Discovery of N-(4-Fluoro-3-methoxybenzyl)-6-(2-(((2S,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methylpyrimidine-4-carboxamide. A Highly Selective and Orally Bioavailable Matrix Metalloproteinase-13 Inhibitor for the Potential Treatment of Osteoarthritis

Article abstract of DOI:10.1021/acs.jmedchem.5b01434

Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.

Full text of DOI:10.1021/acs.jmedchem.5b01434

Article
pubs.acs.org/jmc
Discovery of N‑(4-Fluoro-3-methoxybenzyl)-6-(2-(((2S,5R)‑5-
(hydroxymethyl)-1,4-dioxan-2-yl)methyl)‑2H‑tetrazol-5-yl)-2-
methylpyrimidine-4-carboxamide. A Highly Selective and Orally
Bioavailable Matrix Metalloproteinase-13 Inhibitor for the Potential
Treatment of Osteoarthritis
Peter G. Ruminski,† Mark Massa,† Joseph Strohbach,† Cathleen E. Hanau,† Michelle Schmidt,†
Jeffrey A. Scholten,† Theresa R. Fletcher,† Bruce C. Hamper,† Jeffery N. Carroll,† Huey S. Shieh,†
Nicole Caspers,† Brandon Collins,† Margaret Grapperhaus,† Katherine E. Palmquist,† Joe Collins,†
John E. Baldus,† Jeffrey Hitchcock,† H. Peter Kleine,† Michael D. Rogers,† Joseph McDonald,†
Grace E. Munie,‡ Dean M. Messing,§ Silvia Portolan,§ Laurence O. Whiteley,∥ Teresa Sunyer,‡
and Mark E. Schnute*,†
†Worldwide Medicinal Chemistry and ‡Inflammation and Immunology Research, Pfizer, 610 Main Street, Cambridge, Massachusetts
02139, United States
§Pharmacokinetics, Dynamics, and Metabolism, Pfizer, 700 North Main Street, Cambridge, Massachusetts 02139, United States
∥Drug Safety, Pfizer, 1 Burtt Road, Andover, Massachusetts 01810, United States
S
* Supporting Information
ABSTRACT: Matrix metalloproteinase-13 (MMP-13) is a
zinc-dependent protease responsible for the cleavage of type II
collagen, the major structural protein of articular cartilage.
Degradation of this cartilage matrix leads to the development of
osteoarthritis. We previously have described highly potent and
selective carboxylic acid containing MMP-13 inhibitors;
however, nephrotoxicity in preclinical toxicology species
precluded development. The accumulation of compound in
the kidneys mediated by human organic anion transporter 3
(hOAT3) was hypothesized as a contributing factor for the
finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid
leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable
exposures.
A hallmark of OA disease onset is the degradation of articular
cartilage, and therefore the prevention of this process is a
promising strategy to limit the progression of disease. The major
structural protein of articular cartilage is type II collagen. Matrix
metalloproteinase-13 (MMP-13), a member of a family of zinc-
dependent proteases, shows a high specificity for the cleavage of
type II collagen. In preclinical models of OA disease, transgenic
mice lacking MMP-13 as well as MMP-13 inhibitors have shown
reduced cartilage damage compared with control animals.5−7
Several broad spectrum metalloproteinase inhibitors have
been investigated in human clinical trials; however, a tendinitis-
like stiffening of the joints referred to as muscular skeletal
syndrome (MSS) has limited development.8 The lack of this
finding in the phenotype of the MMP-13 null mouse as well as
INTRODUCTION
■
Osteoarthritis (OA) is a highly debilitating disease characterized
by the degeneration of synovial joints especially of the hand,
knee, hip, and spine leading to pain and reduced mobility. Based
on 2005 data, an estimated 27 million people in the US suffer
from OA.1 As the proportion of elderly in the population and
obesity rates are increasing, both risk factors for disease,2 the
incidence of OA disease is expected to continue to rise. At this
time, available treatment strategies have been limited to
symptomatic pain relief; however, many of these agents have
undesired side effects, and they do not address the cause of the
disease.3 The goal of developing a disease modifying osteo-
arthritis drug (DMOAD) able to halt disease progression has
been elusive. However, continued understanding of the under-
lying biology and advances in clinical trial design are offering new
opportunities for potential treatment options.4
Received: September 15, 2015
© XXXX American Chemical Society
A
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Figure 1. Representative non-zinc binding, highly selective MMP-13 inhibitors.
individuals possessing a missense mutation of the MMP-13 gene
has suggested the poor selectivity profile of the agents studied
was a factor leading to MSS development.9,10
contacts between the amide and two residues, Ala-217 and Thr-
224, as well as a π−π interaction between His-201 and the benzyl
ring.
As described in our previous reports, compounds 2, 3, and 6
were highly potent and selective MMP-13 inhibitors that
demonstrated promising in vivo cartilage protection and safety
in preclinical rodent studies.6,12,13 Further studies of all three
inhibitors in cynomolgus monkeys however revealed the
consistent observation of nephrotoxicity characterized by diffuse
tubular epithelial cell degeneration−regeneration.18 Since these
compounds are organic anions, their ability to be transported by
human organic anion transporter 1 and 3 (hOAT1/3) was
evaluated.18 Indeed compounds 2, 3, and 6 inhibited the uptake
of hOAT3 radiolabeled substrate in transfected cell lines (IC50 =
18, 59, and 6 μM, respectively) and were found to be
accumulated in cultured proximal tubular cells. These results
suggested that all three compounds are substrates for the hOAT3
transporter. Carboxylic acids also present the risk of generating
reactive metabolites through protein conjugation of the resulting
acyl glucuronide.19 It was hypothesized that the nephrotoxicity
finding observed was the consequence of compound accumu-
lation in the kidney epithelial cells followed by a cytotoxic event
mediated through the acyl glucuronide metabolite. Compound 6
was specifically selected for its reduced propensity to undergo
glucuronide acyl migration to form the reactive species
(rearrangement T1/2 = 67 h compared with T1/2 = 0.5 h for
diclofenac). Unfortunately, nephrotoxicity was still observed. As
a result, a chemistry strategy was undertaken to identify a potent
and highly selective MMP-13 inhibitor based on 6 lacking an
organic anion functionality to mitigate the transporter mediated
accumulation risk.
Significant progress in the design of MMP-13 selective
inhibitors has occurred over the last 15 years such that several
highly selective compounds have been reported in the literature,
Figure 1. The first reports of MMP-13 specific inhibitors
included compound 1 from workers at Aventis11 and compound
2 from workers at Pfizer.6 Both compounds were very potent
inhibitors of MMP-13 (IC50 = 8 and 0.67 nM, respectively) while
showing greater than 10 000-fold selectivity against a panel of
related MMP enzymes. Compound 2 was reported to
significantly reduce cartilage lesion areas when dosed orally in
a rabbit anterior cruciate ligament transection/partial meniscec-
tomy model while not showing MMS-like fibroplasia in rats.6
The high selectivity was hypothesized to arise by the compound
adopting a non-zinc binding mode with the inhibitor occupying a
deep region of the S1′ binding pocket unique to MMP-13.
Additional reports of selective MMP-13 inhibitors have appeared
from Pfizer (3 and 6), Takeda (4), Alantos Pharmaceuticals (5),
and Boehringer Ingelheim (7), Figure 1.12−17 The evolution of
highly selective MMP-13 inhibitors has led to several core
structures; however, with the exception of compound 1, a
terminal carboxylic acid has been maintained as a design element.
The preference for the acidic substituent is understandable due
to its proximity to Lys-119 in the binding pocket and the
potential to form an energetically favorable hydrogen-bond or
salt bridge, Figure 2.13,14 Additional important protein−ligand
contacts observed for compound 6 include hydrogen bond
CHEMISTRY
■
Pyridine tetrazole 12 was utilized as a versatile intermediate
allowing for diversity exploration to either the left- or right-hand
side of the molecules, Scheme 1. Compound 12 was prepared
from pyridine carboxylic acid 8 by first transformation of the
carboxylic acid to the nitrile via dehydration of the tert-
butylamide 9. Sodium azide addition to cyanopyridine 10
afforded tetrazole 11, which was subjected to palladium-
mediated carbonylation to provide ester 12. In order to facilitate
a systematic survey of substituents to the tetrazole, ester 12 was
condensed with 3-methoxybenzylamine to provide amide 13,
Scheme 2. Alkylation of the tetrazole occurred with high
Figure 2. Key protein−ligand interactions between MMP-13 and
compound 6.13 Blue shading indicates ligand exposed regions; purple
indicates polar residues; green indicates hydrophobic residues.
B
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a
employing cyanopyrimidine 35 as a key intermediate, Scheme
8. Dichloropyrimidine 35 was hydrolyzed under acidic
conditions to afford compound 36. The resulting desymmetriza-
tion allowed for the effective monocarbonylation of compound
35 through the sequence of palladium mediated carbonylation
resulting in compound 37 followed by chlorination yielding ester
38. Tetraethylammonium cyanide was found to efficiently effect
displacement of the chloride to provide cyanopyrimidine 39.
Condensation of compound 39 with either 3-methoxybenzyl-
amine or 4-fluoro-3-methoxybenzylamine afforded amides 40a
and 40b, respectively, Scheme 9. Tetrazoles 41a and 41b were
provided by heating a water suspension of the nitriles with
sodium azide and zinc bromide. In contrast to the pyridine
tetrazole series where alkylation was regioselective at the 2-
position, alkylation of 41a,b by tosylate 16 derived from trans-
2,5-bis(hydroxymethyl)-1,4-dioxane20 resulted in a mixture of
regioisomers (1/1, N2-desired/N1). The pure 2-substituted
tetrazoles 42 or 43a,b could be obtained after chromatographic
separation. It was subsequently found that the use of metal
additives could alter the alkylation regioisomeric ratio to favor
the 2-position presumably through chelate formation with the
pyrimidine nitrogen. The best selectivity obtained was 5/1 (N2-
desired/N1) employing Co(acac)2 in DMA. The absolute
stereochemistry of compounds 43a,b (and by inference tosylate
16) was established by single crystal X-ray analysis of the
bromomethyldioxane 44 (Supporting Information) obtained by
halogenation of 43a with polymer-supported triphenylphos-
phine and carbon tetrabromide. Oxidation of 43a by pyridinium
dichromate afforded the corresponding carboxylic acid 45.
Scheme 1. Synthesis of Pyridine Tetrazole 12
a
Reagents and conditions: (a) oxalyl chloride, DMF, CH2Cl2; t-
butylamine; (b) POCl3, toluene, 110 °C; (c) Et3N·HCl, NaN3,
toluene, 110 °C; (d) Et3N, dppf, Pd(OAc)2, MeOH, CO, 100 °C.
a
Scheme 2. General Synthesis of 14a−k
RESULTS AND DISCUSSION
■
a
The carboxylic acid functionality in compound 6 had been found
to impart favorable properties in previous MMP-13 inhibitor
SAR studies including good potency, high metabolic stability,
and excellent oral bioavailability. We anticipated several
challenges by departing from the carboxylic acid in order to
test the transporter-driven toxicology hypothesis. The carboxylic
acid provided excellent potency through interactions with Lys-
119; therefore an appropriate surrogate would need to be
identified or potency enhancement elsewhere in the molecule
utilized. Also, carboxylic acid based analog 6 demonstrated high
metabolic stability with low clearance and excellent oral
bioavailability in rat (F = 100%, CL = 14 mL min−1 kg−1). Due
to the relatively high lipophilicity of the scaffold in the absence of
the acid (cLogP = 5.2), metabolic stability concerns were
anticipated. Lastly, the potential for low aqueous solubility of a
non-carboxylic acid series was another design concern since salt
formation by the core scaffold would be more limited.
Initial efforts focused on the identification of an appropriate
replacement for the cyclohexane carboxylic acid motif found in 6,
which maintained MMP-13 potency and selectivity, Table 1. For
the tetrazole substituent, the structural motif of a methylene
linker to a six-membered ring was desired to be maintained due
to the importance of this substituent to be accommodated in the
tunnel region beneath the MMP-13 S1′ loop region, Figure 2. For
this initial survey, we also chose the 3-methoxybenzylamide for
the left-hand substituent as this was one of the most potent
substitution patterns in the previous carboxylic acid series (14a,
MMP-13 Ki = 0.16 nM).6 As anticipated, truncation of the
carboxylic acid to afford the unsubstituted cyclohexyl analog 14b
resulted in a significant but potentially optimizable loss of
potency (Ki = 33.6 nM). We were further pleased to see that
oxygen heterocycles including tetrahydropyrans (14c−e) and
Reagents and conditions: (a) 3-methoxybenzylamine, 100 °C; (b)
PS−PPh3, DBAD, ROH, THF; or in the case of 14e and 14i, ROSO2Y
(Y = 4-toluenyl, 4-nitrophenyl), Et3N, DMA, 85 °C.
regioselectivity at the 2-position to provide analogs such as 14a−
k (Table 1) employing the reaction with alcohols under
Mitsunobu conditions or with arylsulfonate esters in the case
of 14e and 14i. Additional examples of 4-substituted
methylenecyclohexane analogs were prepared as described in
Schemes 3−5. Compound 13 was alkylated by methyl trans-4-
(hydroxymethyl)cyclohexane carboxylate using Mitsunobu
conditions to provide ester 17. The resulting ester was then
converted to the primary amide 18 and subsequently through
dehydration to the nitrile 19. Similarly, 4-amino-substituted
analogs were prepared by alkylation of 13 with trans-or cis-
alcohols 20 and 24 using Mitsunobu conditions, Schemes 4 and
5. Subsequent deprotection afforded the corresponding amines
(22 and 26), which through either acylation or sulfonylation
provided trans-isomers 23a,b and cis-isomers 27a,b. Piperidine
analogs were also prepared through Mitsunobu alkylation of 13
with alcohol 28, Scheme 6. Deprotection of the resulting product
(29) followed by acylation or sulfonylation provided piperidine
amide 31a or sulfonamide 31b.
In order to survey substitution to the benzylamide, carboxylic
acid 33 was employed as the common intermediate, Scheme 7.
Compound 13 was alkylated with trans-cyclohexane-1,4-
dimethanol using Mitsunobu conditions to provide ester 32,
which was subsequently saponified to provide acid 33. Amide
analogs 34a−k (Table 2) were then prepared by coupling of the
corresponding substituted benzylamine with compound 33.
The synthesis of pyrimidine tetrazole analogs followed a
similar synthetic sequence to that of the pyridine series
C
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a
Table 1. SAR of Non-carboxylic Acid Substituents
a
All values are the mean of two or more independent assays. ND, not determined.
D
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a
a
Scheme 3. Synthesis of 18 and 19
Scheme 6. Synthesis of 31
a
Reagents and conditions: (a) PS−PPh3, DBAD, methyl trans-4-
(hydroxymethyl)cyclohexanecarboxylate, THF; (b) HCONH2, THF,
80 °C, NaOMe; (c) PS−PPh3, CCl4, DCE, 80 °C.
a
Scheme 4. Synthesis of 23
a
Reagents and conditions: (a) PS−PPh3, DBAD, 13, THF; (b) TFA,
CH2Cl2; (c) Et3N, silica-bound DMAP, CH2Cl2; CH3SO2Cl or Ac2O.
a
Scheme 7. General Synthesis of 34a−k
a
Reagents and conditions: (a) PS−PPh3, DBAD, 13, THF; (b) TFA,
CH2Cl2; (c) Et3N, CH3SO2Cl or Ac2O, silica-bound DMAP, CH2Cl2.
a
Scheme 5. Synthesis of 27
a
Reagents and conditions: (a) PS−PPh3, DBAD, trans-cyclohexane-
1,4-dimethanol, THF; (b) LiOH, THF, water; (c) 1-hydroxybenzo-
triazole, DMF, N-methylmorpholine, EDCI, corresponding benzyl-
amine.
a
Table 2. SAR of Benzylamide Substituent
b
compound
X
Y
MMP-13/FL Ki (nM) CYP2C9 inh (%)
34a
34b
34c
34d
34e
34f
H
H
F
H
F
119
32.9
110
38.5
78
56
c
a
Reagents and conditions: (a) PS−PPh3, DBAD, 13, THF; (b) TFA,
H
H
H
H
H
F
CH2Cl2; (c) Et3N, CH3SO2Cl or Ac2O, silica-bound DMAP, CH2Cl2.
Cl
c
CH3
CF3
OCH3
F
47.2
23.8
2.1
93
94
66
83
90
96
90
39
the dioxane (14f) offered comparable potency to the cyclo-
hexane ring and allowed for a potential strategy to reduce the
lipophilicity of this group (vide infra). Substitution of the
cyclohexane ring at the 4-position by either hydroxymethyl
(14g,h) or hydroxyl (14j) provided a significant improvement in
potency consistent with the potential for hydrogen bond
interactions with Lys-119 or Asn-194. Transformation of the
carboxylic acid to the primary carboxamide 18 again resulted in
significant loss of potency, while the nitrile 19 allowed for a
partial recovery of potency similar to that observed with
hydroxymethyl substitution (14g). N-Acyl and sulfonyl deriva-
14g
34g
34h
34i
29.3
4.0
Cl
F
CH3
CF3
OCH3
F
3.8
34j
F
6.3
34k
F
1.2
a
All values are the mean of two or more independent assays.
Inhibition of the metabolism of probe substrate diclofenac (5 μM) at
an inhibitor test concentration of 3 μM after 8 min incubation with
pooled HLM. Not determined.
b
c
E
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a
than in human was consistent with the in vivo observation, and as
a result a closer inspection of compounds in assays for both
species was warranted. Although amide and sulfonamide
derivatives 23a,b avoid the potential direct glucuronide
elimination pathway possible with 14g, the modest cLogP
reduction (ΔcLogP = 0.7−0.9) predicted by modifying the
terminal substituent was not sufficient to impact clearance. In
order to further reduce the lipophilicity we needed to turn our
attention to the cyclohexyl ring.
Scheme 8. Synthesis of Cyanopyrimidine 39
Metabolic liability of cyclohexane-containing compounds is
commonly encountered due to the high relative lipophilicity/
atom count of the cyclohexane ring and the susceptibility of all six
carbon atoms to oxidative metabolism; therefore, a common
strategy has been to replace the cyclohexyl ring with oxygen
containing isosters.22 Guided by the predicted cLogP, the
replacement of either one or two cyclohexane carbon atoms with
oxygen afforded compounds with lower measured lipophilicity as
in the case of tetrahydropyran 14c (ΔLogD = −1.15) and
dioxane 14f (ΔLogD = −1.88). Although compound 14c still
showed high clearance, results for dioxane 14f were promising,
Table 3. Compound 14f had low clearance in human liver
microsomes and even though rat liver microsomal clearance
remained high, it showed the lowest rat in vivo clearance to date
in the series. Saturated heterocycles are common elements in
medicinal chemistry with nitrogen heterocycles being ubiquitous
and oxygen heterocycles such as oxetanes and tetrahydropyrans
finding growing utility.23,24 The dioxane ring however has not
been widely utilized in drug design.25−27 Nonetheless,
encouraged by the trend, we next combined the dioxane ring
with a hydroxymethyl substituent (14i) to further reduce the
lipophilicity (LogD = 2.04) and block the potentially
metabolically labile carbon on the dioxane ring. Compound
14i maintained good MMP-13 potency (Ki = 11.7 nM) and now
achieved low clearance in human and rat liver microsomes.
Likewise, 14i demonstrated low rat in vivo clearance (7.7 mL
min−1 kg−1) after intravenous injection and good oral
bioavailability (F = 48%). Even though the primary metabolic
pathway in rat hepatocytes was found to be similar for 14g and
14i, O-glucuronidation and hydroxymethyl oxidation, the
proximity of the dioxane ring to the hydroxymethyl moiety has
a remarkable suppressive effect on the overall metabolism.
To further reduce the lipophilicity, we also considered the
transformation of the pyridine core to a pyrimidine (predicted
a
Reagents and conditions: (a) 13 M H2SO4, rt; (b) Pd(dppf)Cl2,
iPr2EtN, MeOH, CO, 85 °C; (c) oxalyl chloride, DMA, CH2Cl2,
reflux; (d) Et4N(CN), DABCO, CH2Cl2.
tives of 4-amino substituted cyclohexane (23a,b, 27a,b) also
showed good potency with the sulfonamide trans-isomer 23a
achieving MMP-13 potencies comparable to that achieved with a
carboxylic acid (Ki = 0.25 nM). Moving the nitrogen atom into
the ring as in acetyl- (31a) and methylsulfonylpiperidines (31b)
was not as favorable.
The preferred substitution pattern of the benzylamide group
was next re-examined using the trans-hydroxymethylcyclohexane
analog 14g as a base example, Table 2. A methoxy group at the 3-
position of the phenyl ring was clearly favored in the case of
monosubstituted analogs (14g, 34a−f). The replacement of
hydrogen by fluorine at the 4-position of the phenyl ring (34g−
k) consistently improved the potency compared with the
monosubstituted analogs with 3-methoxy derivative 34k being
the most potent. Several benzyl substitution patterns however
were found to present elevated risk of CYP2C9 inhibition, Table
2. Consequently, from this survey 3-methoxybenzyl and 4-fluoro-
3-methoxybenzyl were identified as having the best combination
of potency and reduced drug−drug interaction liability.
As earlier feared, profiling of select cyclohexyl-based leads for
metabolic stability and rat in vivo clearance confirmed that
achieving acceptable pharmacokinetics in the non-carboxylic acid
series would be a hurdle, Table 3. Compounds 14g, 23a, and 23b
each showed moderate to low in vitro intrinsic clearance in
human liver microsomes (HLM); however, in vivo clearance
after intravenous administration to rats was unacceptably high. In
the case of 14g, a higher clearance in rat liver microsomes (RLM)
a
Scheme 9. Synthesis of 42, 43, and 45
a
Reagents and conditions: (a) corresponding benzylamine, iPr2EtN, MeOH, 35 °C; (b) NaN3, ZnBr2, water, reflux; (c) 16, Et3N, DMA, 85 °C; (d)
43a, pyridinium dichromate, DMF, rt.
F
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a
Table 3. In Vitro and Rat In Vivo Pharmacokinetic Data for Select Pyridine Tetrazoles
b
c
d
d
compound
cLogP
LogD
HLM (μL min−1 mg−1
)
RLM (μL min−1 mg−1
)
rat CL (mL min−1 kg−1
)
e
e
e
e
e
g
g
14b
14c
14f
14g
14i
5.03
3.14
1.80
3.56
1.11
2.63
2.84
4.37
3.22
2.49
3.57
2.04
f
41.7
53.1
<8.0
32.7
<8.0
<8.0
98.5
192
74.0
94.9
15.7
f
63.5
54.5
25.8
76.6
7.7
23a
23b
42.6
77.6
f
24.7
f
a
b
c
All in vitro values are the mean of two or more independent assays. Calculated LogP using ACD version 12. Measured pH 7.4 LogD by shake-
d
e
flask method. HLM or RLM in vitro intrinsic CL calculated from metabolic stability data as described in ref 21. Intravenous bolus administration of
1.0 mg/kg (N = 2). Not determined. Intravenous bolus administration of 0.2 mg/kg in cassette PK format (5 compounds, N = 2).
f
g
a
Table 4. In Vitro ADME Property Comparison of Pyridine 14i and Pyrimidine 42
b
c
d
compound
LogD
MMP-13/FL Ki (nM)
MMP-3/CD Ki (nM)
HLM (μL min−1 mg−1
)
sol (μM)
hPPB (%)
14i
42
2.04
1.30
11.7
5.8
>10000
>10000
<8.0
<8.0
28
86
69
237
a
b
c
All values are the mean of two or more independent assays. Measured pH 7.4 LogD by shake-flask method. HLM in vitro intrinsic CL calculated
d
from metabolic stability data as described in ref 21. Thermodynamic solubility of crystalline solid in pH 7.4 phosphate buffer.
ΔcLogP = −0.58). Compound 42 was prepared to enable direct
comparison with pyridine analog 14i, Table 4. Indeed as
predicted, pyrimidine 42 demonstrated a lower measured
lipophilicity compared with the pyridine (ΔLogD = −0.74).
The pyrimidine analog also maintained good MMP-13 potency
(Ki = 5.8 nM) and high selectivity against MMP-3. While
clearance in human liver microsomes remained low for 42, other
pharmaceutical properties were improved compared with the
pyridine analog. Compared with pyridine 14i, aqueous solubility
of pyrimidine 42 was improved by more than 8-fold (237 vs 28
μM) and human plasma protein binding was also reduced (69%
vs 86%). Rat pharmacokinetics for 42 was comparable to the
pyridine analog with low clearance (10.6 mL min−1 kg−1) and
good oral bioavailability (65%), Table 5. To further explore
oral absorption profile compared with the pyridine analog
showing a more than 8-fold increase in free Cmax exposure and a
2.6-fold increase in free AUC possibly driven by the improved
aqueous solubility. Therefore, for the minimal increase in
molecular weight by one mass unit, the transformation of
pyridine to pyrimidine resulted in a significant improvement in
lipophilic ligand efficiency (ΔLIPE = 1.05, LIPE = 6.94) and
pharmaceutical properties such as solubility and oral absorption
while maintaining MMP-13 potency, selectivity, and metabolic
stability.
Utilizing learnings from the benzylamide substituent survey,
the corresponding 4-fluoro-3-methoxybenzyl analog in the
pyrimidine series was next targeted with the intent to further
improve MMP-13 potency while maintaining the favorable
pharmacokinetic properties. Both enantiomers (2S,5R)-43a and
(2R,5S)-43b demonstrated excellent MMP-13 potency with 43a
being the more potent of the two (MMP-13 Ki = 1.5 nM), Table
6. High selectivity was maintained against MMP-3, and 43a was
further found to be highly selective against a panel of 14 matrix
metalloproteinases, TACE, ADAMTS-4, and ADAMTS-5,
Figure 3. Both enantiomers also showed favorable in vitro and
rat in vivo pharmacokinetic profiles demonstrating low clearance
and very high oral bioavailability, Table 6. The pharmacokinetics
of enantiomer 43a in dog and cynomolgus monkey was also
favorable with low clearance (8.2 and 2.1 mL min−1 kg−1,
respectively) and high oral bioavailability (64% and 88%,
respectively). In in vitro metabolic drug−drug interaction assays,
the IC50 of compound 43a was >30 μM for the inhibition of
CYPs 1A2, 2C9, 2D6, and 3A4.
Table 5. Pharmacokinetics of Pyridine 14i and Pyrimidine 42
a
b
rat
dog
CL
F
(%)
PPB
(%)
Cmaxfree
(nM)
compound (mL min−1 kg−1
)
AUCfree (nM·h)
14i
42
7.7
48
65
76
63
66
1230
3240
10.6
580
a
Rat: Crossover design, intravenous bolus administration (1.0 mg/kg,
N = 2), oral suspension administration (1.0 mg/kg, N = 2). Complete
rat pharmacokinetic parameters are available in Table S2 (Supporting
b
Information). Dog: Oral suspension administration to male beagle
dogs in cassette dosing format (5 compounds, 1.0 mg/kg each, N = 3).
potential differentiation, both compounds were dosed orally to
dogs in a cassette experiment such that each animal received a
dose equivalent to 1.0 mg/kg of 14i and 42 in a single
experiment. Pyrimidine 42 demonstrated a significantly better
Treatment of human articular cartilage ex-vivo cultures with
cytokines results in the degradation of type II collagen and the
production of a 45-amino acid fragment known as type II
a
Table 6. Comparison of In Vitro and Pharmacokinetic Properties of 43a and 43b
b
c
c
compound
MMP-13/FL Ki (nM)
MMP-3/CD Ki (nM)
HLM (μL min−1 mg−1
)
rat CL (mL min−1 kg−1
)
rat F (%)
43a
43b
1.5
4.2
>10000
>10000
<8.0
<8.0
18.2
9.8
95
113
a
b
All in vitro values are the mean of two or more independent assays. HLM in vitro intrinsic CL calculated from metabolic stability data as described
c
in ref 21. Rat: Crossover design, intravenous bolus administration (1.0 mg/kg, N = 2), oral suspension administration (1.0 mg/kg, N = 2).
Complete rat pharmacokinetic parameters are available in Table S2 (Supporting Information).
G
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demethylation, methyl oxidation, and both a glucuronide and a
sulfate conjugate were observed. These metabolites were also
identified in cynomolgus monkey hepatocyte incubations and in
in vivo cynomolgus monkey urine samples. Since hydroxymethyl
oxidation was not the only observed metabolic pathway in
human and cynomolgus monkey and exposures of 45 were
predicted to be very low, we had sufficient confidence to proceed
to an exploratory toxicology study to assess nephrotoxicity risk.
Compound 43a was dosed orally (50, 100, and 1000 mg kg−1
day−1) to cynomolgus monkeys for 14 days targeting free
exposures that significantly exceeded efficacious levels observed
in the canine OA model and the level where nephrotoxicity was
observed with the previous carboxylic acid based MMP-13
inhibitor 6. The compound was well tolerated and histological
evaluation of the kidneys did not reveal abnormal pathology.
Free exposures of 43a achieved in the study were 22 times the
projected efficacious AUC and 8.5 times an AUC exposure where
nephrotoxicity was observed with compound 6.
Figure 3. Metalloproteinase selectivity (pIC50) of 43a compared with
broad-spectrum agent marimastat (highest concentration tested 10 μM,
pIC50 = 5).
CONCLUSION
■
collagen neoepitope (TIINE). Compound 43a (PF152)
inhibited cytokine-induced TIINE production in a dose
dependent manner.28 The in vivo effect of compound 43a on
joint lesion progression was evaluated in the canine partial medial
meniscectomy model of OA.28 As a result of joint destabilization
induced by the model, TIINE levels are increased and joint
deterioration occurs. Administration of 43a returned elevated
TIINE levels to prestudy baseline suggesting suppression of
MMP-13 mediated cartilage degradation in vivo. A correspond-
ing reduction in the severity of the resulting OA-like joint lesions
based on histological evaluation was also observed.
The hydroxymethyl substituent plays a necessary role in
pharmacologic potency and in suppressing overall metabolism.
However, in the latter it also poses a risk if oxidative metabolism
results in the carboxylic acid metabolite, a species we were
intending to avoid. In both rat and cynomolgus monkey PK
studies, the carboxylic acid metabolite of 43a (45) was present at
very low levels, and the metabolite concentrations and half-life
appeared to be dependent on the parent, Figure 4. As anticipated,
We have shown that the carboxylic acid functional group found
in previously disclosed highly selective MMP-13 inhibitors is not
required to achieve excellent target potency, selectivity, and in
vivo efficacy. The strategy to optimize nonacidic analogs of
compound 6 has successfully identified inhibitor 43a lacking the
preclinical renal toxicity observed with previous lead molecules.
Critical to achieving this milestone was the discovery that
replacing the cyclohexane ring in initial leads with a dioxane ring
could have a profound impact on metabolic susceptibility and
overall pharmacokinetic properties. The use of a substituted
dioxane ring to reduce lipophilicity and thus improve metabolic
stability is an important tool medicinal chemists should consider
when challenged by poor pharmacokinetics.
EXPERIMENTAL SECTION
■
General Methods. All reagents and solvents were used as purchased
without further purification. The purity of the final compounds was
characterized by high-performance liquid chromatography (HPLC)
using a gradient elution (C18 column, acetonitrile/water, 5/95−75/5, 5
min, 0.1% trifluoroacetic acid) and UV-detection (254 nM). The purity
of all final compounds was 95% or greater. Proton (1H) NMR chemical
shifts are referenced to a residual solvent peak.
N-tert-Butyl-2-chloro-6-methylisonicotinamide (9). Oxalyl
chloride (95 mL, 1.10 mol) was added to a solution of 8 (95 g, 0.55
mol) in dichloromethane (950 mL, anhydrous). The mixture was cooled
in an ice/water bath, and DMF (2.0 mL) was slowly added. When gas
evolution subsided, the ice/water bath was removed, and the mixture
was allowed to reach rt and stir for 15 h. The solution was then
concentrated in vacuo and azeotropically distilled twice from toluene
(200 mL) at reduced pressure. The residue was taken into dichloro-
methane (800 mL, anhydrous) and cooled in an ice/water bath, and a
solution of tert-butylamine (175 mL, 1.7 mol) in dichloromethane (300
mL, anhydrous) was added dropwise. The mixture was allowed to reach
rt and stir for 15 h. The reaction mixture was diluted with water (250
mL). The organic layer was washed with water (200 mL) followed by
brine (200 mL), dried (MgSO4), filtered, and concentrated to afford 120
g (96%) of 9 as a beige solid. 1H NMR (400 MHz, DMSO-d6) δ 7.39 (s,
1 H), 7.38 (s, 1 H), 6.05 (s, 1 H), 2.59 (s, 3 H), 1.5 (s, 9 H).
Figure 4. Mean plasma concentration−time profiles of 43a and
carboxylic acid metabolite 45 after oral administration of 43a (1.0 mg/
kg) to female cynomolgus monkeys (N = 3).
2-Chloro-6-methylisonicotinonitrile (10). POCl3 (572 mL, 6.26
mol) was added to 9 (120 g, 0.53 mol) in toluene (2.5 L, anhydrous).
The resulting mixture was refluxed 15 h, cooled to ambient temperature,
and concentrated in vacuo. The residue was dissolved in dichloro-
methane (1.0 L) and aqueous saturated sodium bicarbonate (500 mL)
was carefully added. Solid sodium bicarbonate was then added
portionwise until the aqueous layer was basic. The organic layer was
washed with brine (300 mL), dried (MgSO4), filtered, and concentrated
carboxylic acid 45 has a lower in vitro Ki against human MMP-13
(0.2 nM) than 43a (1.5 nM) and is thus presumed to be
pharmacologically active. In cynomolgus monkey PK studies, the
AUC ratio of parent/metabolite after oral dosing was 1460 at 1
mg/kg and 107 at 100 mg/kg. In in vitro incubations of 43a in
pooled human hepatocytes, at least six primary metabolites were
identified. In addition to 45, the metabolites resulted from
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1
to afford 76 g (94%) of 10 as a pale tan solid. H NMR (400 MHz,
DMSO-d6) δ 7.41 (s, 1 H), 7.37 (s, 1 H), 2.61 (s, 3 H).
(+)-N-(3-Methoxybenzyl)-6-methyl-4-(2-((tetrahydro-2H-
pyran-2-yl)methyl)-2H-tetrazol-5-yl)picolinamide (14d). Isola-
tion of the second eluting isomer from above afforded 260 mg (41%)
of 14d as an oil. Chiralcel OD-H analytical column (4.6 mm × 250 mm;
20/80, ethanol/CO2), 9.99 min, > 99% ee; [α]2D1°C = 19.0° (c = 9.8,
DMF). 1H NMR (400 MHz, DMSO-d6) δ 9.23 (m, 1 H), 8.40 (s, 1 H),
8.05 (s, 1 H), 7.23−7.19 (m, 1 H), 6.89−6.87 (m, 2 H), 6.80−6.77 (m, 1
H), 4.79−4.74 (m, 2 H), 4.48 (d, J = 6.0 Hz, 2 H), 3.93−3.75 (m, 3 H),
3.70 (s, 3 H), 2.65 (s, 3 H), 1.80−1.25 (m, 6 H). MS (ES+) m/z 423 (M
+ H).
rac-N-(3-Methoxybenzyl)-6-methyl-4-(2-((tetrahydro-2H-
pyran-3-yl)methyl)-2H-tetrazol-5-yl)picolinamide (14e). A mix-
ture of 13 (79 mg, 0.23 mmol), 15 (77 mg, 0.25 mmol), and
triethylamine (0.40 mL) in DMA (0.1 mL) was stirred at 85 °C for 15 h.
The reaction mixture was purified by reverse phase preparative HPLC to
afford 62 mg (61%) of 14e as a solid. 1H NMR (400 MHz, DMSO-d6) δ
9.20 (br. s., 1 H), 8.08 (s, 1 H), 7.24 (t, J = 8.1 Hz, 1 H), 6.93 (br. s., 2 H),
6.82 (d, J = 7.7 Hz, 1 H), 4.75 (dq, J = 13.0, 6.8 Hz, 2 H), 4.51 (d, J = 6.2
Hz, 2 H), 3.73 (s, 5 H), 3.68 (br. s., 1 H), 3.41 (br. s., 1 H), 3.37 (d, J =
2.2 Hz, 1 H), 2.68 (s, 3 H), 2.28 (br. s., 1 H), 1.74 (br. s., 1 H), 1.62 (br.
s., 1 H), 1.50 (d, J = 8.8 Hz, 1 H), 1.35 (d, J = 9.5 Hz, 1 H). MS (ES+) m/
z 423 (M + H).
rac-4-(2-((1,4-Dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-N-(3-
methoxybenzyl)-6-methylpicolinamide (14f). Following condi-
tions analogous to those described for 14b, reaction of rac-(1,4-dioxan-
2-yl)methanol (142 mg, 1.2 mmol) afforded 250 mg (59%) of 14f as an
oil. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (t, J = 6.5 Hz, 1 H), 8.44 (s, 1
H), 8.10 (s, 1 H), 7.25 (t, J = 8.1 Hz, 1 H), 6.95−6.90 (m, 2 H), 6.82 (dd,
J = 8.2, 2.6 Hz, 1 H), 4.96−4.85 (m, 2 H), 4.51 (d, J = 6.3 Hz, 2 H),
4.19−4.11 (m, 1 H), 3.92 (dd, J = 11.4, 2.4 Hz, 1 H), 3.74 (s, 5 H), 3.58−
3.40 (m, 3 H), 2.69 (s, 3 H). MS (ES+) m/z 425 (M + H).
2-Chloro-6-methyl-4-(2H-tetrazol-5-yl)pyridine hydrochlor-
ide (11). Triethylamine hydrochloride (72 g, 0.52 mol) and sodium
azide (34 g, 0.52 mol) were added to a solution of 10 (76 g, 0.50 mol) in
toluene (1.2 L, anhydrous). The mixture was refluxed for 4 h behind a
blast shield. CAUTION! Tetrazoles represent an explosion hazard at
elevated temperatures. Adequate blast shielding is recommended. After
cooling to rt, water (1.5 L) was added, and the layers were separated.
The toluene layer was washed with water (500 mL), and the combined
aqueous layers were acidified with 6 N HCl (100 mL). The resulting
precipitate was filtered and dried in vacuo (60 °C) to afford 105 g (91%)
of 11. 1H NMR (400 MHz, DMSO-d6) δ 7.92 (s, 1 H), 7.89 (s, 1 H),
2.79 (s, 3 H).
Methyl 6-Methyl-4-(2H-tetrazol-5-yl)picolinate (12). Triethyl-
amine (120 mL, 1.63 mol), 1,1′-bis(diphenylphosphino)ferrocene (8.0
g, 14 mmol), and Pd(OAc)2 (9.2 g, 40 mmol) were added to a solution
of 11 (88 g, 380 mmol) in methanol (1.5 L, anhydrous) in a pressure
reactor. The reactor was evacuated, purged with carbon monoxide gas
twice, and finally pressurized to 120 PSI and heated to 100 °C for 24 h
(190 PSI final pressure). CAUTION! Tetrazoles represent an explosion
hazard at elevated temperatures. Adequate blast shielding is recommended.
After cooling to rt, the mixture was filtered through Celite and
concentrated in vacuo. Water (700 mL) and aqueous saturated sodium
bicarbonate (200 mL) were added to dissolve all solids. HCl (3 N) was
added until a pH of 2 was reached. The resulting solid was filtered and
dried in vacuo (50 °C) to afford 78 g (94%) of 12 as a solid. 1H NMR
(400 MHz, DMSO-d6) δ 8.48 (s, 1 H), 8.15 (s, 1 H), 3.95 (s, 3 H), 2.67
(s, 3 H).
N-(3-Methoxybenzyl)-6-methyl-4-(2H-tetrazol-5-yl)-
picolinamide (13). A mixture of 12 (3.29 g, 15.0 mmol) and 3-
methoxybenzylamine (6.17 g, 45.0 mmol) was heated at 100 °C with
removal of methanol in a stream of nitrogen for 1 h. A solution of 1 N
HCl (50 mL) was added. The resulting precipitate was filtered and
washed with ethyl ether. The solid was suspended in a solution of 2.5 N
sodium hydroxide (30 mL)/water (50 mL) and extracted with EtOAc (2
× 50 mL). The aqueous layer was cooled in an ice bath and acidified to
pH 1 with concentrated HCl. The resulting precipitate was filtered and
dried to afford 4.69 g (96%) of 13 as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ 9.28 (t, J = 6.3 Hz, 1 H), 8.49 (d, J = 0.8 Hz, 1 H), 8.13 (d, J
= 1.2 Hz, 1 H), 7.24 (t, J = 8.1 Hz, 1 H), 6.94−6.90 (m, 2 H), 6.82 (dd, J
= 8.0, 2.1 Hz, 1 H), 4.51 (d, J = 6.3 Hz, 2 H), 3.73 (s, 3 H), 2.68 (s, 3 H).
MS (ES+) m/z 325 (M + H).
4-(2-(Cyclohexylmethyl)-2H-tetrazol-5-yl)-N-(3-methoxy-
benzyl)-6-methylpicolinamide (14b). Polymer supported triphe-
nylphosphine (698 mg, 1.5 mmol, 1.5 equiv), 13 (324 mg, 1.0 mmol),
and cyclohexylmethanol (137 mg, 1.2 mmol, 1.2 equiv) were suspended
in THF (16 mL). DBAD (345 mg, 1.5 mmol, 1.5 equiv) was added. The
mixture was allowed to stir for 18 h. The reaction mixture was filtered,
and the resin was washed with THF (20 mL). The filtrate was
concentrated. The crude product was purified by column chromatog-
raphy (heptane/EtOAc, 6/1; 4/1) to afford 248 mg (49%) of 14b an oil.
1H NMR (400 MHz, DMSO-d6) δ 9.29 (t, J = 6.4 Hz, 1 H), 8.43 (s, 1 H),
N-(3-Methoxybenzyl)-4-(2-(((trans)-4-(hydroxymethyl)-
cyclohexyl)methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide
(14g). Following conditions analogous to those described for 14b,
reaction of trans-cyclohexane-1,4-dimethanol (178 mg, 1.23 mmol)
1
afforded 102 mg (37%) of 14g as a white solid. H NMR (400 MHz,
DMSO-d6) δ 9.23 (m, 1 H), 8.39 (s, 1 H), 8.05 (s, 1 H), 7.12−7.28 (m, 1
H), 6.94−6.84 (m, 2 H), 6.82−6.72 (m, 1 H), 4.63 (d, J = 7.0 Hz, 2 H),
4.47 (d, J = 6.4 Hz, 2 H), 3.70 (s, 3 H), 3.21−3.08 (m, 2 H), 2.64 (s, 3 H),
2.04−1.81 (m, 1 H), 1.78−1.50 (m, 4 H), 1.43−1.16 (m, 2 H), 1.15−
0.71 (m, 4 H). MS (ES+) m/z 451 (M + H).
N-(3-Methoxybenzyl)-4-(2-(((cis)-4-(hydroxymethyl)-
cyclohexyl)methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide
(14h). Following conditions analogous to those described for 14b,
reaction of cis-cyclohexane-1,4-dimethanol (89 mg, 0.62 mmol) afforded
62 mg (45%) of 14h as an oil. 1H NMR (400 MHz, DMSO-d6) δ 9.26 (t,
J = 6.4 Hz, 1 H), 8.42 (d, J = 0.9 Hz, 1 H), 8.08 (d, J = 1.1 Hz, 1 H), 7.24
(t, J = 8.1 Hz, 1 H), 6.93−6.90 (m, 2 H), 6.82 (ddd, J = 8.2, 2.5, 0.9 Hz, 1
H), 4.76 (d, J = 7.7 Hz, 2 H), 4.51 (d, J = 6.4 Hz, 2 H), 4.37 (t, J = 5.4 Hz,
1 H), 3.73 (s, 3 H), 3.33 (dd, J = 6.4, 5.5 Hz, 2 H), 2.68 (s, 3 H), 2.31−
2.21 (m, 1 H), 1.65−1.27 (m, 9 H). MS (ES+) m/z 351 (M + H).
rac-N-(3-Methoxybenzyl)-4-(2-(((2S*,5R*)-5-(hydroxymeth-
yl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-6-methylpicolina-
mide (14i). Tosylate 16 (1.4 g, 4.3 mmol) was added to a mixture of 13
(1.0 g, 3.1 mmol) and triethylamine (0.4 g, 4.0 mmol) in anhydrous
DMA (1 mL). The mixture was stirred at 85 °C overnight and then was
purified by reverse phase preparative HPLC to afford 1.04 g (70%) of
14i as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.20 (t, J = 6.2
Hz, 1 H), 8.43 (s, 1 H), 8.08 (s, 1 H), 7.24 (t, J = 8.1 Hz, 1 H), 6.93 (br. s.,
2 H), 6.83 (d, J = 1.5 Hz, 1 H), 4.80−4.95 (m, 2 H), 4.65 (t, J = 5.7 Hz, 1
H), 4.52 (d, J = 6.2 Hz, 2 H), 4.05−4.15 (m, 1 H), 3.99 (dd, J = 11.3, 1.8
Hz, 1 H), 3.77 (d, J = 11.3 Hz, 1 H), 3.73 (s, 3 H), 3.30−3.50 (m, 4 H),
2.68 (s, 3 H). MS (ES+) m/z 455 (M + H).
8.09 (s, 1 H), 7.25 (t, J = 8.0 Hz, 1 H), 6.96−6.89 (m, 2 H), 6.82 (dd, J =
8.2, 2.4 Hz, 1 H), 4.67 (d, J = 7.0 Hz, 2 H), 4.51 (d, J = 6.3 Hz, 2 H), 3.74
(s, 3 H), 2.68 (s, 3 H), 2.08−1.99 (m, 1 H), 1.74−1.54 (m, 5 H), 1.29−
1.00 (m, 4 H). MS (ES+) m/z 421 (M + H).
(−)-N-(3-Methoxybenzyl)-6-methyl-4-(2-((tetrahydro-2H-
pyran-2-yl)methyl)-2H-tetrazol-5-yl)picolinamide (14c). Follow-
ing conditions analogous to those described for 14b, reaction of
tetrahydropyran-2-methanol (0.348 mL, 3.08 mmol) afforded the
racemic product, which was resolved by SFC (Chiralcel OD-H, 30 mm
× 250 mm, 25/75 ethanol/CO2, 70 mL/min). Isolation of the first
eluting isomer afforded 250 mg (39%) of 14c as an oil. Chiralcel OD-H
analytical column (4.6 mm × 250 mm; 20/80, ethanol/CO2), 8.06 min,
N-(3-Methoxybenzyl)-4-(2-(((trans)-4-hydroxycyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (14j). Following
conditions analogous to those described for 14b, reaction of 4-
(hydroxymethyl)cyclohexanol (614 mg, 4.7 mmol) afforded a mixture
of isomers, which was purified by reverse phase preparative HPLC
(acetonitrile/water, 35/65−55/45). Isolation of the first eluting isomer
afforded 298 mg (22%) of 14j. 1H NMR (400 MHz, DMSO-d6) δ 9.19
> 99% ee; [α]2D1°C = −22.7° (c = 8.3, DMF). H NMR (400 MHz,
1
DMSO-d6) δ 9.23 (m, 1 H), 8.40 (s, 1 H), 8.05 (s, 1 H), 7.23−7.19 (m, 1
H), 6.89−6.87 (m, 2 H), 6.80−6.77 (m, 1 H), 4.79−4.74 (m, 2 H), 4.48
(d, J = 6.0 Hz, 2 H), 3.93−3.75 (m, 3 H), 3.70 (s, 3 H), 2.65 (s, 3 H),
1.80−1.25 (m, 6 H). MS (ES+) m/z 423 (M + H).
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(t, J = 6.4 Hz, 1 H), 8.40 (s, 1 H), 8.05 (s, 1 H), 7.22 (t, J = 8.1 Hz, 1 H),
6.87−6.92 (m, 2 H), 6.77−6.82 (m, 1 H), 4.63 (d, J = 7.0 Hz, 2 H), 4.49
(d, J = 6.2 Hz, 2 H), 3.71 (s, 3 H), 2.65 (s, 3 H), 2.52 (s, 1 H), 1.89−1.98
(m, 1 H), 1.77−1.85 (m, 2 H), 1.53−1.61 (m, 2 H), 1.37 (d, J = 7.3 Hz, 2
H), 1.03−1.17 (m, 2 H). MS (ES+) m/z 437 (M + H).
N-(3-Methoxybenzyl)-4-(2-(((cis)-4-hydroxycyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (14k). Isolation
of the second eluting isomer from the reaction above afforded 520 mg
(39%) of 14k. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (t, J = 6.4 Hz, 1
H), 8.40 (s, 1 H), 8.05 (s, 1 H), 7.21 (t, J = 8.1 Hz, 1 H), 6.87−6.92 (m, 2
H), 6.79 (dd, J = 8.4, 1.5 Hz, 1 H), 4.65 (d, J = 7.0 Hz, 2 H), 4.48 (d, J =
6.6 Hz, 2 H), 4.30 (br. s., 1 H), 3.73 (br. s., 1 H), 3.71 (s, 3 H), 2.65 (s, 3
H), 2.52 (s, 1 H), 2.04 (br. s., 1 H), 1.54−1.63 (m, 2 H), 1.27−1.48 (m, 5
H). MS (ES+) m/z 437 (M + H).
N-(3-Methoxybenzyl)-4-(2-(((trans)-4-cyanocyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (19). Polymer
supported triphenylphosphine (500 mg, 1.07 mmol) was added to 18
(77 mg, 0.16 mmol) in a mixture of carbon tetrachloride (0.5 mL) and
DCE (4.5 mL), and the mixture was heated at 80 °C for 2 h. The
reaction mixture was filtered, and the resin was washed with DCE. The
filtrate was concentrated, and the residue was recrystallized from
methanol to afford 34 mg (48%) of 19 as a white solid. 1H NMR (400
MHz, DMSO-d6) δ 9.29 (t, J = 6.6 Hz, 1 H), 8.43 (s, 1 H), 8.09 (s, 1 H),
7.25 (t, J = 8.0 Hz, 1 H), 6.90−6.94 (m, 2 H), 6.83 (dd, J = 8.1, 2.3 Hz, 1
H), 4.69 (d, J = 6.8 Hz, 2 H), 4.51 (d, J = 6.5 Hz, 2 H), 3.74 (s, 3 H), 2.68
(s, 3 H), 2.59−2.67 (m, 1 H), 1.98−2.13 (m, 3 H), 1.65 (d, J = 13.1 Hz, 2
H), 1.45−1.56 (m, 2 H), 1.07−1.19 (m, 2 H). MS (ES+) m/z 446 (M +
H).
tert-Butyl (trans)-4-((5-(2-((3-Methoxybenzyl)carbamoyl)-6-
m e t h y l p y r i d i n - 4 - y l ) - 2 H - t e t r a z o l - 2 - y l ) m e t h y l ) -
cyclohexylcarbamate (21). Following conditions analogous to those
described for 14b, reaction of tert-butyl trans-(4-hydroxymethyl)-
cyclohexylcarbamate 20 (1.38 g, 6.0 mmol) provided a crude product,
which was purified by silica column chromatography (CH2Cl2/
methanol, 100/1; 100/2) to afford 1.824 g (68%) of 21 as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (t, J = 6.3 Hz, 1 H), 8.40
(d, J = 0.8 Hz, 1 H), 8.05 (d, J = 1.1 Hz, 1 H), 7.21 (t, J = 8.1 Hz, 1 H),
6.90−6.86 (m, 2 H), 6.79 (dd, J = 8.2, 1.9 Hz, 1 H), 6.68 (d, J = 7.8 Hz, 1
H), 4.63 (d, J = 7.0 Hz, 2 H), 4.48 (d, J = 6.3 Hz, 2 H), 3.70 (s, 3 H), 3.13
(br. s., 1 H), 2.64 (s, 3 H), 1.90 (br. s., 1 H), 1.77−1.71 (m, 2 H), 1.59−
1.53 (m, 2 H), 1.33 (s, 9 H), 1.14−1.06 (m, 4 H). MS (ES+) m/z 536
(M + H).
N-(3-Methoxybenzyl)-4-(2-(((trans)-4-aminocyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (22). Trifluoro-
acetic acid (5.05 mL, 68.0 mmol) was added to a solution of 21 (1.82 g,
3.40 mmol) in dichloromethane (20 mL). The mixture was allowed to
stir for 2 h and was then concentrated. The residue was dissolved in
dichloromethane (150 mL) and washed with 1 N NaOH solution (2 ×
40 mL). The organic layer was dried (Na2SO4) and concentrated. The
residue was triturated with heptane and filtered to afford 1.41 g (95%) of
22 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (t, J = 6.3 Hz,
1 H), 8.39 (d, J = 0.8 Hz, 1 H), 8.05 (d, J = 1.2 Hz, 1 H), 7.21 (t, J = 8.1
Hz, 1 H), 6.90−6.86 (m, 2 H), 6.79 (dd, J = 8.2, 1.9 Hz, 1 H), 4.62 (d, J =
7.1 Hz, 2 H), 4.48 (d, J = 6.3 Hz, 2 H), 3.70 (s, 3 H), 2.64 (s, 3 H), 2.45−
2.37 (m, 1 H), 1.96−1.84 (m, 1 H), 1.71 (d, J = 12.0 Hz, 2 H), 1.53 (d, J
= 12.2 Hz, 2 H), 1.37 (br. s., 2 H), 1.07 (q, J = 12.8 Hz, 2 H), 1.01−0.88
(m, 2 H). MS (ES+) m/z 436 (M + H).
rac-(Tetrahydro-2H-pyran-3-yl)methyl 4-Nitrobenzenesulfo-
nate (15). 4-Nitrobenzene-1-sulfonyl chloride (0.62 g, 2.8 mmol) and
pyridine (253 mg, 3.20 mmol) were added to a solution of rac-
(tetrahydro-2H-pyran-3-yl)methanol (0.31 g, 2.67 mmol) in dichloro-
methane (5 mL), and the mixture was stirred at rt for 15 h. The reaction
mixture was diluted with dichloromethane (10 mL) and washed with
water (2 × 10 mL) and brine (10 mL), dried over sodium sulfate,
filtered, and concentrated. The residue was purified on silica gel column
chromatography (heptane/EtOAc, 100/0−50/50) to afford 0.65 g
(81%) of 15 as an oil. MS (ES+) m/z 302 (M + H).
rac-((2R*,5R*)-5-(Hydroxymethyl)-1,4-dioxan-2-yl)methyl 4-
Methylbenzenesulfonate (rac-16). trans-2,5-Bis(hydroxymethyl)-
1,4-dioxane (342.9 g, 2.31 mol),20 p-toluene sulfonyl chloride (441.0 g,
2.31 mol), triethylamine (471.5 g, 4.66 mol), and dichloromethane (3
L) were combined in a 5 L round-bottomed flask equipped with a
mechanical stirrer under nitrogen. The mixture was maintained at 35 °C
for 30 min with water bath cooling (CAUTION! Exothermic reaction)
and then allowed to stir at rt overnight. The mixture was diluted with 3 N
HCl (1 L), and the resulting solids were filtered. The organic phase of
the filtrate was separated, washed with 3 N HCl (1 L) followed by
saturated ammonium chloride solution (500 mL), dried (MgSO4), and
concentrated to afford 338.3 g (48%) of rac-16 as a white solid. 1H NMR
(400 MHz, CDCl3) δ 7.77 (d, J = 8.1 Hz, 2 H), 7.33 (d, J = 8.1 Hz, 2 H),
3.90−4.02 (m, 2 H), 3.37−3.86 (m, 9 H), 2.43 (s, 3 H). MS (ES+) m/z
303 (M + H).
Chiral Resolution of rac-16. Chromatographic separation of rac-
16 by chiral SFC (AD-H; 30% methanol/dichloromethane, 10/1; 200
mL/min) was performed. Isolation of the first eluting isomer afforded
(2R,5R)-16 (>99% ee) as a white solid. Isolation of the second eluting
isomer afforded (2S,5S)-16 (>99% ee) as a white solid.
N-(3-Methoxybenzyl)-6-methyl-4-(2-(((trans)-4-
(methylsulfonamido)cyclohexyl)methyl)-2H-tetrazol-5-yl)-
picolinamide (23a). Triethylamine (0.096 mL, 0.690 mmol) and
methane sulfonyl chloride (0.021 mL, 0.276 mmol) were added to a
solution of 22 (100.0 mg, 0.230 mmol) in dichloromethane (2 mL). The
mixture was stirred for 2 h at rt and then concentrated. The residue was
purified by reverse phase preparative HPLC to afford 28 mg (24%) of
23a as a solid. 1H NMR (400 MHz, DMSO-d6) δ 9.31−9.04 (m, 1 H),
8.41 (s, 1 H), 8.05 (s, 1 H), 7.29−7.12 (m, 1 H), 6.93 (s, 1 H), 6.90 (m, 2
H), 6.82−6.77 (m, 1 H), 4.64 (d, J = 7.3 Hz, 2 H), 4.49 (d, J = 5.9 Hz, 2
H), 3.71 (s, 3 H), 2.86 (s, 3 H), 2.65 (s, 3 H), 2.04−1.78 (m, 3 H), 1.69−
1.49 (m, 2 H), 1.32−0.95 (m, 4 H). MS (ES+) m/z 514 (M + H).
N-(3-Methoxybenzyl)-4-(2-(((trans)-4-acetamidocyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (23b). Acetic
anhydride (0.033 mL, 0.344 mmol) and silica-bound DMAP (833.0
mg, 0.575 mmol, 0.69 mmol/g loading) were added to a solution of 22
(50.1 mg, 0.115 mmol) in dichloromethane (2 mL). The reaction
mixture was stirred overnight at rt, filtered, and washed with DMF. The
filtrate was concentrated, and the residue was purified by reverse phase
preparative HPLC to afford 22.4 mg (41%) of 23b. 1H NMR (400 MHz,
DMSO-d6) δ 9.26−9.09 (m, 1 H), 8.41 (s, 1 H), 8.05 (s, 1 H), 7.63 (s, 1
H), 7.30−7.13 (m, 1 H), 6.98−6.86 (m, 2 H), 6.83−6.75 (m, 1 H), 4.64
(d, J = 7.3 Hz, 2 H), 4.49 (d, J = 5.9 Hz, 2 H), 3.71 (s, 3 H), 2.65 (s, 3 H),
2.06−1.89 (m, 1 H), 1.81−1.74 (m, 3 H), 1.73 (s, 3 H), 1.60 (m, 2 H),
1.28−0.99 (m, 4 H). MS (ES+) m/z 478 (M + H).
(trans)-Methyl 4-((5-(2-((3-Methoxybenzyl)carbamoyl)-6-
m e t h y l p y r i d i n - 4 - y l ) - 2 H - t e t r a z o l - 2 - y l ) m e t h y l ) -
cyclohexanecarboxylate (17). Following conditions analogous to
those described for 14b, reaction of methyl trans-4-(hydroxymethyl)-
cyclohexanecarboxylate (2.12 g, 12.33 mmol) afforded 1.58 g (54%) of
17 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.26 (t, J = 6.44
Hz, 1 H), 8.42 (s, 1 H), 8.07 (s, 1 H), 7.24 (t, J = 8.19 Hz, 1 H), 6.97−
6.88 (m, 2 H), 6.86−6.77 (m, 1 H), 4.67 (d, J = 6.98 Hz, 2 H), 4.51 (d, J
= 6.18 Hz, 2 H), 3.73 (s, 3 H), 3.57 (s, 3 H), 2.67 (s, 3 H), 2.31−2.19 (m,
1 H), 2.08−1.95 (m, 1 H), 1.95−1.84 (m, 2 H), 1.70−1.58 (m, 2 H),
1.39−1.25 (m, 2 H), 1.21−1.06 (m, 2 H). MS (ES+) m/z 479 (M + H).
N-(3-Methoxybenzyl)-4-(2-(((trans)-4-carbamoylcyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (18). Forma-
mide (120 mg) was added to a solution of 17 (100 mg, 0.2 mmol) in
anhydrous tetrahydrofuran (1 mL) and the mixture was heated to reflux.
A 25% sodium methoxide solution in methanol (120 mg) was added,
and the reaction was stirred at reflux for 2 h. The reaction mixture was
allowed to cool to rt and then diluted with methanol (2 mL). The
resulting precipitate was filtered and washed with a small amount of
methanol, and the solid was dried in a vacuum desiccator to afford 78 mg
(85%) of 18 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (t, J
= 6.6 Hz, 1 H), 8.4 (s, 1 H), 8.05 (s, 1 H), 7.21 (t, J = 8.2 Hz, 1 H), 7.15
(s, 1 H), 6.88 (m, 2 H), 6.79 (d, J = 8.1 Hz, 1 H), 6.61 (s, 1 H), 4.62 (d, J
= 7.4, 2 H), 4.46 (d, J = 7.4, 2 H), 3.70 (s, 3 H), 2.62 (s, 3 H), 2.02−1.90
(m, 2 H), 1.68, (d, J = 13.6, 2 H), 1.60 (d, J = 13.1, 2 H), 1.35−1.20 (m, 2
H), 1.15−1.00 (m, 2 H). MS (ES+) m/z 464 (M + H).
tert-Butyl (cis)-4-((5-(2-((3-Methoxybenzyl)carbamoyl)-6-
m e t h y l p y r i d i n - 4 - y l ) - 2 H - t e t r a z o l - 2 - y l ) m e t h y l ) -
cyclohexylcarbamate (25). Following conditions analogous to those
J
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Article
described for 14b, reaction of tert-butyl cis-(4-hydroxymethyl)-
cyclohexylcarbamate 24 (1.41 g, 6.17 mmol) provided a crude product,
which was purified by reverse phase preparative HPLC to afford 1.65 g
(50%) of 25 as a solid. MS (ES+) m/z 480 (M − C4H8).
N-(3-Methoxybenzyl)-6-methyl-4-(2-((1-(methylsulfonyl)-
piperidin-4-yl)methyl)-2H-tetrazol-5-yl)picolinamide (31b).
Compound 31b was prepared analogous to 31a employing methane
sulfonyl chloride. Yield 55%. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s,
1 H), 8.41 (s, 1 H), 8.05 (s, 1 H), 7.27−7.15 (m, 1 H), 6.95−6.86 (m, 2
H), 6.83−6.73 (m, 1 H), 4.76 (d, J = 7.3 Hz, 2 H), 4.49 (d, J = 6.6 Hz, 2
H), 3.71 (s, 3 H), 3.60−3.47 (m, 2 H), 2.81 (s, 3 H), 2.65 (s, 3 H), 2.27−
2.07 (m, 1 H), 1.72−1.61 (m, 2 H), 1.48−1.24 (m, 4 H). MS (ES+) m/z
500 (M + H).
Methyl 4-(2-(((trans)-4-(hydroxymethyl)cyclohexyl)methyl)-
2H-tetrazol-5-yl)-6-methylpicolinate (32). DBAD (4.6 g, 20.0
mmol) was added to a mixture of 13 (2.19 g, 10.0 mmol), polymer
supported triphenylphosphine (9.3 g, 20.0 mmol), and trans-cyclo-
hexane-1,4-dimethanol (2.88 g, 20.0 mmol) in THF (200 mL) cooled to
0 °C. The mixture was allowed to warm to rt. After 18 h, the reaction
mixture was filtered, and the resin was washed with THF (100 mL). The
filtrate was concentrated. The residue was dissolved in EtOAc (200 mL)
and washed with water (50 mL). The organic layer was dried (Na2SO4)
and concentrated. The crude product was purified by silica column
chromatography (CH2Cl2/methanol, 100/1−100/4) to afford 2.3 g
(67%) of 32 as an oil. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 0.8
Hz, 1 H), 8.12 (d, J = 1.1 Hz, 1 H), 4.67 (d, J = 7.0 Hz, 2 H), 4.35 (t, J =
5.3 Hz, 1 H), 3.93 (s, 3 H), 3.19 (t, J = 5.8 Hz, 2 H), 2.66 (s, 3 H), 2.04−
1.90 (m, 1 H), 1.74 (d, J = 10.6 Hz, 2 H), 1.61 (d, J = 10.6 Hz, 2 H),
1.37−1.23 (m, 1 H), 1.08 (dq, J = 12.6, 2.7 Hz, 2 H), 0.87 (dq, J = 12.8,
3.1 Hz, 2 H). MS (ES+) m/z 346 (M + H).
N-(3-Methoxybenzyl)-4-(2-(((cis)-4-aminocyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (26). Trifluoro-
acetic acid was added to a solution of 25 (1.65 g, 3.1 mmol) in
dichloromethane, and the mixture was allowed to stir for 30 min at rt.
The reaction mixture was concentrated. The residue was dissolved in
dichloromethane, and MP-carbonate resin was added. The mixture was
agitated for 1 h, filtered, and washed with dichloromethane and
methanol. The filtrate was concentrated to afford 1.09 g (81%) of 26 as a
solid. 1H NMR (400 MHz, DMSO-d6) δ 9.28−9.19 (m, 1 H), 8.41 (s, 1
H), 8.09−8.03 (m, 1 H), 7.93 (br. s., 2 H), 7.27−7.17 (m, 1 H), 6.93−
6.85 (m, 2 H), 6.84−6.76 (m, 1 H), 4.72 (d, J = 7.3 Hz, 2 H), 4.48 (d, J =
6.4 Hz, 2 H), 3.70 (s, 3 H), 3.28−3.18 (m, 1 H), 2.65 (s, 3 H), 2.31−2.17
(m, 1 H), 1.86−1.33 (m, 8 H). MS (ES+) m/z 436 (M + H).
N - ( 3 - M e t h o x y b e n z y l ) - 6 - m e t h y l - 4 - ( 2 - ( ( ( c i s ) - 4 -
(methylsulfonamido)cyclohexyl)methyl)-2H-tetrazol-5-yl)-
picolinamide (27a). Compound 27a was prepared analogous to 23a
1
from compound 26. Yield 57%. H NMR (400 MHz, DMSO-d6) δ
9.25−9.12 (m, 1 H), 8.41 (s, 1 H), 8.05 (s, 1 H), 7.28−7.13 (m, 1 H),
6.97−6.84 (m, 3 H), 6.84−6.73 (m, 1 H), 4.67 (d, J = 6.6 Hz, 2 H), 4.49
(d, J = 6.6 Hz, 2 H), 3.71 (s, 3 H), 3.48−3.36 (m, 1 H), 2.87 (s, 3 H), 2.65
(s, 3 H), 2.22−1.99 (m, 1 H), 1.79−1.29 (m, 4 H). MS (ES+) m/z 514
(M + H).
N-(3-Methoxybenzyl)-4-(2-(((cis)-4-acetamidocyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (27b). Com-
pound 27b was prepared analogous to 23b from compound 26. Yield
49%. 1H NMR (400 MHz, DMSO-d6) δ 9.23−9.14 (m, 1 H), 8.41 (s, 1
H), 8.05 (s, 1 H), 7.68−7.60 (m, 1 H), 7.25−7.17 (m, 1 H), 6.94−6.86
(m, 2 H), 6.82−6.76 (m, 1 H), 4.68 (d, J = 7.3 Hz, 2 H), 4.49 (d, J = 6.6
Hz, 2 H), 3.82−3.74 (m, 1 H), 3.71 (s, 3 H), 3.31−3.23 (m, 1 H), 2.69−
2.62 (m, 3 H), 2.21−2.07 (m, 1 H), 1.80 (s, 3 H), 1.66−1.31 (m, 3 H).
MS (ES+) m/z 478 (M + H).
tert-Butyl 4-((5-(2-((3-methoxybenzyl)carbamoyl)-6-methyl-
pyridin-4-yl)-2H-tetrazol-2-yl)methyl)piperidine-1-carboxylate
(29). A mixture of 28 (1.00 g, 3.10 mmol), tert-butyl 4-(hydroxymethyl)
piperidine-1-carboxylate (0.81 g, 3.8 mmol), and polymer supported-
triphenylphosphine (2.90 g, 6.21 mmol) were suspended in anhydrous
THF (32 mL). The mixture was cooled to 0 °C in an ice bath for 15 min,
and then DBAD (1.11 g, 4.82 mmol) was added. The mixture was
allowed to warm to rt while stirring over 15 h. The reaction mixture was
filtered, and the filtrate was concentrated. The residue was purified by
silica gel chromatography (20 g, 0−50% gradient EtOAc/heptane) to
afford 1.52 g (94%) of 29 as a white solid. 1H NMR (400 MHz, DMSO-
d6) δ 9.22 (t, J = 8.0 Hz, 1 H), 8.40 (s, 1 H), 8.08 (s, 1 H), 7.22 (t, J = 8.2
Hz, 1 H), 6.90 (m, 2 H), 6.80 (m, 1 H), 4.78 (d, J = 7.0 Hz, 2 H), 4.50 (d,
J = 7.0 Hz, 2 H), 4.00−3.90 (m, 2 H), 3.75 (s, 3 H), 2.68 (s, 3 H), 2.25−
2.15 (m, 1 H), 1.60−1.50 (m, 2 H), 1.38 (s, 9 H), 1.20−1.10 (m, 2 H).
MS (ES+) m/z 544 (M + Na).
N-(3-Methoxybenzyl)-6-methyl-4-(2-(piperidin-4-ylmethyl)-
2H-tetrazol-5-yl)picolinamide (30). Trifluoroacetic acid (6 mL) was
added to a solution of 29 (1.51 g, 2.89 mmol) in dichloromethane (20
mL). The reaction mixture was stirred for 1 h at rt and was then
concentrated. The residue was purified by reverse phase preparative
HPLC to afford 0.83 g (56%) of 30 as the trifluoroacetate salt. MS (ES+)
m/z 422 (M + H).
N-(3-Methoxybenzyl)-4-(2-((1-acetylpiperidin-4-yl)methyl)-
2H-tetrazol-5-yl)-6-methylpicolinamide (31a). Acetic anhydride
(0.034 mL, 0.356 mmol) and silica-bound DMAP (862.0 mg, 0.595
mmol, 0.69 mmol/g loading) were added to a solution of 30 (50.2 mg,
0.119 mmol) in dichloromethane (2 mL), and the mixture was agitated
overnight at rt. The reaction mixture was filtered, and the resin was
washed with DMF. The filtrate was concentrated, and the residue was
purified by reverse phase preparative HPLC to afford 24.5 mg (44%) of
31a as a solid. 1H NMR (400 MHz, DMSO-d6) δ 9.27−9.11 (m, 1 H),
8.41 (s, 1 H), 8.05 (s, 1 H), 7.28−7.14 (m, 1 H), 6.95−6.86 (m, 2 H),
6.80 (m, 1 H), 4.71 (d, J = 7.3 Hz, 2 H), 4.49 (d, J = 6.6 Hz, 2 H), 3.71 (s,
3 H), 2.65 (s, 3 H), 2.57−2.44 (m, 2 H), 2.37−2.16 (m, 1 H), 1.94 (s, 3
H), 1.57 (m, 2 H), 1.35−0.98 (m, 4 H). MS (ES+) m/z 464 (M + H).
4-(2-(((trans)-4-(Hydroxymethyl)cyclohexyl)methyl)-2H-tet-
razol-5-yl)-6-methylpicolinic Acid (33). Lithium hydroxide (479
mg, 20.0 mmol) was added to a solution of 32 (2.3 g, 6.68 mmol)
dissolved in a mixture of THF/water (40 mL, 1/1), and the mixture was
stirred at rt for 3 h. The solvent was removed in vacuo, and the residue
was diluted with water (20 mL). The solution was made acidic with 1 N
HCl solution (20 mL), and the resulting precipitate was filtered to afford
1.03 g (47%) of 33 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
13.36 (br, 1 H), 8.40 (d, J = 0.8 Hz, 1 H), 8.09 (d, J = 1.1 Hz, 1 H), 4.66
(d, J = 7.1 Hz, 2 H), 4.35 (br. s., 1 H), 3.19 (d, J = 6.2 Hz, 2 H), 2.65 (s, 3
H), 2.03−1.90 (m, 1 H), 1.74 (d, J = 10.5 Hz, 2 H), 1.61 (d, J = 10.9, 2
H), 1.35−1.25 (m, 1 H), 1.08 (dq, J = 12.6, 2.8 Hz, 2 H), 0.87 (dq, J =
12.8, 3.2 Hz, 2 H). MS (ES+) m/z 332 (M + H).
General Procedure for Synthesis of Amides 34a−l. A mixture of
33 (33 mg, 0.10 mmol) and 1-hydroxybenzotriazole (18.9 mg, 0.14
mmol) in DMF (0.5 mL) was shaken for 15 min. N-Methylmorpholine
(44 μL, 0.40 mmol) was added followed by the corresponding
benzylamine (0.12 mmol) in DMF (0.5 mL). 1-Ethyl-3-(3′-
dimethylaminopropyl)carbodiimide hydrochloride (26.8 mg, 0.14
mmol) was then added, and the mixture was shaken for 18 h. The
reaction mixture was purified by reverse phase preparative HPLC to
afford the title compound.
N-Benzyl-4-(2-(((trans)-4-(hydroxymethyl)cyclohexyl)methyl)-2H-
tetrazol-5-yl)-6-methylpicolinamide (34a). 76% yield. 1H NMR (400
MHz, DMSO-d6) δ 9.26 (t, J = 6.4 Hz, 1 H), 8.39 (s, 1 H), 8.04 (s, 1 H),
7.18−7.35 (m, 5 H), 4.62 (d, J = 7.0, 2 H), 4.50 (d, J = 6.2 Hz, 2 H), 4.32
(t, J = 5.3 Hz, 1 H), 3.14 (t, J = 5.9 Hz, 2 H), 2.63 (s, 3 H), 1.99−1.86 (m,
1 H), 1.70 (d, J = 11.7 Hz, 2 H), 1.57 (d, J = 12.1 Hz, 2 H), 1.32−1.20
(m, l H). 1.03 (q, J = 12.0 Hz, 2 H), 0.83 (q, J = 12.0 Hz, 2 H). MS (ES+)
m/z 421 (M + H).
N-(4-Fluorobenzyl)-4-(2-(((trans)-4-(hydroxymethyl)cyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34b). 52% yield. 1H
NMR (400 MHz, DMSO-d6) δ 9.35 (t, J = 6.4 Hz, 1 H), 8.43 (s, 1 H),
8.09 (s, 1 H), 7.39 (dd, J = 8.4, 5.7 Hz, 2 H), 7.16 (t, J = 8.9 Hz, 2 H), 4.67
(d, J = 7.0 Hz, 2 H), 4.52 (d, J = 6.3 Hz, 2 H), 4.37 (t, J = 5.3 Hz, 1 H),
3.19 (t, J = 5.7 Hz, 2 H), 2.68 (s, 3 H), 1.91−2.02 (m, 1 H), 1.71−1.78
(m, 2 H), 1.58−1.64 (m, 2 H), 1.24−1.37 (m, 1 H), 1.02−1.14 (m, 2 H),
0.81−0.94 (m, 2 H). MS (ES+) m/z 439 (M + H).
N-(3-Fluorobenzyl)-4-(2-(((trans)-4-(hydroxymethyl)cyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34c). 20% yield. 1H
NMR (400 MHz, DMSO-d6) δ 9.37 (t, J = 6.4 Hz, 1 H), 8.43 (d, J = 0.9
Hz, 1 H), 8.09 (d, J = 1.1 Hz, 1 H), 7.40−7.34 (m, 1 H), 7.21−7.13 (m, 2
H), 7.07 (dt, J = 8.5, 2.4 Hz, 1 H), 4.66 (d, J = 6.9 Hz, 2 H), 4.55 (d, J =
6.4 Hz, 2 H), 4.35 (t, J = 5.3 Hz, 1 H), 3.19 (t, J = 5.8 Hz, 2 H), 2.66 (s, 3
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H), 2.04−1.91 (m, 1 H), 1.74 (d, J = 11.0 Hz, 2 H), 1.62 (d, J = 12.0 Hz,
2 H), 1.35−1.25 (m, 1 H), 1.08 (q, J = 13.2 Hz, 2 H), 0.87 (q, J = 11.2
Hz, 2 H). MS (ES+) m/z 439 (M + H).
(m, 4 H), 1.54−1.39 (m, 1 H), 1.19−1.05 (m, 2 H), 1.04−0.91 (m, 2 H).
99%. MS (ES+) m/z 469 (M + H).
6-Chloro-2-methylpyrimidin-4-ol (36). 4,6-Dichloro-2-methyl-
pyrimidine 35 (475 g, 2.91 mol) was added portionwise to a solution of
13 M sulfuric acid (1.2 L, 15.6 mol) at 0 °C. The resulting mixture was
allowed to warm to rt over 1.5 h. The solution was poured into a well
stirred mixture of ice and 6 N NaOH (3.6 L, 21.6 mol). The resulting
solids were then collected by vacuum filtration, washed with warm water
(3 × 1 L), and dried at 35 °C under high vacuum to provide 393.4 g
(93%) of 36 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.81
(br. s, 1 H), 6.30 (s, 1 H), 2.26 (s, 3 H). MS (ES+) m/z 145 (M + H).
Methyl 6-Hydroxy-2-methylpyrimidine-4-carboxylate (37). A
solution of 36 (36.7 g, 254 mmol), [1,1′-bis(diphenylphosphino)-
ferrocene]palladium(II) dichloride (10.4 g, 12.7 mmol), and
diisopropylethylamine (49.2 g, 381 mmol) in methanol (254 mL) was
heated at 85 °C in the presence of carbon monoxide (50 psi) for 24 h.
The reaction mixture was allowed to cool to rt. The resulting solids were
collected by vacuum filtration, rinsed with methanol (100 mL) followed
by diethyl ether (2 × 100 mL), and dried under vacuum to provide 38.7
g (91%) of 37 as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 12.79
(br. s, 1 H), 6.68 (s, 1 H), 3.79 (s, 3 H), 2.29 (s, 3 H). MS (ES+) m/z 169
(M + H).
N-(3-Chlorobenzyl)-4-(2-(((trans)-4-(hydroxymethyl)cyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34d). 35% yield. 1H
NMR (400 MHz, DMSO-d6) δ 9.37 (t, J = 6.4 Hz, 1 H), 8.38 (s, 1 H),
8.05 (s, 1 H), 7.26−7.37 (m, 4 H), 4.62 (d, J = 7.0 Hz, 2 H), 4.49 (d, J =
6.6 Hz, 2 H), 4.33 (t, J = 5.3 Hz, 1 H), 3.14 (t, J = 5.7 Hz, 2 H), 2.64 (s, 3
H), 1.97−1.87 (m, 1 H), 1.70 (d, J = 10.6 Hz, 2 H), 1.57 (d, J = 11.3 Hz,
2 H), 1.31−1.21 (m, 1 H), 1.03 (q, J = 12.4 Hz, 2 H), 0.83 (q, J = 12.4
Hz, 2 H). MS (ES+) m/z 455 (M + H).
N-(3-Methylbenzyl)-4-(2-(((trans)-4-(hydroxymethyl)cyclohexyl)-
methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34e). 77% yield. 1H
NMR (400 MHz, DMSO-d6) δ 9.21 (t, J = 6.4 Hz, 1 H), 8.39 (s, 1 H),
8.04 (s, 1 H), 7.17 (t, J = 7.5 Hz, 1 H), 7.07−7.13 (m, 2 H), 7.02 (d, J =
7.3 Hz, 1 H), 4.62 (d, J = 7.0 Hz, 2 H), 4.46 (d, J = 6.2 Hz, 2 H), 4.32 (t, J
= 5.1 Hz, 1 H), 3.14 (t, J = 5.9 Hz, 2 H), 2.63 (s, 3 H), 2.24 (s, 3 H),
1.97−1.87 (m, 1 H), 1.70 (d, J = 11.0 Hz, 2 H), 1.57 (d, J = 11.3 Hz, 2
H), 1.30−1.20 (m, 1 H), 1.04 (q, J = 13.2 Hz, 2 H), 0.83 (q, J = 12.2 Hz,
2 H). MS (ES+) m/z 435 (M + H).
N-(3-(Trifluoromethyl)benzyl)-4-(2-(((trans)-4-(hydroxymethyl)-
cyclohexyl)methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34f).
1
77% yield. H NMR (400 MHz, DMSO-d6) δ 9.43 (t, J = 6.2 Hz, 1
Methyl 6-Chloro-2-methylpyrimidine-4-carboxylate (38). Ox-
alyl chloride (18.6 mL, 213 mmol) and DMA (3.5 mL, 44 mmol) were
added to a solution of 37 (30.0 g, 178 mmol) in dichloromethane (446
mL). The mixture was heated at reflux for 2 h. After cooling to rt, the
mixture was filtered. The filtrate was partially concentrated and purified
by silica gel chromatography (35/65, EtOAc/heptane) to provide 26.6 g
(80%) of 38 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s,
1 H), 3.88 (s, 3 H), 2.65 (s, 3 H). MS (ES+) m/z 187 (M + H).
Methyl 6-Cyano-2-methylpyrimidine-4-carboxylate (39). Tet-
raethylammonium cyanide (27.6 g, 177 mmol) and DABCO (3.60 g,
32.0 mmol) was added to a solution of 38 (30.0 g, 161 mmol) in
dichloromethane (460 mL), and the mixture was stirred for 30 min at rt.
The reaction mixture was washed with 1 N NaOH (3 × 100 mL), water
(2 × 100 mL), and brine (200 mL). The organic layer was dried
(Na2SO4), filtered, and concentrated. The crude product was purified by
silica gel chromatography (2/1, EtOAc/heptane) to provide 24.4 g
(86%) of 39 as a white crystalline solid. 1H NMR (400 MHz, DMSO-d6)
δ 8.38 (s, 1 H), 3.88 (s, 3 H), 2.75 (s, 3 H). MS (ES+) m/z 178 (M + H).
6-Cyano-N-(3-methoxybenzyl)-2-methylpyrimidine-4-car-
boxamide (40a). Diisopropylethylamine (25.5 g, 198 mmol) was
added to a solution of 3-methoxybenzylamine (9.68 g, 70.6 mmol) and
39 (10.0 g, 56.4 mmol) in DMA (113 mL), and the mixture was heated
at 100 °C for 2 h. After cooling to rt, the mixture was diluted with water
(250 mL) and extracted with EtOAc (3 × 250 mL). The combined
organic layers were washed with brine (250 mL), dried (NaSO4),
filtered, and concentrated. The crude product was purified by silica gel
chromatography (20/80, EtOAc/heptane) to provide 14.6 g (92%) of
40a as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.55 (t, J = 6.2
Hz, 1 H), 8.31 (s, 1 H), 7.19 (t, J = 8.1 Hz, 1 H), 6.74−6.89 (m, 3 H),
4.44 (d, J = 6.3 Hz, 2 H), 3.69 (s, 3 H), 2.75 (s, 3 H). MS (ES+) m/z 283
(M + H).
6-Cyano-N-(4-fluoro-3-methoxybenzyl)-2-methylpyrimi-
dine-4-carboxamide (40b). A mixture of 4-fluoro-3-methoxybenzyl-
amine hydrochloride (13.5 g, 70.6 mmol) and diisopropylethylamine
(29.2 g, 226 mmol) in methanol (56 mL) was stirred for 15 min at rt.
Compound 39 (10.0 g, 56.4 mmol) was added, and the reaction mixture
was heated at 35 °C for 1 h. The methanol was removed in vacuo, and
the residue was partitioned between EtOAc (500 mL) and 1 N HCl (500
mL). The aqueous layer was extracted with EtOAc (2 × 250 mL). The
combined organic layers were washed with saturated aqueous sodium
bicarbonate (2 × 250 mL) followed by brine (250 mL), dried (Na2SO4),
filtered, and concentrated. The crude product was purified by silica gel
chromatography (1/2, EtOAc/heptane) to provide 11.7 g (69%) of 40b
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.58 (t, J = 6.2 Hz, 1
H), 8.32 (s, 1 H), 7.04−7.19 (m, 2 H), 6.79−6.90 (m, 1 H), 4.44 (d, J =
6.2 Hz, 2 H), 3.78 (s, 3 H), 2.76 (s, 3 H). MS (ES+) m/z 301 (M + H).
N-(3-Methoxybenzyl)-2-methyl-6-(2H-tetrazol-5-yl)-
pyrimidine-4-carboxamide (41a). A suspension of 40a (14.2 g, 50.3
H), 8.37 (s, 1 H), 8.04 (s, 1 H), 7.69−7.48 (m, 4 H), 4.61 (d, J = 7.0 Hz, 2
H), 4.56 (d, J = 6.2 Hz, 2 H), 4.31 (t, J = 5.1 Hz, 1 H), 3.13 (t, J = 5.7 Hz,
2 H), 2.63 (s, 3 H), 1.98−1.85 (m, 1 H), 1.68 (d, J = 11.7 Hz, 2 H), 1.56
(d, J = 11.3 Hz, 2 H), 1.31−1.19 (m, 1 H), 1.02 (q, J = 12.8 Hz, 2 H),
0.82 (q, J = 12.4 Hz, 2 H). MS (ES+) m/z 489 (M + H).
N-(3,4-Difluorobenzyl)-4-(2-(((trans)-4-(hydroxymethyl)-
cyclohexyl)methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34g).
1
74% yield. H NMR (400 MHz, DMSO-d6) δ 9.36 (t, J = 6.4 Hz, 1
H), 8.38 (s, 1 H), 8.05 (s, 1 H), 7.39−7.30 (m, 2 H), 7.13−7.18 (m, 1
H), 4.62 (d, J = 7.0 Hz, 2 H), 4.47 (d, J = 6.6 Hz, 2 H), 4.32 (t, J = 5.3 Hz,
1 H), 3.14 (t, J = 5.9 Hz, 2 H), 2.64 (s, 3 H), 1.97−1.87 (m, 1 H), 1.70 (d,
J = 11.0 Hz, 2 H), 1.57 (d, J = 12.1 Hz, 2 H), 1.30−1.18 (m, 1 H), 1.03
(q, J = 12.8 Hz, 2 H), 0.83 (q, J = 11.3 Hz, 2 H). MS (ES+) m/z 457 (M
+ H).
N-(3-Chloro-4-fluorobenzyl)-4-(2-(((trans)-4-(hydroxymethyl)-
cyclohexyl)methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34h).
1
53% yield. H NMR (400 MHz, DMSO-d6) δ 9.40 (t, J = 8.4 Hz, 1
H), 8.42 (d, J = 0.9 Hz, 1 H), 8.09 (d, J = 1.2 Hz, 1 H), 7.56 (d, J = 7.7 Hz,
1 H), 7.39−7.35 (m, 2 H), 4.66 (d, J = 7.1 Hz, 2 H), 4.51 (d, J = 6.4 Hz, 2
H), 4.35 (t, J = 5.3 Hz, 1 H), 3.19 (t, J = 5.8 Hz, 2 H), 2.68 (s, 3 H), 2.03−
1.90 (m, 1 H), 1.74 (d, J = 10.6 Hz, 2 H), 1.61 (d, J = 10.7 Hz, 2 H),
1.35−1.24 (m,·1 H), 1.08 (dq J = 12.4, 2.4 Hz, 2 H), 0.87 (dq, J = 12.6,
3.0 Hz, 2 H). MS (ES+) m/z 473 (M + H).
N-(4-Fluoro-3-methylbenzyl)-4-(2-(((trans)-4-(hydroxymethyl)-
cyclohexyl)methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34i).
1
79% yield. H NMR (400 MHz, DMSO-d6) δ 9.25 (t, J = 6.2 Hz, 1
H), 8.38 (s, 1 H), 8.04 (s, 1 H), 7.21 (d, J = 7.3 Hz, 1 H), 7.17−7.13 (m, 1
H), 7.03 (t, J = 9.2 Hz, 1 H), 4.62 (d, J = 7.0 Hz, 2 H), 4.43 (d, J = 6.2 Hz,
2 H), 4.32 (t, J = 5.1 Hz, 1 H), 3.14 (t, J = 5.7 Hz, 2 H), 2.63 (s, 3 H), 2.17
(s, 3 H), 1.97−1.87 (m, 1 H), 1.70 (d, J = 11.0 Hz, 2 H), 1.57 (d, J = 13.2
Hz, 2 H), 1.30−1.20 (m, 1 H), 1.03 (q, J = 10.6 Hz, 2 H), 0.83 (q, J =
12.4 Hz, 2 H). MS (ES+) m/z 453 (M + H).
N-(4-Fluoro-3-(trifluoromethyl)benzyl)-4-(2-(((trans)-4-
(hydroxymethyl)cyclohexyl)methyl)-2H-tetrazol-5-yl)-6-methylpico-
linamide (34j). 59% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.43 (t, J =
6.2 Hz, 1 H), 8.37 (s, 1 H), 8.05 (s, 1 H), 7.73 (d, J = 6.2 Hz, 1 H), 7.71−
7.66 (m, 1 H), 7.47−7.40 (m, 1 H), 4.62 (d, J = 7.0 Hz, 2 H), 4.53 (d, J =
6.2 Hz, 2 H), 4.32 (t, J = 5.3 Hz, 1 H), 3.14 (t, J = 5.5 Hz, 2 H), 2.64 (s, 3
H), 1.97−1.87 (m, 1 H), 1.69 (d, J = 10.6 Hz, 2 H), 1.57 (d, J = 11.3 Hz,
2 H), 1.30−1.20 (m, 1 H), 1.03 (q, J = 12.8 Hz, 2 H), 0.83 (q, J = 12.4
Hz, 2 H). MS (ES+) m/z 507 (M + H).
N-(4-Fluoro-3-methoxybenzyl)-4-(2-(((trans)-4-(hydroxymethyl)-
cyclohexyl)methyl)-2H-tetrazol-5-yl)-6-methylpicolinamide (34k).
28% yield.1H NMR (400 MHz, CDCl3) δ 8.70 (s, 1 H), 8.42 (t, J =
6.2 Hz, 1 H), 8.03 (d, J = 1.3 Hz, 1 H), 7.06−6.94 (m, 2 H), 6.92−6.85
(m, 1 H), 4.62 (d, J = 6.4 Hz, 2 H), 4.52 (d, J = 7.0 Hz, 2 H), 3.86 (s, 3
H), 3.48−3.38 (m, 3 H), 2.61 (s, 3 H), 2.14−2.00 (m, 1 H), 1.90−1.66
L
DOI: 10.1021/acs.jmedchem.5b01434
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Journal of Medicinal Chemistry
Article
mmol), sodium azide (3.60 g, 55.3 mmol), and zinc bromide (11.3 g,
50.3 mmol) in water (112 mL) was heated at reflux for 16 h. CAUTION!
Tetrazoles represent an explosion hazard at elevated temperatures. Adequate
blast shielding is recommended. After the mixture cooled to rt, 6 N HCl
(180 mL) was added, and the mixture was stirred vigorously for 1 h. The
resulting solids were collected by vacuum filtration, rinsed with 6 N HCl
(3 × 100 mL) followed by water (2 × 100 mL), and dried at 55 °C under
vacuum to provide 15.0 g (92%) of 40a as a white solid. 1H NMR (400
MHz, DMSO-d6) δ 9.53 (t, J = 6.4 Hz, 1 H), 8.42 (s, 1 H), 7.21 (t, J = 8.2
Hz, 1 H), 6.86−6.91 (m, 2 H), 6.76−6.82 (m, 1 H), 4.48 (d, J = 6.6 Hz, 2
H), 3.70 (s, 3 H), 2.82 (s, 3 H). MS (ES+) m/z 326 (M + H).
dichloromethane. The combined organic layers were dried (Na2SO4)
and concentrated. The resulting oil was purified by reverse phase HPLC.
The acetonitrile was removed in vacuo, and the aqueous residue was
extracted with dichloromethane (2 × 50 mL). The combined organic
layers were concentrated. The crude product was triturated with ethyl
ether (3×) to afford 361 mg (37%) of 45 as a white solid. 1H NMR (400
MHz, DMSO-d6) 13.03 (s, 1 H), 9.58 (t, J = 6.4 Hz, 1 H), 8.42 (s, 1 H),
7.21−7.17 (m, J = 8.5, 1.5 Hz, 1 H), 7.18−7.11 (dd, J = 11.6, 8.4 Hz, 1
H), 6.93−6.88 (m, 1 H), 4.98 (dd, J = 14.3, 3.8 Hz, 1 H), 4.91 (dd, J =
14.2, 7.9 Hz, 1 H), 4.50 (d, J = 6.3 Hz, 2 H), 4.17−4.05 (m, 3 H), 3.95
(dd, J = 11.8, 2.9 Hz, 1 H), 3.82 (s, 3 H), 3.49 (dt, J = 18.1, 10.8 Hz, 2 H),
2.83 (s, 3 H). MS (ES+) m/z 488 (M + H).
Collagenase Activity. Apparent inhibition constants (Ki,app) were
determined using the synthetic quenched fluorescent peptide substrate
MCA-Arg-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-Glu-Arg-NH2 as previously
described.29 Full-length recombinant human proteins were used for
MMPs 1, 2, 9, and 13. The catalytic domain of the protein was used for
MMPs 3, 7, 8, 10, 12, 14, 15, 16, 20, 24, 25, and 26. Selectivity against
ADAM-17 (TACE), ADAMTS-4 (aggrecanase 1), and ADAMTS-5
(aggrecanase 2) was determined as previously described.29
N-(4-Fluoro-3-methoxybenzyl)-2-methyl-6-(2H-tetrazol-5-
yl)pyrimidine-4-carboxamide (41b). Following conditions analo-
gous to those described for 41a, reaction of 40b (4.80 g, 16.0 mmol)
1
provided 5.45 g (99%) of 41b as a white solid. H NMR (400 MHz,
DMSO-d6) δ 9.55 (t, J = 6.4 Hz, 1 H), 8.42 (s, 1 H), 7.06−7.20 (m, 2 H),
6.81−6.94 (m, 1 H), 4.47 (d, J = 6.4 Hz, 2 H), 3.79 (s, 3 H), 2.82 (s, 3 H).
MS (ES+) m/z 344 (M + H).
rac-N-(3-Methoxybenzyl)-6-(2-(((2S*,5R*)-5-(hydroxymeth-
yl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methylpyrimi-
dine-4-carboxamide (42). A mixture of rac-16 (4.3 g, 14.0 mmol),
41a (3.2 g, 9.8 mmol), and triethylamine (2.0 g, 19.7 mmol) in
anhydrous DMA (4 mL) was heated at 85 °C for 18 h. The reaction
mixture was purified by reverse phase preparative HPLC to afford 1.38 g
(31%) of 42 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.52−
9.44 (m, 1 H), 8.42 (s, 1 H), 7.24 (t, J = 8.1 Hz, 1 H), 6.95−6.89 (m, 2
H), 6.83 (d, J = 8.1 Hz, 1 H), 4.93 (d, J = 3.3 Hz, 1 H), 4.88 (d, J = 7.7 Hz,
1 H), 4.51 (d, J = 5.9 Hz, 3 H), 4.08 (d, J = 3.3 Hz, 1 H), 3.99 (d, J = 11.3
Hz, 1 H), 3.78 (d, J = 11.3 Hz, 1 H), 3.74 (s, 3 H), 3.48−3.36 (m, 4 H),
3.31 (d, J = 2.2 Hz, 1H), 2.83 (s, 3 H). MS (ES+) m/z 456 (M + H).
N-(4-Fluoro-3-methoxybenzyl)-6-(2-(((2S,5R)-5-(hydroxy-
methyl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methyl-
pyrimidine-4-carboxamide (43a). A mixture of (2R,5R)-16 (12.4 g,
41.0 mmol), 41b (10.0 g, 29.1 mmol), and triethylamine (7.04 g, 69.7
mmol) in anhydrous DMA (31 mL) was heated at 85 °C for 18 h. After
cooling to rt, the mixture was diluted with water (300 mL) and extracted
with dichloromethane (3 × 200 mL). The combined organic layers were
washed with 1 N NaOH (2 × 100 mL), 1 M HCl (2 × 100 mL), water
(100 mL), and brine (250 mL). The organic layer was dried (Na2SO4),
filtered, and evaporated. The residue was purified by silica gel
chromatography (heptane/EtOAc/methanol, 50/45/5). The resulting
mixture of regioisomers was separated by SFC (OJ-H, 30 mm × 250
mm, 40% methanol, 70 mL/min), and the desired isomer was
recrystallized from EtOAc/ethanol (80/1, 80 mL) to provide 3.5 g
(25%) of 43a as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.53 (t,
J = 6.3 Hz, 1 H), 8.39 (s, 1 H), 7.19−7.06 (m, 2 H), 6.91−6.83 (m, 1 H),
4.98−4.78 (m, 2 H), 4.68 (t, J = 5.7 Hz, 1 H), 4.47 (d, J = 6.4 Hz, 2 H),
4.10−3.99 (m, 1 H), 3.96 (dd, J = 11.4, 2.4 Hz, 1 H), 3.79 (s, 3 H), 3.74
(dd, J = 11.4, 2.2 Hz, 1 H), 3.47−3.19 (m, 5 H), 2.80 (s, 3 H). MS (ES+)
m/z 474 (M + H).
In Vivo Animal Studies. All procedures performed on animals were
in accordance with regulations and established guidelines and were
reviewed and approved by Pfizer Institutional Animal Care and Use
Committee.
For determination of rat in vivo clearance, each test compound was
dissolved in 70% PEG400/10% ethanol/20% normal saline vehicle at
0.5 mg/mL and dosed at 2 mL/kg via IV bolus to N = 2 male Sprague−
Dawley IGS rats weighing 250−300 g. Blood was collected over a 24 h
time course, and following centrifugation the resulting plasma samples
were precipitated with acetonitrile and analyzed for test compound
concentration using an LC-MS/MS procedure. PK parameters were
calculated from plasma concentration−time curves using noncompart-
mental analysis in Watson LIMS. For cassette dose PK, the procedure
was identical except five compounds were dissolved in vehicle at 0.1 mg/
mL each and dosed simultaneously, resulting in a combined total dose
by mass that was the same as for singleton PK.
For dog PK, test compounds were dosed orally as a suspension in
0.5% Methocel/0.1% TWEEN80 to fasted male beagle dogs.
Compounds were dosed at 5 mL/kg by gavage in a cassette experiment
(N = 5/cassette) such that each animal received a dose equivalent to 1.0
mg/kg of each compound in a single experiment. Blood collection,
sample processing, concentration determination, and PK calculations
were identical to the procedure used for rat PK.
In cynomolgus monkeys, compound 43a was administered once daily
by oral gavage using a 50% drug load spray-dried dispersion formulation
in a 50% by weight aqueous sucrose solution. Doses of 50, 100, and 1000
mg kg−1 day−1 were administered for 14 days (one male and one female
per dose group). Gross necropsy observations, clinical pathology, and
histopathology of the kidneys were evaluated.
N-(4-Fluoro-3-methoxybenzyl)-6-(2-(((2R,5S)-5-(hydroxy-
methyl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methyl-
pyrimidine-4-carboxamide (43b). Analogous to the procedures for
43a, (2S,5S)-16 (5.2 g, 17.2 mmol) was reacted with 41b (4.22 g, 12.3
mmol). The minor regioisomer was removed by SFC (OJ-H, 30 mm ×
250 mm, 45% acetonitrile, 70 mL/min), and the desired isomer was
recrystallized from EtOAc to afford 1.5 g (26%) of 43b as a white solid.
1H NMR (400 MHz, DMSO-d6) δ 9.50 (t, J = 6.2 Hz, 1 H), 8.42 (s, 1 H),
7.17−7.08 (m, 2 H), 6.90−6.85 (m, 1 H), 4.95−4.80 (m, 2 H), 4.69 (t, J
= 5.5 Hz, 1 H), 4.47 (d, J = 6.2 Hz, 2 H), 4.09−3.93 (m, 2 H), 3.79 (s, 3
H), 3.77−3.71 (m, 1 H), 3.45−3.20 (m, 5 H), 2.79 (s, 3 H). MS (ES+)
m/z 474 (M + H).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01434.
■
S
Synthesis and X-ray data collection for the crystal structure
of compound 44 and pharmacokinetic parameters of
compounds 14i, 42, and 43b in rat and compound 43a in
rat, dog, and cynomolgus monkey.(PDF)
SMILES representations of compounds with MMP13/FL
(2S,5S)-5-((5-(6-((4-Fluoro-3-methoxybenzyl)carbamoyl)-2-
methylpyrimidin-4-yl)-2H-tetrazol-2-yl)methyl)-1,4-dioxane-2-
carboxylic acid (45). A mixture of 43a (952 mg, 2.0 mmol) and
pyridinium dichromate (4.61 g, 12.0 mmol) in DMF (20.0 mL) was
shaken for 36 h at rt. The reaction mixture was poured into EtOAc (250
mL) and washed with 1 N HCl solution (2 × 50 mL). The organic layer
was dried (Na2SO4) and concentrated. The residue was suspended in 1
N sodium hydroxide solution and extracted with EtOAc. The aqueous
layer was acidified to pH 2 with concentrated HCl and extracted with
Ki (XLSX)
AUTHOR INFORMATION
Corresponding Author
*Phone: 617-674-7362. E-mail: mark.e.schnute@pfizer.com.
■
Notes
The authors declare no competing financial interest.
M
DOI: 10.1021/acs.jmedchem.5b01434
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
loproteinase inhibitor, administered orally to patients with advanced
lung cancer. J. Clin. Oncol. 1998, 16, 2150−2156.
ACKNOWLEDGMENTS
■
We thank Jon Bordner for single crystal X-ray data collection,
members of Pfizer pharmacokinetics and drug metabolism group
for in vivo exposure and metabolite characterization, and
members of Pfizer drug safety group for in vivo toxicology
support.
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ABBREVIATIONS
■
AUC, area under the curve; CD, catalytic domain; CL, clearance;
%F, bioavailability (fraction bioavailable); DABCO, 1,4-
diazabicyclo[2.2.2]octane; DBAD, di-tert-butyl azodicarboxy-
late; DCE, dichloroethane; DMA, N,N-dimethylacetamide;
DMAP, 4-(dimethylamino)pyridine; DMF, N,N-dimethylforma-
mide; DMSO, dimethyl sulfoxide; dppf, 1,1′-bis-
(diphenylphosphino)ferrocene; EDCI, 1-ethyl-3-(3′-
dimethylaminopropyl)carbodiimide hydrochloride; EtOAc,
ethyl acetate; FL, full length; HLM, human liver microsomes;
HPLC, high-pressure liquid chromatography; LC-MS/MS,
liquid chromatography-tandem mass spectrometry; LIPE, lip-
ophilic ligand efficiency; MMP, matrix metalloproteinase; PPB,
plasma protein binding; rt, room temperature; SAR, structure−
activity relationships; SFC, supercritical fluid chromatography;
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Hamper, B. C.; Carroll, J. N.; Patt, W. C.; Shieh, H. S.; Collins, B.;
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