideuteromethyl
analogue would maintain target inhibition and show a reduced propensity for N-demethylation in
in vitro assays with liver microsomes and following oral administration to rats. The N-trideuteromethyl
compound exhibited similar activity as a tyrosine kinase inhibitor as imatinib and similar efficacy as an
antiproliferative in cellular assays. In comparison to imatinib, the trideuterated analogue also showed
reduced N-demethylation upon incubation with both rat and human liver microsomes, consistent with
a deuterium isotope effect. However, the reduced in vitro metabolism did not translate into increased
exposure of the N-trideuteromethyl analogue following intravenous administration of the compound
to rats and no significant difference was observed for the formation of the N-desmethyl metabolite from
either parent drug.
Received 29 November 2012
Revised 1 March 2013
Accepted 11 March 2013
Available online 1 April 2013
Keywords:
Imatinib
Kinetic deuterium isotope effect
Leukemia
Metabolism
Tyrosine kinase
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
hydrogen atom abstraction, followed by radical recombination to
insert oxygen, resulting in the formation of an enzyme-bound
aminoalcohol (Scheme 1).5–7 Subsequent non-enzymatic cleavage
of the -aminoalcohol C–N bond, with loss of formaldehyde, then
affords the demethylated metabolite bearing a free HN-group.
The rate-limiting step in this process is usually the cleavage of
a-
The alteration of the oxidative metabolism of drug candidates to
enhance their pharmacokinetic or toxicological properties is an
important aspect of the drug discovery process. The most common
strategy is to replace metabolically labile protons with fluorine
atoms and this has culminated in the launch of a number of fluori-
nated drugs.1 Recently there has also been a burgeoning interest,
particularly from intellectual property aspects, in impeding drug
metabolism by replacing hydrogen atoms with deuterium to in-
crease the in vivo half-life and hence the efficacy of drugs.2–4 In
contrast to their aromatic counterparts, replacing metabolically la-
bile alkyl protons with fluorine often leads to substantial changes
in size, shape and electronic properties that can affect the intrinsic
activity of the molecules under study. However, substitution by
deuterium does not suffer from such limitations and making such
an approach to modulating drug metabolism attractive.
a
the
bond, the activation energy of this process may be significantly in-
creased by replacing the
C-hydrogen atoms with deuterium.8,9
aC–H bond and, because of the increased strength of the C–D
a
Although to date no deuterated drugs have been registered as a re-
sult of such a kinetic deuterium isotope effect (KDIE) approach,
morphine and amphetamine derivatives, where deuteration re-
duces metabolic N-demethylation, provide classic examples of
the effect.9,10
Imatinib (1; GlivecÒ/GleevecÒ; Fig. 1) is a potent inhibitor of the
tyrosine kinase activity of the BCR-ABL1 oncoprotein, as well as
that of several receptor tyrosine kinases, including the stem cell
factor receptor, KIT, and as such it is an effective therapy for
chronic myeloid leukaemia (CML), as well as gastrointestinal stro-
mal tumours (GIST).11–14 Patient responses and survival rates in
both CML and GIST have been shown to be associated with higher
imatinib plasma trough concentrations.15,16 Consistent with this,
the single 400 mg once-daily oral dose normally administered to
patients can, in the case of suboptimal patient responses, be
Xenobiotics possessing O-alkyl or N-alkyl groups are frequently
metabolised via heme-containing monooxygenase cytochrome
P450 (CYP) enzymes. The initial process in the demethylation of
alkylamines has been shown to involve enzyme catalysed a-carbon
⇑
Corresponding author. Tel.: +41 61 6966878; fax: +41 61 6966246.
E-mail address: paul.manley@pharma.novartis.com (P.W. Manley).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.038

3232
P. W. Manley et al. / Bioorg. Med. Chem. 21 (2013) 3231–3239
HO-Radical
Addition
H-Radical
the extent of metabolic demethylation of imatinib might be related
to patient responses. This N-demethylation of imatinib is also ob-
served in rodents and, at least in the case of mice, is exacerbated
by high imatinib tissue distribution into the liver (Fig. 2).19 A likely
reason for this is that the highly basic imatinib (pKa1 7.8) predom-
inates in a cationic form at physiological pH (71.5% at pH 7.4), and
is largely actively transported into cells via the oxycation trans-
porters (hOCT-1 in humans, ꢀ78% homology with rat OCT1; rat/
mouse ꢀ95% homology), which is highly expressed in liver.20–22
Based upon this information, we compared the activities of
imatinib with that of CGP74588 (2) and, as a potential strategy
to improve the pharmacokinetic profile and hence efficacy of
imatinib, we studied the metabolic stability of imatinib and the
N-deuteromethyl analogue, 3, in rat and human liver microsomes,
which have been shown to recapitulate imatinib metabolism,23
and compared their plasma pharmacokinetic profiles in rats.
H
H
H
C
H
H
H
N
N
N
H
Abstraction
C-N Bond
Cleavage
H
O
H
H
N
+
O
H
Scheme 1. Mechanistic pathway for CYP450 catalysed demethylation of
alkylamines.
N
H
N
H
N
N
N
R
O
N
H3C
2. Methods and results
2.1. Chemistry
N
The synthesis of the compounds used in this study is depicted in
Scheme 2. The aniline 4,24 was benzoylated to give the benzyl chlo-
ride derivative 5, as a crystalline hydrochloride salt. Reaction of 5
1: R = CH3, imatinib
2: R = H, CGP74588
with piperazine then afforded the desmethyl metabolite,
2
3: R = CD3, N-deuteromethyl analogue of imatinib
(CGP74588). Alkylation of 2 with deuteromethyl iodide then gave
the N-trideuteromethyl analogue, 3. Acylation of 2 with either N-
acetyl or N-formylpiperazine provided the potential metabolites,
6 and 7. Propylphosphonic anhydride-mediated amide-coupling
of 4 with the benzoic acid 8, prepared from 4-bromomethylbenzoic
acid and 1-methylpiperazin-2-one, afforded an authentic sample of
the imatinib metabolite B (9). An authentic sample of the imatinib
metabolite A (10), was prepared by N-oxidation of imatinib with 3-
chloroperoxybenzoic acid.
Figure 1. Structures of imatinib (1), the N-demethylated imatinib metabolite,
CGP74588 (2) and the N-trideuteromethyl analogue of imatinib (3).
increased to up to 400 mg twice-daily to improve outcomes.
Although in most patients imatinib is well absorbed with high bio-
availability following oral administration, it is extensively metabo-
lised by liver CYP3A4, 3A5 and 2C8 enzymes,17,18 with the major
metabolite being the N-demethylated compound, CGP74588 (2;
Fig. 1).15 In a phase 3 study in newly diagnosed CML, in the quartile
of patients with lowest imatinib exposure who achieved an inferior
clinical response rate, the mean steady-state trough level of 2 was
32% of that of imatinib trough levels and ranged up to 84%.15 Con-
versely, in the quartile of patients with highest imatinib exposure,
who achieved the best responses, mean steady-state trough levels
of 2 were 21% (range 13–36%). It can therefore be concluded that
2.2. Pharmacology
The effects of compounds on BCR-ABL1 autophosphorylation
and on BCR-ABL1-dependent cell viability were determined in
murine haematopoietic Ba/F3 cells transfected to express human
BCR-ABL1, using a capture enzyme-linked immunosorbant (ELISA)
and a luminescence ATP-detection assay, respectively, as previ-
ously described.25,26 Similarly, human GIST882 cells,27 expressing
Figure 2. Concentration—time profile of imatinib (3) in naive mice following a single peroral dose of 20 mg/kg formulated as a solution in NMP/ PEG 300 (10%/90%, v/v). Filled
diamonds, open squares and open triangles represent mean plasma, liver and muscle concentrations, respectively. Error bars represent SEM; 4 animals per time-point.

P. W. Manley et al. / Bioorg. Med. Chem. 21 (2013) 3231–3239
3233
N
N
N
H
N
H
N
H
H3C
COOH
N
NH2
N
N
N
CH3
8
O
N
O
O
N
H3C
H3C
(v)
N
N
4
9
(i)
Cl
N
H
H
N
H
N
H
N
NH
N
N
N
(ii)
O
O
N
N
H3C
H3C
N
N
5
2
(iii)
(iv)
N
N
H
N
H
N
H
H
N
N
R
N
N
N
N
CD3
O
O
O
N
N
H3C
H3C
6: R = CH3
7: R = H
N
N
3
Scheme 2. Synthesis of the N-demethylated imatinib metabolite, CGP74588 (2), the N-deuteromethyl analogue of imatinib and authentic samples for metabolite
identification. Reagents: (i) 4-ClCH2C6H4COCl, dioxane; (ii) piperidine, DMF; (iii) CD3I, K2CO3, DMF; (iv) N-acetyl- or N-formyl-piperazine, i-Pr2EtNH2, DMF; (v) 8,
propylphosphonic anhydride, Et3N, DMF.
an activating KIT mutation (exon 13, K642E) were used to assess
the effects of compounds on both KIT autophosphorylation and
KIT-dependent cell viability.26 The results are summarised in Table
1.
efficacy against cell proliferation. The imatinib metabolite B (9)
as well as the N-acetyl (6) and the N-formyl compound (7) were
devoid of kinase activity at concentrations <3000 nM.
The desmethyl metabolite, CGP74588 (2) was substantially less
active than imatinib in cell-based assays, both as an inhibitor of
BCR-ABL1 and of KIT. Compound 2 (mean IC50 512 95; n = 5)
was also less active than imatinib (mean IC50 194 7; n = 94)25 in
32D cells transformed to express BCR-ABL1 (data not shown).
The N-trideuteromethyl analogue 3, showed similar potency to
that of imatinib as an inhibitor of the tyrosine kinase activity of
BCR-ABL1 and of KIT, and this translated into similar degrees of
2.3. In vitro metabolism
The in vitro metabolism of imatinib 1 and the N-trideuterom-
ethyl analogue 3 was examined following incubation with liver
microsomes from rat and human. The samples were analysed by
capillary-HPLC/MS and screened for potential metabolites.
Imatinib was metabolised slightly more rapidly by human liver
microsomes compared to those from rat, as reflected by intrinsic
Table 1
Comparison of the effects of compounds on kinase autophosphorylation and kinase-dependent proliferation of either BCR-ABL1 dependent murine Ba/F3 cells or KIT dependent
cells derived from a human GIST (SEM: standard error of mean; n: number of determinations)
Compound
Cell-line
BCR-ABL1
Mean IC50 SEM nM
KIT
Mean IC50 SEM nM
Kinase inhibition
Kinase inhibition
Cell proliferation
Cell proliferation
Imatinib (1)
Ba/F3-BCR-ABL
GIST882
221 31 (n = 14)
678 39 (n = 23)
97 12 (n = 7)
342 139 (n = 2)
157 12 (n = 6)
108 7 (n = 13)
293 13 (n = 2)
115 9 (n = 6)
Desmethyl compound (2)
N-Deutero compound (3)
Ba/F3-BCR-ABL
GIST882
Ba/F3-BCR-ABL
GIST882
2229 33 (n = 3)
436 36 (n = 7)
1558 68 (n = 5)
682 81 (n = 6)

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P. W. Manley et al. / Bioorg. Med. Chem. 21 (2013) 3231–3239
Table 2
Comparison of the in vitro metabolic profiles of imatinib 1 and N-trideuteromethyl analogue of imatinib 3 observed with liver microsomes of rat (RLM) and human (HLM)
Compd
Metabolic stability
Uncorrected peak area of unchanged drug and metabolites (% of total peak area)
a
b
Clint
RLM
t
Parent
2
A
B
C
D
E
F
½
HLM
RLM
HLM
RLM
HLM
RLM
HLM
RLM
HLM
RLM
HLM
RLM
HLM
RLM
HLM
RLM
HLM
RLM
HLM
Imatinib
3
56.6
72.4
66.4
55.6
25
20
21
26
77.2
80.9
67.3
81.9
7.9
3.3
22.6
7.1
8.4
8.1
4.4
3.9
0.3
0.4
0.3
0.4
2.0
2.6
1.4
1.3
1.8
2.4
0.2
0.3
0.4
0.4
1.0
1.0
2.1
2.1
2.9
4.1
a
Mean intrinsic clearance, n = 4 (
Mean half-life, n = 4 (minutes).
l
L minꢁ1 mgꢁ1).
b
Table 3
Characteristic HPLC/MS data of imatinib 1 and [D3]-imatinib 3 and metabolites observed with liver microsomes of rat (RLM) and human (HLM)
HPLC/MS
Metabolites of imatinib 1 and N-trideuteromethyl analogue, 3
Parent
3
2
A
B
C
D
E
F
1
1
3
1
3
1
3
1
3
1
3
1
3
1
3
Retention time
(min)
16.5
494
16.5 16.4
16.4
17.1
17.02
18.9
18.8
15.2
15.1
18.4
18.3
14.1
14.1
513
16.9
508
16.8
MH+
497
480
480
510
513
0.234
220
394
495
410 423
450
2
508
511
510
513
412
412
510
511
0.470
220
408
493
275
290
1
HR-MS (d ppm)
0.172 0.03 0.010 0.052 0.177
0.709 0.763 0.177 0.351 0.562 0.562 0.412 0.468 0.296
Key fragment
a
217
394
ꢁ
220
394
ꢁ
203
394
ꢁ
203
394
ꢁ
217
394
492
410 423
450
2
231
394
490
ꢁ
234
394
493
ꢁ
ꢁ
ꢁ
135
394
394a
ꢁ
135
394
394a
ꢁ
ꢁ
ꢁ
217
408
490
275
290
1
Key fragment b
MH+–H2O
Other
394
ꢁ
394
ꢁ
410
492
ꢁ
410
495
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
395
395
H/D exchange
Confirmed
structures
2
+
2
+
3
+
3
+
2
+
2
ꢁ
2
ꢁ
2
ꢁ
3
ꢁ
3
ꢁ
3
+
3
ꢁ
+
ꢁ
ꢁ
ꢁ
a
Identical to key fragment a.
clearance, half-life and the percentage of unchanged drug (Table 2).
Conversely, 3, was metabolised slightly more rapidly by rat liver
microsomes.
The parent compounds and their respective metabolites were
characterized by +ESI product ion spectra and the number of
exchangeable hydrogen atoms. From the MS data, the probable
structures of the metabolites were elucidated based upon two
consistent with the m/z value of 508 of the parent ion, as well as
the mass shifts for formation of key fragments and b. However,
a
comparison with an authentic sample of 7 indicated that this
was not the structure, and mass spectral analysis of the corre-
sponding metabolite from the N-trideuteromethyl analogue 3,
which showed m/z value of 511, suggested that the structure of
metabolite B was in fact the lactam 9, and this was unambiguously
confirmed by comparison with an authentic sample (Scheme 2).
characteristic key fragments (
compounds and specific fragmentation of the individual metabo-
lites (Table 3). Fragment- is formed by cleavage of the amide
a and b) observed with the parent
The authentic N-formyl compound
7 was also subjected to
a
in vitro incubation with liver microsomes of rat and human and
found to be stable under the conditions applied, further suggesting
that this compound was not a discrete intermediate in the
bond and fragment-b by cleavage of the N-benzyl amine bond
(see Scheme 3). The structures of the desmethyl metabolite (2)
and of metabolites A, B, E and F were unambiguously confirmed
by co-injection of synthesized reference compounds to prove iden-
tical retention time and comparison of their product ion spectra.
The mass spectral data of imatinib 1, the N-trideuteromethyl ana-
logue 3, and the seven most abundant in vitro metabolites are
summarised in Table 3 and the elucidated structures are shown
in Scheme 4. Metabolic N-demethylation gave the desmethyl com-
pound 2, with the MH+ ion showing a mass shift of ꢁ14 Da for
imatinib and ꢁ17 Da for the N-trideuteromethyl analogue 3, lead-
ing to m/z 480 with identical fragmentation pattern of the product
ion spectra. The structure of 2 was further confirmed by compari-
son with the authentic synthesised compound.
N
H
N
H
N
N
N
R
N
O
H3C
R= CH3 = Imatinib, 1
MH+ = m/z 494
R= CD3 = 3
N
MH+ = m/z 497
CID
CID
N-oxidation of the N-methylpiperazine side-chain leads to
metabolites A and C, with the ion spectra for both showing a mass
C+
H
N
H
N
N
shifts of +16 Da on key fragment
a and 0 Da on key fragment b;
N
N
the number of exchangeable H-atoms were unchanged from those
of the parents, further supporting N-oxidation of the piperazine
moieties. The structure of imatinib metabolite A was unambigu-
ously confirmed by comparison with the synthetic standard, thus
allowing exact allocation of the sites of N-oxidation for both
metabolites.
R
N
O
O+
H3C
Key Fragment-alpha
R= CH3 = m/z 217
R= CD3 = m/z 220
Key Fragment-beta
m/z 394
N
Initially, the structure of the imatinib metabolite B was tenta-
tively assigned as being the N-formyl compound 7, which was
Scheme 3. Key fragments observed for imatinib
analogue 3.
1
and N-trideuteromethyl

P. W. Manley et al. / Bioorg. Med. Chem. 21 (2013) 3231–3239
3235
N
N
H
N
N
H
N
H
N
H
N
H
N
H
N
N+
O
N
CR3
N
N
N
N
CR3
H
O
O
O
O
N
N
N
H3C
H3C
H3C
2a
Aa
Ba
N
N
N
O
N+
N
OH
H
N
H
N
H
N
H
N
H
N
H
N
N
N
N
N
N
CR3
CR3
O
O
O
N
N
N
H3C
H3C
H3C
N
N
N
Cb
Db
R = H; 1 (imatinib)
R = D; 3 (N-deuteromethylimatinib)
N
N
H
H
N
H
H
N
N
N
N
N
N
N
CR3
CR3
O
O
N
N
HC
O
OH
Ea
Fb
N
N
Scheme 4. Metabolite pattern for imatinib 1 and N-trideuteromethyl analogue 3. aConfirmed structures; bproposed structures.
N-demethylation of imatinib. The N-acetyl compound 6, which is
also a potential secondary metabolite of imatinib and 3, was not
detected in any incubates.
the extent of metabolic dealkylation of 3–2, was less in rat liver
microsomes (3.3%) than in human. The extent of metabolism of 3
to the N-oxide A, was very similar to that of imatinib, in both rat
and human liver microsomes.
The formation of metabolite D, characterised by having an iden-
tical m/z value at 412 for MH+ ions arising from both imatinib and
3, and a mass shift of ꢁ82 Da for imatinib and ꢁ85 Da for [3D]-
2.4. Pharmacokinetics
imatinib on key fragment a, is probably caused by an initial oxida-
tive event on the N-benzyl moiety (probably N-oxidation to C) with
subsequent cleavage to the corresponding benzylalcohol.
To determine the pharmacokinetic behavior in vivo, imatinib
(1) and the N-trideuteromethyl analogue (3) were administered
intravenously to rats at 1 mg/kg. The blood was analysed by LC/
MS/MS for the concentration of 1, 3, and their common N-desm-
ethyl metabolite, 2. The concentration-time profiles of 1 and 3 in
rats following a single dose are illustrated in Figure 3 and the phar-
macokinetic parameters, calculated by non-compartmental analy-
sis, are summarised in Table 4. The pharmacokinetic profiles of
both parent compounds are almost identical in rat, in terms of
exposure, clearance and half-life. Circulating levels of the desmeth-
yl metabolite 2, were very low following administration of either
imatinib (AUC ratio 2.4%) or 3 (AUC ratio 1.0) and although the
trend was consistent with a kinetic isotope effect, the difference
was not significant.
Metabolite E is formed by aliphatic C-hydroxylation of the
methylphenyl moiety indicated by abundant loss of water and a
mass shift of +16 Dalton on key fragment b relative to the parent
compound. Subsequent metabolism of E (oxidation) then lead to
formation of the corresponding aldehyde, F, the structure of which
was supported by the mass of the molecular ions, of fragments
a
and b, and the number of exchangeable H-atoms (Table 2).
Phase II metabolites such as glucuronides and/or glutathione
adducts were not observed. The metabolite pattern is shown on
Scheme 4. All peak areas were calculated without consideration
of the response factor between 1, 3 and respective metabolites
(see Table 2).
In the case of imatinib, the desmethyl compound 2 was the ma-
jor metabolite (22.6% of parent) with human liver microsomes,
whereas with rat microsomes 2 (7.9%) and the N-oxide A (8.4%;
4.4% with human) were formed to a similar extent. In comparison,
although 2 (7.1%) was also the major metabolite of the N-deuter-
omethyl analogue 3 with human liver microsomes, it was less pre-
dominant than in the case of imatinib. As in the case of imatinib,
3. Discussion
Here we show that the N-demethylated metabolite of imatinib,
2 (CGP74588), is significantly less active (ꢂthreefold) than the
parent drug, as an inhibitor of the tyrosine kinase activity of both

3236
P. W. Manley et al. / Bioorg. Med. Chem. 21 (2013) 3231–3239
1000
100
10
N-trideuteromethyl analogue 3 with both human (7.1%) and rat
[D3]-Imatinib, i.v. 1 mg/kg
Imatinib, i.v. 1 mg/kg
(3.3%) microsomes, which is supportive of a kinetic deuterium iso-
tope effect operating in this process. The reasons for this species
difference is unclear. In rat microsomes, the reduced extent of
demethylation was partially compensated by an increase in piper-
azine N-oxidation (metabolite C)/piperazine cleavage (metabolite
D). Otherwise there was no evidence for metabolite switching,4,8
with neither the extent, nor the pattern of formation of the other
imatinib metabolites being substantially influenced by the pres-
ence of the N-deuteromethyl group of 3.
Several studies,30–34 have reported N-formyl and N-acetyl con-
jugates of piperazines, which can themselves be deacylated by li-
ver microsomes,34,35 but these (i.e., compounds 6 and 7) do not
appear to have been formed in the present study and therefore
the extent of N-demethylation with either human or rat micro-
somes does not appear to have been masked by the formation of
secondary metabolites.
1
0.1
1234
6
8
24
48
Time postadministration (hr)
Although the extent of the kinetic isotope effect observed par-
ticularly in human microsomes is not insubstantial compared to
other studies with trialkylamines,36 this did not translate into an
increased exposure of the N-trideuteromethyl analogue 3 com-
pared to that of imatinib, following intravenous administration to
rats, where the pharmacokinetic properties of both parent com-
pounds were almost identical. This is probably related to the rela-
tively low extent of demethylation seen in rat microsomes, coupled
with the low level of clearance of both parent compounds seen in
vivo.
The success of other approaches to improve patient responses
in CML by optimising drug binding to BCR-ABL and thereby
increasing potency, as in the case of nilotinib,37 circumvented the
need for the metabolically labile N-methylpiperazine as a pharma-
cophore element and consequently deuteromethyl-analogues of
imatinib were not pursued further. However, based upon the
results of this study, trideuteromethyl analogues of pharmacologi-
cally active N-methylpiperazine compounds still merit consider-
ation when selecting candidates for drug development.
Figure 3. Mean plasma concentration—time profiles of imatinib (1; open circles)
and the N-trideuteromethyl analogue (3; filled circles) in rats following a single
intravenous bolus dose of 1 mg/kg formulated as a solution in NMP/ PEG 300 (30%/
70%, v/v). Error bars represent SEM; 4 animals per time-point.
Table 4
Comparison of mean in vivo PK parameters of imatinib (1) and N-trideuteromethyl-
analogue (3) in rat. Data are given as mean SD (AUC: mean area under the curve,0-
tlast)
Compound
Imatinib, 1
3
Number of animals
3
17
4
19
Clearance (mL minꢁ1 kgꢁ1
)
0
5
VSS (L kgꢁ1
)
5.5 0.5
4.6 0.3
5.4 0.4
1968 52
1423
5.6 0.9
4.2 0.2
5.0 0.4
1848 414
1151
Terminal t (h)
½
MRT (h)
AUC0– (nmol h Lꢁ1
)
AUCParent
(nmol h Lꢁ1
)
0–48
AUCMetabolite
(nmol h Lꢁ1
)
33.9
11.0
0–48
AUCparent: AUCmetabolite (%)
2.4
1.0
BCR-ABL1 and of KIT. This may be due to the methyl group favour-
ing a distribution of solution conformations (in particular that
where the piperazine ring adopts a chair conformation with the
methyl substituent in the equatorial position) that includes the
binding mode observed in crystallographic studies,11 together with
the higher desolvation cost of the secondary amine.28 Given the re-
duced activity of 2, the high level of imatinib N-demethylation
which has been seen to correlate with low plasma levels of imati-
nib, probably contributes to the poor prognosis of those patients
having relatively low steady-state plasma concentrations of imati-
nib, together with relatively high levels of the metabolite.15,16 Con-
sequently, a potential strategy to improve patient outcomes would
be to administer an analogue of imatinib that was not subject to
such metabolic inactivation. Theoretically, such a compound might
be the N-trideuteromethyl analogue, 3, which has been evaluated
in the study reported here. Although deuteration is known to in-
crease the basicity of amines,29 and the N-methylpiperazine group
is a critical pharmacophore element of imatinib,11 the in vitro po-
tency of compound 3 was equivalent to that of imatinib.
4. Experimental
4.1. Synthesis
4.1.1. General
Chromatography was performed using silica gel (E. Merck,
Grade 60, particle size 0.040–0.063 mm, 230–400 mesh ASTM)
with the eluent indicated. Melting points were determined on a
Leitz Kofler hot-stage apparatus and are uncorrected. Proton
NMR spectra were recorded at 298 K with either a Bruker Avance
600 MHz or a Varian Mercury 400 MHz instrument. Chemical anal-
yses, indicated by the symbols of the elements, were performed by
Solvias AG (Basel) and results obtained were within 0.4% of the
theoretical values.
4.1.2. 4-(Chloromethyl)-N-[4-methyl-3-[[4-(3-pyridinyl)-2-
pyrimidinyl]amino]phenyl]-benzamide, hydrochloride (5)
A stirred solution of 4-methyl-N-3-[4-(3-pyridinyl)-2-pyrimidi-
nyl]-1,3-benzenediamine (4; 110.9 g, 400 mmol)24 in dioxane
(1000 mL) was treated with a solution of 4-(chloromethyl)benzoyl
chloride (75.6 g, 400 mmol) in dioxane (700 mL) at 22 °C over
15 min. The temperature rose to 34 °C and stirring was continued
for 150 min. The product was filtered, washed (dioxane) and
recrystallised from EtOH to afford 5 as a yellow crystalline solid
(184 g, 99%): mp 278–282 °C; NMR (DMSO-d6) d 2.22 (s, 3H), 4.6
(br s, 1H), 4.83 (s, 2H), 7.20 (d, 1H), 7.44 (dd, 1H), 7.53 (d, 1H),
7.57 (d, 2H), 7.87 (m, 1H), 7.96 (d, 2H), 8.13 (s, 1H), 8.58
(d, 1H), 8.85 (d, 1H), 8.87 (dt, 1H), 9.12 (s, 1H), 9.41 (d, 1H),
10.25 (s, 1H).
In in vitro metabolism studies imatinib was found to be metab-
olised more rapidly by human as compared to rat liver micro-
somes, with the N-demethylated compound
2 being the
predominant metabolite with human microsomes and a major
metabolite with rat microsomes. In contrast, the N-trideuterom-
ethyl analogue of imatinib, 3, was metabolised more rapidly by
rat microsomes, although it showed a similar pattern of metabo-
lism to that of imatinib with both human and rat liver microsomes.
The extent of formation of the N-demethylated metabolite 2 from
imatinib with both human (22.6%) and rat (7.9%) liver microsomes,
was much larger than the extent of formation of 2 from the

P. W. Manley et al. / Bioorg. Med. Chem. 21 (2013) 3231–3239
3237
4.1.3. N-[4-Methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-
phenyl]-4-(1-piperazinylmethyl)benzamide (2)
J = 8.0 Hz, 1H), 8.50 (d, J = 5.3 Hz, 1H), 8.67 (d, J = 4.3 Hz, 1H),
8.95 (s, 1H), 9.26 (m, 1H), 10.15 (s, 1H). Anal. Calcd for
The benzyl chloride 5 (12.9 g, 30 mmol) was added to a solution
of piperazine (25.8 g, 300 mmol) in 50% aqueous EtOH (50 mL) and
the mixture was heated under reflux for 14 h. The mixture was fil-
tered, diluted with water at 60 °C and cooled to afford the product,
which was filtered, washed and dried. The free-base (4.8 g) was
dissolved in hot EtOH (20 mL) and treated with methanesulphonic
acid (0.99 g) to give the methanesulphonate salt of 2 as a yellow
crystalline solid, mp 242–245 °C; NMR (600 MHz; DMSO-d6) d
2.22 (s, 3H), 2.31 (s, 3H), 2.57 (br s, 4H), 3.10 (br s, 4H), 3.63 (s,
2H), 7.21 (d, J = 8.28 Hz, 1H), 7.44 (d, J = 5.08 Hz, 1H), 7.45–7.50
(m, 3H), 7.52 (dd, J = 7.81, 4.80 Hz, 1H), 7.94 (d, J = 7.91 Hz, 2H),
8.08 (s, 1H), 8.48 (d, J = 8.09 Hz, 1H), 8.51 (d, J = 5.08 Hz,
2H), 8.68 (d, J = 4.52 Hz, 1H), 9.01 (s, 1H), 9.28 (s, 1H), 10.19
C29H29N7O2: C, 68.62; H, 5.76; N, 19.32; O, 6.30. Found: C, 68.32;
H, 5.63; N, 19.08; O, 6.50.
4.1.7. 4-[(4-Methyl-3-oxo-1-piperazinyl)methyl]-N-[4-methyl-
3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide (9)
A solution of propylphosphonic anhydride in DMF (1.3 mL of
50%, 2.25 mmol) was added dropwise to a stirred mixture of 4
(416 mg, 1.5 mmol),19 4-[(4-methyl-3-oxo-1-piperazinyl)methyl]-
benzoic acid (372 mg, 1.5 mmol) and triethylamine (1.7 mL,
12 mmol) in DMF (4 mL). The mixture was stirred for 17 h at room
temperature and then treated with saturated aqueous sodium
hydrogen carbonate solution (100 mL) and extracted with EtOAc.
The combined extracts were washed with water, dried and the
solvent was evaporated off under reduced pressure to give the
product which was recrystallised from MeOH to give a yellow crys-
talline solid, mp 187–192 °C; 1H NMR (500 MHz, DMSO-d6) d 2.21
(s, 3H), 2.63 (t, J = 5.5 Hz, 2H), 2.80 (s, 3H), 2.96 (s, 2H), 3.26 (t,
J = 5.5, 2H), 3.61 (s, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 5.1 Hz,
1H), 7.46 (d, J = 8.0 Hz, 2H), 7.48 (m, 1H), 7.51 (dd, J = 7.9, 4.7 Hz,
1H), 7.91 (d, J = 8.0 Hz, 2H), 8.07 (s, 1H), 8.47 (d, J = 7.9 Hz, 1H),
8.50 (d, J = 5.1 Hz, 1H), 8.67 (d, J = 4.7 Hz, 1H), 8.99 (s, 1H), 9.26
(d, J = 2 Hz, 1H), 10.18 (s, 1H). Anal. Calcd for C29H29N7O2: C,
68.62; H, 5.76; N, 19.32. Found: C, 68.29; H, 5.74; N, 18.92.
.
(s, 1H). Anal. Calcd for C28H29N7O.CH4SO3 0.62H2O: C, 59.36;
H, 5.88; N, 16.70. Found: C, 59.23; H, 5.89; N, 16.68.
4.1.4. 4-[[4-(Methyl-d3)-1-piperazinyl]methyl]-N-[4-methyl-3-
[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide (3)
A stirred solution of 2 (240 mg, 0.50 mmol) in DMF (9 mL) was
treated with powdered anhydrous K2CO3 (69 mg, 0.50 mmol) fol-
lowed by trideuteromethyl iodide (0.31 mL, 0.50 mmol) and stirred
at 22 °C for 90 min. In order to acetylate any remaining starting
material acetic anhydride (0.01 mL, 0.10 mmol) was then added
and the mixture was stirred for a further 20 min. Water (82 mL)
was then added and the mixture was evaporated to dryness under
reduced pressure. The crude product was purified by column chro-
matography (silica gel, aq ammonia/CH3OH/CH2Cl2–1:9:90) and
recrystallised from EtOAc to afford 3 as a colourless crystalline so-
lid (104 mg, 42%): mp 205–207 °C; NMR (DMSO-d6) d 2.21 (s, 4H),
2.33 (d, J = 24.83 Hz, 6H), 3.51 (s, 2H), 7.19 (d, J = 8.28 Hz, 1H), 7.42
(d, J = 6.26 Hz, 3H), 7.47 (d, J = 8.28 Hz, 1H), 7.51 (dd, J = 7.87,
4.84 Hz, 1H), 7.89 (d, J = 8.07 Hz, 2H), 8.06 (s, 1H), 8.47 (d,
J = 8.07 Hz, 1H), 8.50 (d, J = 5.25 Hz, 1H), 8.67 (d, J = 4.64 Hz, 1H),
4.1.8. 4-[(4-Methyl-3-oxo-1-piperazinyl)methyl]benzoic acid (8)
A mixture of 3-bromomethylbenzoic acid (4.30 g, 20 mmol), 1-
methylpiperazin-2-one (2.3 g, 20 mmol),38 and powdered potas-
sium carbonate (2.76 g, 20 mmol) in ethanol (50 mL) was stirred
for 17 h at room temperature. The solvent was evaporated off un-
der reduced pressure to give a residue which was treated with HCl
(10 mL of 2 M) and extracted with EtOAc. The combined extracts
were washed with water, dried and the solvent was evaporated
off under reduced pressure to give 8, which was recrystallised from
EtOAc/hexane to give a cream crystalline solid, mp 161–166 °C; 1H
NMR (DMSO-d6) d 2.63 (t, J = 5.5 Hz, 2H), 2.80 (s, 3H), 2.96 (s, 2H),
3.26 (t, J = 5.5, 2H), 3.61 (s, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.90 (d,
J = 8.0 Hz, 2H), 12.9 (br s, 1H).
8.98 (s, 1H), 9.26 (d, J = 1.41 Hz, 1H), 10.16 (s, 1H). Anal. Calcd for
.
C
29H27N7OD3 0.2H2O: C, 69.63; N, 19.60; O, 3.84. Found: C, 69.32;
N, 19.51; O, 3.41.
4.1.5. 4-[(4-Acetyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-
pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamide (6)
4.1.9. 4-[(4-Methyl-4-oxido-1-piperazinyl)methyl]-N-[4-
methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-
phenyl]benzamide (10)
A stirred solution of 5 (466 mg, 1.0 mmol) and i-Pr2EtNH2
(0.70 mL, 4.0 mmol) in CH2Cl2 (15 mL), was treated with a solution
of 1-acetylpiperazine (192 mg, 1.5 mmol) in DMF (5 mL) and
heated at 50 °C in a sealed glass vial for 18 h. The mixture was
evaporated to dryness under reduced pressure, dissolved in DMF
and purified by MPLC reverse phase chromatography (Merck
LiChroprepÒ RP-18, acetonitrile/water 0.1% trifluoroacetic acid)
and recrystallised from EtOH to afford 6 as a yellow crystalline so-
lid (274 mg, 52%): mp 189–191 °C. NMR (DMSO-d6) d 1.96 (s, 3H),
2.20 (s, 2H), 2.30 (m, 2H), 2.36 (m, 2H), 3.41 (m, 4H), 3.55
(s, 2H), 7.19 (d, J = 8.60 Hz, 1H), 7.4–7.6 (m, 5H), 7.90 (d,
J = 7.82 Hz, 2H), 8.06 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.50
(d, J = 5.3 Hz, 1H), 8.67 (d, J = 4.3 Hz, 1H), 8.95 (s, 1H), 9.26
A stirred solution of 4-[[4-(methyl)-1-piperazinyl]methyl]-N-
[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benz-
amide (1; 2 g, 4.05 mmol) in CH2Cl2 (70 mL) at ꢁ20 °C was treated
with MCPBA (2.06 g of 55%, 4.27 mmol) and stirred for 72 h at
room temperature. The mixture was evaporated to dryness under
reduced pressure and the crude product was purified by column
chromatography (silica gel, water/CH3OH/CH2Cl2–5:30:70) and
recrystallised from EtOH to afford 10 as a yellow crystalline solid,
mp 154–158 °C; NMR (600 MHz; DMSO-d6) d 2.22 (s, 3H), 2.79–
2.89 (m, 4H), 3.03 (s, 3H), 3.33–3.41 (m, 4H), 3.58-3.64 (m, 2H),
7.20 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 5.05 Hz, 1H), 7.44-7.47 (m,
2H), 7.48 (dd, J = 8.1, 1.5 Hz, 1H), 7.52 (dd, J = 8.0, 4.7 Hz, 1H),
7.91 (d, J = 7.9 Hz, 2H), 8.07 (s, 1H), 8.47 (d, J = 8.1 Hz, 1H), 8.51
(d, J = 5.2 Hz, 1H), 8.68 (dd, J = 4.6, 1.1 Hz, 1H), 9.01 (s, 1H), 9.27
(d, J = 1.6 Hz, 1H), 10.20 (s, 1H).
.
(d, J = 1.96 Hz, 1H), 10.15 (s, 1H). Anal. Calcd for C30H31N7O2 0.1H2O
C, 68.84; H, 6.01; N, 18.73; O, 6.42. Found: C, 68.99; H, 6.12; N,
18.44; O, 6.63.
4.1.6. 4-[(4-Formyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-
(3-pyridinyl)-2pyrimidinyl]-amino]phenyl]benzamide (7)
Compound 7 was prepared following the procedure used for 6
to afford the product as a cream crystalline solid, mp 233–235 °C.
NMR (DMSO-d6) d 2.20 (s, 2H), 2.31 (m, 2H), 2.37 (m, 2H), 3.3–
3.4 (m, 4H), 3.57 (s, 2H), 7.19 (d, J = 8.20 Hz, 1H), 7.3–7.6 (m,
5H), 7.90 (d, J = 7.80 Hz, 2H), 7.97 (s, 1H), 8.08 (s, 1H), 8.46 (d,
4.2. Metabolism studies in liver microsomes
4.2.1. In vitro incubation for calculation of intrinsic clearances
Assessment of the microsomal stability and calculation of
in vitro metabolic clearance rates with rat and human liver micro-
somes was conducted as described.39

3238
P. W. Manley et al. / Bioorg. Med. Chem. 21 (2013) 3231–3239
4.2.2. In vitro incubation for metabolite identification
were administered a single bolus dose of 1 mg/kg test substance
in 0.5 mL/kg vehicle (30% NMP, 70% PEG200) into the femoral vein.
Sequential blood samples (approx. 50 lL) were collected from each
Stock solutions of imatinib 1, the N-trideuteromethyl analogue
3 and N-formyl-derivative 7 (2 mmol/L) were prepared in DMSO.
Solutions of alamethicin and uridine 50-diphospho-glucuronic acid
(UDPGA, 24 mmol/L) were prepared in water (0.125 mmol/L) and
phosphate buffer (100 mM pH 7.4), respectively. Incubations were
performed with the respective parent compound at 37 °C for up to
60 min with liver microsomes from rat and human. For this pur-
animal via the femoral artery for up to 48 h after administration
and were stored at ꢁ20 °C. To determine the concentrations of
the test substances, aliquots (30 lL) of blood samples were used
and proteins were precipitated with 200
a structural analogue of the test substance as internal standard.
The supernatants (2 L) were injected directly onto the LC/MS/
lL of CH3CN containing
pose, 3
mixed with 417
60 L UDPGA stock solution, respectively. To this reaction mixture
L stock solution (2 mM in DMSO) of the parent compound was
added and pre-incubated for 3 min at 37 °C. The metabolic reaction
was started by addition of 60 L of the NADPH-regenerating sys-
l
L liver microsomes, containing 20 mg protein/mL, were
l
l
L phosphate buffer, 60 L of alamethicin and
l
MS system for analysis. The test article and its internal standard
l
were separated with a Phenomenex POLAR RP (50 ꢃ 2 mm ID,
3
l
2.5 lm). The mobile phase consisted of Solvent A with 1% formic
acid in H2O and Solvent B with 1% formic acid in CH3OH. At a con-
l
stant flow rate of 350 L/min, a linear mobile phase gradient from
l
tem, consisting of isocitrate-dehydrogenase (1 U/mL), NADP
(1 mmol/L) and isocitrate (5 mmol/L). In a further incubation,
50 lL of a solution of 50 mmol/L glutathione (GSH) in phosphate
buffer (100 mM, pH 7.4) was added as second co-factor for moni-
toring of reactive intermediates. After 60 min, reactions were
95% Solvent A to 75% Solvent B over a 5-min period was applied. A
Sciex API 4000 Q-Trap (Applied Biosystems, Concord, Ontario, Can-
ada) mass spectrometer fitted with an electrospray ionization
interface (ESI) was used for the mass detection. Under these exper-
imental conditions, the lower limit of quantification was 0.4 ng mL.
The pharmacokinetic parameters were estimated using a non-com-
partmental approach (WinNonlin, Version 4.0; Pharsight Corp.,
Mountain View, CA).
stopped with 600
samples were subsequently centrifuged (10,000 g, 5 min) and
100 L of supernatant was diluted with 400 L water, re-centri-
L were used for HPLC/MS analysis.
lL ice-cold CH3CN and stored at ꢁ80 °C. Thawed
l
l
fuged and aliquots of 5–10
l
References and notes
4.2.3. HPLC/MS(n) analysis
In order to protect the analytical column and to enrich the tar-
get compounds, the samples were injected via a pre-column in
back-flush mode (column swithing). The analytical system con-
sisted of an automated injection device with cycle-composer
(HTS PAL; CTC Analytics, Zwingen, Switzerland) and a trapping col-
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trapping column was pre-conditioned with 70 L H2O/CH3CN
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lm). The
l
l
lL
H2O/CH3CN (97.5/2.5). Desorption of the analytes was achieved
with the mobile phase of the HPLC pump during 15 min. After
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Beckenried, Switzerland) and a capillary column, 150 ꢃ 0.3 mm,
filled with X-Bridge BEH130, C18, particle size 3.5 lm (Waters
Corp, Milford, MA). Gradient mobile phase programming was used
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l
95) + 0.1% CH3COONH4 + 0.02% TFA. Eluent B was CH3CN/CH3OH/
H2O (47.5/47.5/5) + 0.1% CH3COONH4 + 0.02% TFA. The mobile
phase was held isocratic for 2 min at 5% B, followed by a linear gra-
dient from 5% B to 95% B over 25 min and a 5 min isocratic phase at
95% B. The column temperature was kept at 45 °C. The H/D ex-
change experiment was performed by replacement of water/meth-
anol with deuterated water/deuteromethanol (CH3OD).
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of a linear ion trap (LTQ XL, Thermo, San Jose, CA). The ionisation
technique employed was positive electrospray (+ES). The mass
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ters: Isolation width 2.0 Da; normalised collision energy 20–25%;
activation time 30 ms. Sheath gas setting of 15 units and auxiliary
gas of 1 unit was used and a spray voltage of 3.5 kV applied. The
heated metal capillary was maintained at 275 °C. Data acquisition
and was supported by Xcalibur 2.1.0.
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