Potent and selective nonpeptidic inhibitors of procollagen C-proteinase

Article abstract of DOI:10.1021/jm061010z

6-Cyclohexyl-N-hydroxy-3-(1,2,4-oxadiazol-5-yl)hexanamides were previously disclosed as inhibitors of procollagen C-proteinase (PCP) culminating in the identification of amide 1. Our objective was to discover a second inhibitor that would have improved affinity for PCP and to optimize properties for transepidermal delivery (TED) to intact skin. Further investigation of this template identified a number of potent PCP inhibitors (IC₅₀ values of 2-6 nM) with improved TED flux. Sulfonamide 56 had excellent PCP enzyme activity when measured with a peptide substrate (K_i 8.7 nM) or with the endogenous substrate procollagen (IC₅₀ 3.4 nM) and demonstrates excellent selectivity over MMPs involved in wound healing (>10 000-fold). In the fibroplasia model, 56 inhibited deposition of insoluble collagen by 76 ± 2% at 10 μM and was very effective at penetrating human skin in vitro with a TED flux of 1.5 μg/cm²/h, which compares favorably with values for agents that are known to penetrate skin well in vivo. Based on this profile, 56 (UK-421,045) was selected as a candidate for further preclinical evaluation as a topically applied, dermal anti-scarring agent.

Full text of DOI:10.1021/jm061010z

3442
J. Med. Chem. 2007, 50, 3442-3456
Potent and Selective Nonpeptidic Inhibitors of Procollagen C-Proteinase
Paul V. Fish,*,† Gillian A. Allan,‡ Simon Bailey,† Julian Blagg,† Richard Butt,§ Michael G. Collis,§ Doris Greiling,§
Kim James,† Jackie Kendall,† Andrew McElroy,† Dawn McCleverty,| Charlotte Reed,| Robert Webster,‡ and Gavin A. Whitlock†
Departments of DiscoVery Chemistry, Pharmacokinetics and Drug Metabolism, DiscoVery Biology, and Pharmaceutical Sciences Research and
DeVelopment, Pfizer Global Research and DeVelopment, Sandwich, Kent, CT13 9NJ, United Kingdom
ReceiVed August 22, 2006
6-Cyclohexyl-N-hydroxy-3-(1,2,4-oxadiazol-5-yl)hexanamides were previously disclosed as inhibitors of
procollagen C-proteinase (PCP) culminating in the identification of amide 1. Our objective was to discover
a second inhibitor that would have improved affinity for PCP and to optimize properties for transepidermal
delivery (TED) to intact skin. Further investigation of this template identified a number of potent PCP
inhibitors (IC50 values of 2-6 nM) with improved TED flux. Sulfonamide 56 had excellent PCP enzyme
activity when measured with a peptide substrate (Ki 8.7 nM) or with the endogenous substrate procollagen
(IC50 3.4 nM) and demonstrates excellent selectivity over MMPs involved in wound healing (>10 000-
fold). In the fibroplasia model, 56 inhibited deposition of insoluble collagen by 76 ( 2% at 10 µM and was
very effective at penetrating human skin in vitro with a TED flux of 1.5 µg/cm2/h, which compares favorably
with values for agents that are known to penetrate skin well in vivo. Based on this profile, 56 (UK-421,045)
was selected as a candidate for further preclinical evaluation as a topically applied, dermal anti-scarring
agent.
Introduction
occur across joints. Unsightly scars, particularly on the face or
hands, can cause significant psychological distress in many
cases.
Hence, we identified PCP as an attractive point at which to
intervene in the collagen deposition pathway and initiated a
research program aimed at identifying small, reversible inhibitors
of this enzyme suitable for topical application as antiscarring
agents.
In the context of dermal scars, selectivity over inhibition of
several other proteolytic enzymes that are found in the wound
environment was felt to be important. For instance, matrix
metalloproteinase-1 (MMP-1) is expressed in keratinocytes at
the migrating front of acute wounds and is required, along with
MMP-2 and -9, for keratinocyte migration (i.e., wound re-
epithelialisation in vivo). MMP-2 is expressed in fibroblasts and
endothelial cells in acute wounds and is required for fibroblast
and endothelial migration (i.e., granulation and angiogenesis).
MMP-9 has also been suggested to be involved in cell migration,
while MMP-14 is involved in the activation of MMP-2.
Therefore, an agent designed to prevent dermal scarring without
effects on the rate or quality of wound healing would need to
be selective over MMPs.8
Fibrosis is a complex process that is characterized by
excessive accumulation of extracellular matrix (ECMa), prin-
cipally type I collagen. Recent advances in the understanding
of the molecular mechanisms of fibrosis have revealed that,
rather than being a distinct pathological event, fibrotic ECM
deposition results from disregulation of normal tissue repair
processes.1,2 In the case of wounds to the skin, dermal fibroblasts
respond to activation by transforming growth factor-â (TGF-
â1) by releasing soluble procollagens and procollagen C-
proteinase (PCP;3,4 EC 3.4.24.19, also known as Bone Mor-
phogenetic Protein-1, BMP-1).5,6 Proteolytic cleavage of the
C-terminal propeptide of types I, II, and III procollagens by
PCP at Gly-Asp and Arg-Asp sites affords collagen, which
undergoes self-assembly into fibrils, followed by cross-linking
to give an insoluble collagen matrix. The cross-linking process
is primarily driven by prolyl hydroxylase-mediated hydroxyla-
tion of the proline-rich collagen molecules. In addition to
processing procollagens, PCP is also able to cleave latent prolyl
oxidase to its active form and, therefore, may contribute further
to ECM formation.7
While this work was in progress, two types of low molecular
weight inhibitors of PCP have been reported with good
inhibition of enzyme activity (Chart 1). These inhibitors
incorporate either a hydroxamic acid (e.g., 3-5) or phosphinate
(e.g., 6) as the zinc binding ligand in the catalytic site of the
enzyme.9 Our own efforts identified succinyl hydroxamates 1
(UK-383,367) and 2 as potent and selective inhibitors of PCP.10
The continuing results of these efforts are presented in this
article. The compounds described herein are potent, competitive,
and reversible inhibitors of PCP enzymatic activity, with high
selectivity for PCP relative to MMPs.
The excessive accumulation of ECM by these processes leads
to the formation of scar tissue, which can range in severity from
being grossly disfiguring to cosmetically unpleasant. Clinically
significant scars include hypertrophic/keloid scars and those that
occur following burns. These latter categories can often lead to
functional impairment due to skin contraction, especially if they
* To whom correspondence should be addressed. Tel.: +44 (0)1304
644589. Fax: +44 (0) 1304 651987. E-mail: paul.fish@pfizer.com.
† Discovery Chemistry.
‡ Pharmacokinetics and Drug Metabolism.
§ Discovery Biology.
| Pharmaceutical Sciences Research and Development.
a Abbreviations: ECM, extracellular matrix; MMP, matrix metallopro-
teinase; PCP, procollagen C-proteinase; TED, transepidermal delivery; CDI,
1,1-carbonyldiimidazole; DCM, dichloromethane; DIPE, diisopropyl ether;
DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; THF, tetrahy-
drofuran; TFA, trifluoroacetic acid.
Chemistry
The general method for the synthesis of the oxadiazole
hydroxamates disclosed in Tables 1-6 (compounds 7-71) is
outlined in Scheme 1. Nitriles 72 were reacted with HONH2 to
10.1021/jm061010z CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/26/2007

Inhibitors of Procollagen C-Proteinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3443
Table 2. Heteroaromatic Substituted Oxadiazoles
Chart 1. PCP Inhibitors
PCP IC50
cmpd
R
(nM)
7
14
15
16
17
18
19
20
21
22
23
24
25
Ph
116
25
23
2
30
23
14
14
74
39
92
46
10
2-Py
3-Py
4-Py
6-methyl-3-pyridazinyl
2-pyrimidinyl
5-pyrimidinyl
2-pyrazinyl
3-pyrazolyl
4-pyrazolyl
1-methyl-2-imidazolyl
2-furanyl
5-uracilyl
Table 3. Substituted Pyridyl Oxadiazoles
Table 1. Substituted Aryl Oxadiazoles
PCP IC50
(nM)
cmpd
R
2-Pyridyl
26
27
28
29
4-CO2H
4-CONH2
4-CONHMe
4-CONMe2
6
17
30
17
a
PCP IC50
(nM)
cmpd
R
3-Pyridyl
7
8
9
10
11
12
13
H
116
154
32
75
3
30
31
32
33
34
35
36
5-CO2H
6- CO2H
6-NH2
6-NHMe
17
59
18
13
28
8
4-CO2Et
4-CO2H
4-SO2NMe2
4-NHSO2Me
3-CO2Et
6-NMe2
184
6
6-(4-methyl-1-piperazinyl)
6-NHSO2Me
3-CO2H
27
a Mean of at least two experiments.
give the corresponding N-hydroxy amidines 73, which were then
coupled with (R)-succinic acid mono-ester 7411 to give (presum-
ably) the O-acyl derivatives 75. Thermal cyclodehydration of
75 in xylene created the 1,2,4-oxadiazoles 76 and deprotection
of the t-butyl esters gave carboxylic acids 77. Finally, activation
of the carboxylic acids 77 with either 1,1-carbonyldiimidazole
(CDI) or i-BuOCOCl, followed by reaction with TMSONH2
and methanolic workup, gave compounds 7, 8, 12, 14-25, 37-
39, 50, 56, 65, and 66. Oxadiazole esters 8 and 12 were
converted to the corresponding carboxylic acids 9 and 13,
respectively, by mild base hydrolysis.
The remaining inhibitors were also prepared by the general
synthetic sequence outlined in Scheme 1, except that additional
functional group transformations were undertaken of appropriate
intermediates (Schemes 2-7).
Arylsulfonamides 10 and 11 were prepared from aniline 80
(Scheme 2). Reduction of 79 with SnCl2 gave aniline 80 and
then deprotection of the t-butyl ester with TFA gave 81. The
aniline 81 was then converted to reversed sulfonamide 82 via
a diazonium salt. Thus, diazotization of 81 with NaNO2/cHCl,
followed by treatment with SO2 in the presence of CuCl2 gave
the corresponding sulfonyl chloride, which then reacted with
HNMe2 to give 82. Acid 82 was then converted to hydroxamate
10 by standard methods. Alternatively, reaction of aniline 80
with MeSO2Cl gave sulfonamide 83, and then reaction with TFA
and introduction of the hydroxamate gave 11.
2-Pyridyl acid 26 and amides 27-29 were prepared from ester
85 (Scheme 3). Acid 26 was prepared in a similar manner to
benzoic acid 9 by introduction of the hydroxamate and then
mild base hydrolysis of the ethyl ester. Ester 85 was treated
with NH3, NH2Me or NHMe2 to yield the corresponding
carboxamides 86-88, which were then converted to 27-29 by
standard methods.
The synthesis of the 3-pyridyl acid 30 was prepared by the
exact same procedure as 2-pyridyl acid 26 except that ethyl
5-cyanonicotinate was used as the starting material. The
synthesis of the 3-pyridyl compounds 31-36 is described in
Scheme 4. Pd-mediated ethoxycarbonylation of 6-chloropyridine

3444 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Table 4. Pyridylmethyl and Aminomethyl Oxadiazoles
Fish et al.
Table 6. Oxadiazole Acylsulphonamides
PCP IC50
(nM)
PCP IC50
(nM)
cmpd
R
cmpd
R
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
2-Py
3-Py
4-Py
NH2
6
6
67
68
69
70
71
H
4-Me
4-OMe
3,4-(OMe)2
4-F
7
14
17
1.8
33
28
122
23
21
29
10
6
6
8
31
24
5
NHMe
NHEt
NH-i-Pr
NH-t-Bu
NH-c-Pr
NH-c-Bu
NHC(Me)2CH2OH
NMe2
1-pyrrolidinyl
Scheme 1. General Method for the Synthesis of Inhibitorsa
4-morpholinyl
4-hydroxy-1-piperidinyl
13
Table 5. Substituted Aminomethyl Oxadiazoles
PCP IC50
(nM)
cmpd
R1
R2
COMe
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
H
Me
H
H
H
Me
H
H
H
H
19
3
COMe
CONH2
SO2NH2
SO2Me
SO2Me
SO2Et
SO2-i-Pr
SO2Ph
SO2-2-Py
SO2-3-Py
SO2-4-pyrazole
324
98
11
38
26
21
26
107
26
88
15
20
3
H
H
-SO2CH2CH2CH2-
89 gave the corresponding 6-ester 90, and then conversion to
acid 31 followed standard methods. The 6-chloropyridine
derivative 89 (or 92) was reacted with a range of amines to
give the corresponding 6-aminopyridines 93-95. However, the
attempted aminolysis of 89 with NH3 was unsuccessful, and
so, amine 91 was prepared according to Scheme 1, where the
6-aminopyridine group was installed early in the sequence (73:
R ) 6-amino-3-pyridyl). Aminopyridines 91 and 93-95 were
converted to 32-35 by standard methods. Amine 91 was
converted to sulfonamide 36 by reaction with MeSO2Cl to give
the bissulfonamide 96, and NaOH hydrolysis then, simulta-
neously, removed one of the MeSO2- groups and cleaved the
t-Bu ester to give acid 97.
a Reagents and conditions: (a) HONH2; (b) CDI; (c) xylene, 130 °C;
(d) TFA; (e) i-BuOCOCl or CDI, then TMSONH2, then MeOH.
followed by tosylation of the resulting alcohol, gave 99. Reaction
of tosylate 99 with ammonia in a sealed vessel at 50 °C cleanly
gave amine 100 in high yield. To convert 100 into 40, it was
necessary to protect the primary amino group (as 101) to prevent
self-condensation with the activated acid during the reaction
with HONH2 used to introduce the hydroxamate group. It proved
unnecessary to protect sec- and tert-amines in this reaction.
Thus, tosylate 99 underwent reaction with a variety of alkyl
amines and amino alcohols to give amines 102, which were
then converted to 41-49 and 51 by standard methods.
3-(Aminomethyl)oxadiazoles 40-49 and 51 were all prepared
Oxadiazole amines 100 and 103 proved to be useful inter-
mediates and were converted to carboxamides (52, 53), urea
from tosylate 99 (Scheme 5). Hydride reduction of ester 98,10

Inhibitors of Procollagen C-Proteinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3445
Scheme 2. Synthesis of Compounds 10 and 11a
SO2(CH2)3Cl), treatment with NaOMe simultaneously created
the sultam and cleaved the t-butyl ester to yield 105 (R, X )
SO2(CH2)3), which was converted to 64 by standard methods.
The synthesis of acyl sulfonamide derivatives 67-71 proved
to be problematic, and our preferred method is shown in Scheme
7. Treatment of amide 106 (which is readily available from 98)10
with 2 equiv of NaH at 0 °C gave the carboxamide anion, and
reaction in situ with an arylsulphonyl chloride gave the
corresponding acyl sulfonamide 107, which were converted to
67-71 by standard methods.
Compound Design
At the outset of the work described in this article, no small
molecule inhibitors of PCP have been described in the literature.
to identify new leads, compounds from the Pfizer file, prepared
in previous metalloproteinase projects, were screened for their
ability to inhibit PCP (Figure 1). From this targeted screening,
a number of inhibitors with IC50 values < 10 µM were identified
and, of particular interest, were compounds I and II, which are
derived from the classical succinic acid group of metalloprotease
inhibitors.11
Compound I displayed only modest activity against PCP and
was inversely selective against MMP-2. However, it was noted
that replacement of the phenyl ring of I with a cyclohexane, to
give II, gave a 10-fold increase in potency against PCP and
also led to a significant drop off in activity against MMP-2. A
second hit from the screening was III, which demonstrated
moderate and balanced potency against both PCP and MMP-2.
Oxadiazole III can also be considered as a succinic acid
derivative in which the C1-carboxy terminus has been masked
as a heterocycle. Hence, combining these two pieces of SAR,
as an initial venture, we prepared IV as the direct analogue of
III, where the phenyl ring was replaced by cyclohexyl.
Remarkably, this simple modification gave a 180-fold increase
in inhibitory activity against PCP and further attenuated MMP-2
activity. Hence, it was clear that the (cyclohexyl)propyl side
chain of IV played an important role in regulating affinity for
PCP and confirming selectivity over MMP-2. Finally, replace-
ment of the chemically and metabolically vulnerable ester
group of IV with 3-carboxamides on the oxadiazole ring
identified primary amide 1 as a potent and selective inhibitor
of PCP.10
a Reagents and conditions: (a) CDI, 74; (b) xylene, 130 °C; (c) SnCl2;
(d) TFA; (e) (i) cHCl, NaNO2, -10 °C; (ii) SO2, CuCl2, -10 °C; (iii)
NHMe2; (f) i-BuOCOCl or CDI, then TMSONH2, then MeOH; (g)
MeSO2Cl.
Scheme 3. Synthesis of Compounds 26-29a
With compound 1 selected for early clinical development as
a topically applied dermal anti-scarring agent, our objective was
to discover a second agent that would have improved affinity
for PCP and would optimize physicochemical properties suitable
for the transepidermal delivery (TED) to intact skin.12 It was
also important that we retain desirable properties, such as high
selectivity over the MMPs, crystallinity, hydrolytic stability, and
to enhance solubility in formulations suitable for TED.
In the first instance, target compounds were tested for their
ability to inhibit the PCP-mediated cleavage of a fluorogenic
substrate and key physicochemical properties were measured.
Selected compounds were then screened for selectivity against
MMP-2 and, if required, additional MMPs. Finally, preferred
compounds were assessed in an in vitro cell-based model of
collagen deposition (fibroplasia model; vide infra) and for their
ability to penetrate human skin in vitro.
a Reagents and conditions: (a) CDI, 74; (b) xylene, 130 °C; (c) TFA;
(d) i-BuOCOCl or CDI, then TMSONH2, then MeOH; (e) LiOH; (f)
NHR1R2, 23-60 °C.
The ability of a compound to penetrate the stratum corneum
and epidermis of human skin (flux, J; µg/cm2/h) is a function
of the intrinsic permeability of the compound (IP) and the
concentration of the compound in the vehicle (Co; eq 1).13
(54), sulfonyl urea (55), sulfonamides (57-63), and sultam (64;
Scheme 6). The amines 100 and 103 underwent reaction with
a suitable reagent (see the scheme) to yield amino derivatives
104, which were then converted to 52-55 and 57-63 by
standard methods. Amine 100 was coupled with Cl(CH2)3SO2-
Cl to give the corresponding sulfonamide 104 (R ) H; X )
J ) IP × Co
(1)

3446 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Scheme 4. Synthesis of Compounds 31-36a
Fish et al.
a Reagents and conditions: (a) Pd cat., CO, EtOH; (b) TFA; (c) i-BuOCOCl or CDI, then TMSONH2, then MeOH; (d) LiOH; (e) NHR1R2, 90 °C; (f)
MeSO2Cl; (g) NaOH.
Intrinsic permeability is related to the physicochemical
properties of the molecule, such as molecular size (as measured
by molecular weight, (mw)), lipophilicity (Log D7.4), H-bond
donor/acceptor ability, charge, and degree of ionization (pKa).14-16
In general, intrinsic permeability will increase with increasing
lipophilicity, but will decrease as molecular weight, H-bonding
ability, or degree of ionization are increased.14-16 A second
important parameter in defining TED flux is the concentration/
solubility of the compound in the vehicle, which is again a
function of physicochemical properties of the compound. We
opted to try to maximize solubility in both aqueous and organic
media so as to ensure flexibility in developing a formulation
suitable for topical application and clinical development. Our
objective was to seek an agent with TED flux >0.1 µg/cm2/h
in vitro, which would compare favorably with values for agents
that are known to penetrate skin well in vivo.14,17
Hence, the design of inhibitors with suitable physicochemical
properties was a key aspect of the program. We were seeking
to identify potent PCP-i (IC50 < 10 nM) that were crystalline
solids (mp > 110 °C) with good aqueous solubility (>50 µg/
mL) and satisfactory aqueous stability under autoclave condi-
tions (121 °C). An inhibitor that met these criteria would
compare favorably with amide 1 (IC50 44 nM; mw 324; pKa
9.0 -CONHOH; Log D7.4 2.6; aq. sol. 33 µg/mL; mp 134-
140 °C). Any inhibitor with greatly improved solubility (>1000
µg/mL) was likely to offer a significant advantage in improving
TED flux. The preferred values of other physicochemical
parameters, such as molecular weight, lipophilicity, H-bonding
ability, charge, or degree of ionization were less clearly defined
and, wherever possible, it was our intention to assess their
influence experimentally.
The 6-cyclohexyl-N-hydroxyhexanamide backbone of this
class of PCP inhibitors confers good affinity for PCP and
selectivity over MMPs. It also confers quite a burden in terms
of lipophilicity and tends to dominate the physicochemical
properties of the inhibitors. Our attempts to modify this
backbone by introduction of polar groups, rigidifying the many
rotatable bonds or downsizing the lipophilic chain, resulted with
a significant reduction in affinity for PCP.10 Hence, as there
was scope to further explore the 3-position of the oxadiazole
ring, we decided to investigate a wider range of substituents
beyond carboxamides. In terms of affinity for PCP, our
experience from the carboxamide series had shown that a variety
of amide substitutions were tolerated, however, it proved
difficult to improve activity below a plateau of IC50 <20-40
nM. In terms of physicochemical properties, there proved to
be a fine balance between compounds that were crystalline
solids and those that were amorphous solids, low melting waxes,
or gums. Hence, it was evident that these substituents would
need to be quite polar in nature to furnish inhibitors with
appropriate physicochemical properties for TED and clinical
development.

Inhibitors of Procollagen C-Proteinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3447
Scheme 6. Synthesis of Compounds 52-55 and 57-64a
Scheme 5. Synthesis of Compounds 40-49 and 51a
a Reagents and conditions: (a) AcCl; (b) OdC(OSuc)2, NH3; (c)
ClSO2NHtBu; (d) R′SO2Cl; (e) Cl(CH2)3SO2Cl; (f) TFA; (g) i-BuOCOCl
or CDI, then TMSONH2, then MeOH; (h) NaOMe.
Scheme 7. Synthesis of Compounds 67-71a
a Reagents and conditions: (a) NaBH4; (b) TsCl; (c) NH3, 50 °C; (d)
TFA; (e) BOC-on; (f) i-BuOCOCl or CDI, then TMSONH2, then MeOH;
(g), NHR1R2, 40 °C.
Our earlier studies with the carboxamides highlighted op-
portunities for suitable starting points. The incorporation of
heterocycles, acidic groups, and basic groups linked through
the amide were tolerated, and these classes of compounds tended
to have more acceptable physicochemical properties. Glycine
derivative 2 (IC50 21 nM; mp 141-142 °C) was one of the
more potent compounds from this series, and so, we decided
to prepare a series of carboxylic acids and acid surrogates
whereby the amide linker was to be replaced by a phenyl ring
(Table 1).
The first analogue, compound 7, demonstrated modest
potency for PCP (IC50 116 nM) and potency was further
enhanced by the incorporation of a carboxylic acid at either
the 4- (9) or more preferably the 3-position (13, IC50 6 nM).
The replacement of the carboxylic acid with a weakly acidic
isostere such as sulfonamide 11 (IC50 3 nM; calculated pKa 9.5
-NHSO2Me) was also tolerated. These preliminary results gave
encouragement that our objective of significantly increasing
activity for PCP inhibition was achievable. However, none of
these phenyl derivatives combined sufficient potency against
PCP with a suitable solid form. Hence, the next step was to
replace the phenyl ring by heterocycles, with the aim of
improving solid form by the introduction of more polar groups
capable of promoting crystallinity through favorable H-bonding
interactions.
a Reagents and conditions: (a) NaH, RC6H4SO2Cl; (b) TFA; (c)
i-BuOCOCl or CDI, then TMSONH2, then MeOH.
most of these heterocyclic derivatives were either noncrystalline
or the few examples that were solids had very low aqueous
solubility. Uracil 25 had the best balance of improved affinity
for PCP and solubility (IC50 10 nM; mw 391; pKa 7.6
-CONHCO-, 9.4 -CONHOH; Log D7.4 2.1; aq. sol. 72 µg/
mL; mp 210 °C) when compared to amide 1 and so was selected
for further evaluation. The pyridyl analogues 14 and 15 also
represented suitable structures for further modification
(Table 3).
It had been shown that substitution of a phenyl ring with an
appropriately positioned carboxylic acid had resulted in an
increase in affinity (cf. 9, 13 vs 7) and a similar tactic was
applied in the 2- and 3-pyridyl series. The effects with the
pyridyl acids (26 vs 14; 30 vs 15) were not so pronounced as
with the phenyl acids, although, once again, a meta-arrangement
The phenyl ring was successfully replaced with a variety of
6- and 5-ring heterocycles directly attached to the oxadiazole
(14-25, Table 2), with the six-membered rings demonstrating
a clear advantage in affinity (e.g., 16, IC50 2 nM). However,

3448 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Fish et al.
with crystallinity (mw 372; pKa 4.4 -PyH+, 9.4 -CONHOH;
Log D7.4 3.3; aq. sol. 27 µg/mL; mp 112 °C), although no
improvement in aqueous solubility was achieved with these
weakly basic compounds. The preparation of a series of alkyl
amines 40-51 demonstrated good affinity, with toleration of a
wide variety of groups. The amino group could be substituted
with small linear (41, 42), branched (43, 44, 47), or cyclic (45,
46) alkyl groups. The alkyl groups could be further substituted
with an O atom as either alcohols (47, 51) or ethers (50). The
amines could be secondary (41-47), tertiary (48), or incorpo-
rated into a ring (49-51). Only the primary amine 40 had
relatively poor affinity (IC50 122 nM). Amino alcohol 47 had
the best balance of affinity for PCP (IC50 8 nM) and physico-
chemical properties (mw 382; pKa 6.4 -CH2N+HHR, 9.0
-CONHOH; Log D7.4 2.6; aq. sol. 1080 µg/mL at pH 7.1; mp
49 °C as free base) and so was selected for further evaluation.
Unfortunately, it was subsequently discovered that all the amines
37-51 failed to demonstrate satisfactory aqueous stability under
autoclave conditions (121 °C, 15 min, pH 3-6), with the major
route of decomposition being hydrolysis of the hydroxamate to
the corresponding carboxylic acid. All the other inhibitors
disclosed in Tables 1-3, 5, and 6 were quite stable under these
conditions.
A series of derivatives of the methylamino group were then
prepared with the aim of improving hydrolytic stability of this
class (Table 5). It has been shown that carboxamides (e.g., 1)
had excellent aqueous stability under these conditions, and so,
we thought that increasing the sp2 character of the N atom would
confer additional aqueous stability. Initially, a few representative
examples were prepared; carboxamides 52 and 53 had good
affinity but were not of a suitable solid form, and urea 54 and
sulfonyl urea 55 were not of sufficient potency. Only sulfona-
mide 56 had good activity for PCP inhibition (IC50 11 nM)
combined with improved aqueous solubility and a crystalline
solid form (mw 388; pKa 8.9 -CONHOH, 10.2 -NHSO2Me;
Log D7.4 2.3; aq. sol. 130 µg/mL; mp 115 °C). The preparation
of a series of sulfonamide analogues by alkylation of the N
atom (57), variation of the sulfonyl group (58-63), or tying
the sulfonamide into a ring (sultam 64) failed to identify a
compound with a superior profile to 56. However, modification
of the linker between the oxadiazole and the sulfonamide was
successful; piperidine sulfonamide 66 was the most potent
compound in the sulfonamide series (IC50 3 nM; mw 442; Log
D7.4 > 2; aq. sol. 21 µg/mL; mp 148 °C) although no
improvement in aqueous solubility was achieved. Compounds
56 and 66 demonstrated excellent aqueous stability and were
selected for further evaluation.
Figure 1. Identification of lead series and compound 1.
between the oxadiazole and acid substituent was preferred (26,
30 vs 31). Pyridyl acid 26 had good affinity for PCP (IC50
6
nM) combined with excellent aqueous solubility (mw 402; pKa
2.8 -COOH, 9.05 -CONHOH; Log D7.4 0.6; aq. sol. > 60 000
µg/mL at pH 7.0; mp 117 °C) and so was selected for further
evaluation. The conversion of 2-pyridyl acid 26 to a series of
amides (27-29) resulted in a small drop in affinity for PCP. A
more beneficial modification was through the introduction of
an amino group as was observed with aminopyridines (32-
35). A 6-(1-piperazinyl) group was the preferred substituent in
the 3-pyridine series 35 (IC50 8 nM), and this compound
demonstrated that a basic center was well tolerated (calculated
pKa 8.4 R2N+HMe). The introduction of a sulfonamide 36 as
an acidic isostere was also tolerated in the pyridine series (IC50
27 nM; pKa 6.5 -NHSO2Me, 9.3 -CONHOH), but was inferior
to the phenyl analogue 11.
The result that inhibitors containing a basic group were well
tolerated prompted further exploration of compounds of this
type, because the introduction of a basic amino group at an
appropriate position should furnish compounds with improved
aqueous solubility. The amine group was attached to the
oxadiazole, employing a -CH2- linker so as to maintain the
basic nature of the N by reducing the influence of the electron-
withdrawing oxadiazole group and to mimic a similar spatial
arrangement of the N atom of the carboxamides (e.g., 1). The
corresponding homologues 3-(2-aminoethyl)oxadiazoles were
avoided, as these have been shown to be unstable to rearrange-
ment.18 The first examples to be prepared were the pyridylm-
ethyl analogues 37-39 (Table 4), as directly linked pyridines
had been quite potent (e.g., 16). All three isomers showed good
affinity, with 38 having the best balance of potency (IC50 6 nM)
To further explore the scope around the sulfonamide series,
a small set of acyl sulfonamides 67-71 were prepared (Table
6). These compounds were designed as hybrids of carboxamide
1 and sulfonamide 60, with the result that the acyl sulfonamide
67 would incorporate an acidic, ionisable group (-CONHSO2-
Ph) that should enhance aqueous solubility compared to 1 or
60. The acyl sulfonamides 67-71 had good affinity for PCP
and compound 67 was selected for further evaluation (IC50
7 nM; mw 464; pKa 3.2 -CONHSO2Ph, 9.9 -CONHOH;
Log D7.4 1.0; aq. sol. 3980 µg/mL; mp 149 °C) as a representa-
tive lead from this class as it had much improved aqueous
solubility.
The program had identified a number of preferred compounds
with improved affinity for PCP combined with a satifactory
crystalline solid form and increased aqueous solubility; these
included neutral compounds (56, 66), acids (26, 67), weakly
acidic heterocyle (25), weakly basic heterocycle (38), and amine

Inhibitors of Procollagen C-Proteinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3449
Table 7. MMP Selectivity and Efficacy in the Fibroplasia Model
PCP IC50
(nM)
MMP inhibitiona,b,c
IC50 (nM)
fibroplasia
modeld
cmpd
X
1
CONH2
5-uracil
44
MMP-1: >10 000
MMP-2: >10 000
MMP-3: >10 000
MMP-9: >10 000
MMP-14: >10 000
MMP-2: 79 600
76 ( 5% I at 10 µM
25
10
6
56 ( 12% I at 30 µM
MMP-3: 78 000
MMP-9: 50 000
MMP-13: 49 600
MMP-14: >100 000
MMP-1: >100 000
MMP-2: >30 000
MMP-2: >100 000
MMP-1: >100 000
MMP-2: >30 000
MMP-1: >100 000
MMP-2: >100 000
MMP-3: 91 000
e
26
4-carboxy-2-pyridine
38
47
CH2-3-Py
CH2NHC(Me)2CH2OH
6
8
87 ( 11% I at 10 µM
36 ( 8% I at 10 µM
56
CH2NHSO2Me
11
76 ( 2% I at 10 µM
MMP-9: >100 000
MMP-13: >100 000
MMP-14: >100 000
MMP-2: >100 000
66
67
3
7
88 ( 3% I at 10 µM
56 ( 11% I at 30 µM
CONHSO2Ph
MMP-1: >100 000
MMP-2: 92 000
MMP-3: 76 000
MMP-9: 61 000
MMP-13: >100 000
MMP-14: >100 000
a <50% I at 10 µM. b <50% I at 100 µM. c <50% I at 30 µM. d Mean ( sem (n ) 3). e Compound 26 failed to consistently demonstrate reduction in
collagen deposition.
Table 8. In Vitro Penetration through Human Skin in 50% Aqueous Propylene Glycol
satd solubility,
Co (µg/mL)
TED flux
TED flux
ratioc
a
cpmd
X
mw
LogD
2.6
pKa
pH
(µg/cm2/h)b
1
9
25
26
CONH2
324
401
391
402
6.5
4.8
5.0
3.8
4.8
7.5
6.0
4.3
4.6
5.0
6.5
670
260
4100
290
0.304
NFDd
0.209
0.107
NFDd
NFDd
1.33
0.028
0.110
0.128
1.40
0.09
4-carboxy-phenyl
5-uracil
4-carboxy-2-pyridine
3.7
7.6
2.8
2.7e
2.1
0.6
0.70
0.025
570
2970
33 000
4560
610
3900
8260
47
56
66
67
109
CH2NHC(Me)2CH2OH
CH2NHSO2Me
CH(CH2CH2)2NSO2Me
CONHSO2Ph
382
388
442
464
352
2.6
2.3
>2
1.0
6.4
10.2
1.7
0.14
0.06
0.46
1.0
3.2
CONMe2
3.6
a Aqueous pKa of ionized group X. The pKa of the hydroxamic acid was 8.9-9.9 (see text for values). b Mean of 6-10 experiments. c Compared to an
internal standard (109). The disconnection between TED flux and TED flux ratio is a function of the variability of skin samples (see text). d No flux
detected. e pKa measured in 50% aqueous propylene glycol.
(47). These preferred compounds were then evaluated in MMP
selectivity assays (Table 7) in an in vitro cell-based model of
collagen deposition (fibroplasia model)19 (Table 7) and for their
ability to penetrate human skin in vitro (Table 8).
Compounds 25, 26, 38, 47, 56, 66, and 67 all demonstrate
excellent selectivity over the MMP enzymes with at least 5000-
fold selectivity (Table 7). Initial screening was performed with
MMP-2 as a representative member of the MMP family because

3450 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Fish et al.
of the key role it plays in the tissue healing process, and then
additional profiling of other MMPs was performed as necessary.
We believe the principle origin of this selectivity is due to the
lipophilic propyl(cyclohexane) side chain that is likely to bind
in a deep lipophilic pocket adjacent to the catalytic Zn2+ in the
active site of PCP. This binding model is supported by
hydroxamate inhibititors such as 4 and 5, which also incorporate
a very lipophilic side chain. Compounds related to 5 are selective
for PCP over the MMPs9 but not to the same degree as the
compounds listed in Table 7. Compound 56 had a slight
advantage in its selectivity profile with >10 000-fold verses
MMP-1, -2, -9, -13, and -14 and 9000-fold verses MMP-3.
The fibroplasia model was used to approximate the processes
leading to excessive collagen deposition and scar tissue forma-
tion in man. This in vitro cell-based model of collagen
deposition19 uses adult dermal fibroblasts cultured in the
presence of proline, ascorbate, and TGF-â1. Under these
conditions, the fibroblasts proliferate, stratify, and deposit ECM,
including type 1 collagen (Figure 2a,b). The collagen deposition
was quantified by HPLC determination of 4-hydroxyproline
content of the insoluble culture fraction. These biochemical
effects were confirmed by histological analysis and quantifica-
tion of cell counts and cell viability to confirm that test
compounds had no direct effects on the cells other than to inhibit
collagen deposition. It should be noted that a correlation between
the deposition of collagen in the in vitro fibroplasia model and
the process in vivo has not yet been established.
Evaluation of preferred compounds in the fibroplasia model
furnished some unexpected results (Table 7). Interestingly,
inhibitors that incorporate a partially ionized group (at pH 7.4),
whether acidic (e.g., 25, 26, 67) or basic (e.g., 47), were inferior
at inhibiting the deposition of insoluble collagen deposition when
compared to neutral inhibitors (e.g., 1, 38, 56, 66), despite all
compounds having excellent affinity for the PCP enzyme. The
neutral inhibitors were able to reduce collagen deposition by
>75% inhibition at 10 µM, whereas the ionized inhibitors could
only reach a significant level of inhibition (>50%) at a higher
concentration of 30 µM; performance in this model proved to
be a deciding factor in compound selection. Compound 56
inhibited deposition of insoluble collagen in a dose-dependent
manner and by 76 ( 2% at 10 µM (mean ( sem; n ) 3; Figure
2c). In addition, 56 had no effect on cell number in the model,
confirming the effect was not due to inhibition of cell prolifera-
tion or compound cytotoxicity.
Figure 2. Effect of 56 in human dermal fibroplasia model. (a, b)
Histological analysis: images are taken from representative experiments
(×40-fold magnification). (c) Compound 56 inhibited deposition of
insoluble collagen in a dose-dependent manner and by 76 ( 2% at 10
µM (mean ( sem; n ) 3).
The disconnection between enzyme affinity and efficacy in
the fibroplasia model is not fully understood, however, we would
suggest that the following two factors are making an important
contribution. First, the fibroplasia model is a cell-based system,
and so, a significant proportion of the inhibitor concentration
will be lost to binding to the cell culture media. Second, as
PCP is an extracellular but cell surface bound enzyme, it may
reside in a localized lipophilic environment.20 If the inhibitor
binds to the active site in the neutral form to penetrate this
environment, ionized compounds will be at a disadvantage.
Hence, these two factors could combine to significantly reduce
the free concentration of the neutral form of ionized compounds
with the results that they are less effective in this model.
an internal standard (109) to allow comparison of results. The
direct comparison of TED flux results was not appropriate due
to the high variability in the nature of human skin (donor, site,
and condition), and so, the TED flux ratio standardized to an
internal reference was used to assess ability to penetrate skin.
In general, the solubility of the compound in the vehicle was
the most important parameter in determining flux (Figure 3).
This trend was independent of charge as neutral (1, 25, 56, 66,
109), acidic (67), and weakly basic (47) compounds demon-
strated good TED flux ratios. The ability of 56 to demonstrate
improved skin penetration compared to 1 could be attributed to
the higher saturated solubility of 56 in the vehicle as they were
similar in all other respects. The poor flux of acids 9 and 26
was partly attributed to low solubility in the vehicle although
additional factors were involved. The TED flux ratio of 26 was
further investigated by performing experiments at higher pHs,
The ability of preferred compounds to penetrate human skin
in vitro was measured (Table 8). Human cadaver skin was
mounted in a Franz diffusion cell, and a saturated solution of
the test compound in 50% aqueous propylene glycol was added
to the donor compartment. Samples of receptor fluid were taken
at intervals between 0 and 24 h to determine flux. The vehicle
also contained [14C]-mannitol to test for membrane integrity and

Inhibitors of Procollagen C-Proteinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3451
Experimental Section
Biology. PCP Inhibition. To determine potency of PCP inhibi-
tors, a fluorogenic PCP cleavage assay was used. This assay is based
on the method of Beekman21 using a fluorogenic substrate. The
substrate (Dabcyl-Arg-Tyr-Tyr-Arg-Ala-Asp-Asp-Ala-Asn-Val-
Glu(EDANS)-NH2) contains the cleavage site of human PCP.22
Human PCP was purified from supernatant of stable transfected
CHO cells using hydrophobic interaction column followed by
Superdex 200 gel filtration.23 Total protein (4 µg) of this enzyme
preparation was incubated with various concentrations of the
substance to be tested and 3 × 10-6 M substrate in assay buffer
(50 mM Tris-Base, pH 7.6 containing 150 mM NaCl, 5 mM CaCl2,
1 µM ZnCl2, and 0.01% Brij 35). The assay was performed in 96-
well black fluorimeter plates and fluorescence was read continuously
in a fluorimeter over 2.5 h (λex ) 340 nm, λem ) 485 nm) at a
constant 37 °C with shaking. Release of the fluorogenic signal was
in linear correlation to PCP activity. Reading of the mean velocity
from 30 min after start of experiment until 2.5 h was calculated by
the Biolise software.24 IC50 values were calculated by plotting %
inhibition values against compound concentration using Tessela add-
in for Excel spreadsheet. The IC50 values reported in Tables 1-7
are a mean of at least two experiments, and a difference of <2-
fold should not be considered significant. The inhibition constant
Ki was calculated from the IC50 using the Cheng-Prusoff equation
Ki ) IC50/(1 + ([S]/Km)), where the Km of the substrate was 10.29
µM.
MMP Inhibition. The assays for MMPs 1, 2, 3, 9, 13, and 14
have been described previously.11 In brief, the assays for MMPs 2,
3, 9, and 14 were based upon the original protocol described by
Knight et al. (Fed. Euro. Biochem. Soc. 1992, 296 (3), 263-266).
Assays for MMPs 2, 3, and 9 used the Nagase substrate (J. Biol.
Chem. 1994, 269, 20952-20957), whereas the assay for MMP-14
(purchased from Prof. Tschesche, Department of Biochemistry,
Faculty of Chemistry, University of Bielefeld, Germany) used the
substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Bachem Ltd,
Essex, U.K.), as described by Will et al. (J. Biol. Chem. 1996, 271
(29), 17119-17123). The assay for MMP-1 inhibition used the
substrate Dnp-Pro-â-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(N-
Me-Ala)-NH2, as originally described by Bickett et al. (Anal.
Biochem. 1993, 212, 58-64). The assay for MMP-13 inhibition
used the substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-
NH2. Enzyme and substrate concentrations were typically 1 nM
and 5-10 µM, respectively.
Figure 3. Plot of TED flux ratio vs saturated solubility in 50% aqueous
propylene glycol. Compound classes were assigned as follows: acids
(2) and bases (9) have >50% ionization at the pH of the experiment,
whereas neutrals (() are either nonionized or have <50% ionization.
which increased solubility up to 2970 µg/mL; however, these
changes had a detrimental effect on skin penetration. Acid 26
had better flux at lower pH, suggesting increased ionization had
an adverse effect. Comparing 26 with 67, both acids would be
99% ionized at pH 5, and so, the degree of ionization was not
the only factor. The failure of 26 to demonstrate reasonable
skin penetration was probably related to a combination of poor
solubility, degree of ionization, and the high molecular weight
of the template because, in general, small acids have satisfactory
TED flux (cf nicotinic acid, J ) 2.2 µg/cm2/h).14
A more detailed analysis of the ability of 56 to penetrate
human skin was then performed. Compound 56 had excellent
solubility in alternative formulations suitable for TED delivery,
which translated to a significant increase in TED flux. When
propylene glycol-citrate buffer-ethanol (80:10:10) was em-
ployed as the TED vehicle, the saturated solubility increased
to 72 000 µg/mL and the TED flux increased to 1.50 µg/cm2/h
(TED flux ratio 9.19; all measured at pH 4.8).
Conclusion
From these experiments, 56 emerged as having the superior
combination of required properties when compared to the
alternative lead compounds. Sulfonamide 56 had excellent PCP
enzyme activity when measured with a peptide substrate (IC50
11.3 ( 3.7 nM; calcd Ki 8.7 ( 2.9 nM; mean ( sem; n ) 4)
or with the endogenous substrate procollagen (IC50 3.4 ( 1.0
nM; mean ( sem; n ) 3) and demonstrates excellent selectivity
over the MMPs involved in wound healing (>10 000-fold). We
believe the (cyclohexyl)propyl side chain played an important
role in confirming activity for PCP and regulating selectivity
over the MMPs by binding in a deep lipophilic pocket adjacent
to the catalytic Zn2+ in the active site of PCP. In the fibroplasia
model, 56 inhibited deposition of insoluble collagen by 76 (
2% at 10 µM (mean ( sem; n ) 3) and was superior to the
ionized PCP inhibitors 25, 26, 47, and 67, which were less
effective even at higher concentrations; performance in this
model proved to be the deciding factor in compound selection.
Compound 56 was a crystalline solid with good solubility in
both aqueous and organic solvents suitable for TED formulation
and had satisfactory aqueous stability under autoclave condi-
tions. Furthermore, 56 was very effective at penetrating human
skin in vitro with a TED flux of 1.5 µg/cm2/h, which compares
favorably with values for agents that are known to penetrate
skin well in vivo. Based on this profile, 56 (UK-421,045) was
selected as a candidate for further preclinical evaluation as a
topically applied, dermal antiscarring agent.
Procollagen Cleavage Assay. This assay is based on the method
of Greenspan and co-workers.25 PCP activity was assessed by
measuring C-terminal peptide cleavage of the natural substrate,
procollagen-1. The C-terminal peptide was radioactively labeled
on Tryptophan residues, which are only present in this part of
procollagen. Human PCP (16 µg) was incubated with 0.01% bovine
3
serum albumin and H-procollagen (27 µg, specific activity 16.9
× 106 cpm/mg) in 50 mM Tris, pH 7.4, 150 mM NaCl, 15 mM
CaCl2, and 0.02% Brij 35 for 18 h at 37 °C. Procollagen was then
precipitated by incubation with 0.01% sirius red at room temperature
for 30 min. After centrifugation, the radioactivity in the supernatant
was measured by scintillation counting. IC50 values were calculated
by plotting % inhibition values against compound concentrations
using Tessela add-in for Excel spreadsheet.
Fibroplasia Model. This assay is based on that of Clark19a and
utilizes adult human dermal fibroblasts (Clonetics). Cells were
seeded at confluency in 24-well plates (1 × 105 cells/well/mL) and
cultured in Dulbecco’s modified eagle’s medium (Gibco BRL),
supplemented with 10% fetal calf serum (PAA laboratories), 50
IU/mL penicillin, 50 µg/mL streptomycin (Gibco BRL), 300 µM
proline (Sigma), 50 µg/mL ascorbate (Sigma), and 3 ng/mL TGF-
â1(Sigma), at 37 °C and 5% CO2. The inhibitor was added at
varying concentrations and medium was changed twice a week.
Samples for collagen analysis and histology were taken after 8 days
of culture. For collagen analysis, cell pellets were washed twice in
water and then processed for hydroxy-4-proline analysis using
HPLC.

3452 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Fish et al.
In Vitro Transepidermal Penetration Experiments. The ability
of test compounds to penetrate human cadaver skin was assessed
using a Microette Transdermal Diffusion Cell autosampling system
with static Franz diffusion cells. Experiments were routinely
performed at pH 4.3-6.5 to mimic the natural pH of healthy adult
skin. The receptor compartments and reservoirs were filled with
receptor fluid (degassed phosphate buffered saline, pH 7.4) at 37
( 1 °C, resulting in a skin surface temperature of 32 ( 1°C. Split
thickness human cadaver skin (300-500 µm) was sandwiched
between the donor and the receptor compartments, with the stratum
corneum side facing the donor compartment. The apparatus was
left to equilibrate for 1 h prior to application of a saturated solution
of test compound in 50% v/v aqueous propylene glycol (PG; 200
µL). [14C]Mannitol (4 µCi/mL) was added to the formulation to
test for membrane integrity. 5-{(1R)-4-Cyclohexyl-1-[2-(hydroxy-
lamino)-2-oxoethyl]butyl}-1,2,4-oxadiazole-3-carboxamide (109),
which had been shown to flux well across the skin, was added at
a fixed concentration (90% saturated solubility) to the test vehicles
to act as internal reference compound to adjust for variability in
skin donor or experimental conditions. Samples of receptor fluid
from each cell were collected automatically at preprogrammed
intervals between 0 and 24 h. The receptor fluid samples removed
were replaced with fresh phosphate buffered saline from reservoirs.
Concentrations of test compound were measured by LC-MS-MS
analysis and membrane integrity was determined by the concentra-
tion of 14C-mannitol in the receptor fluid. Membranes were
classified as intact when the percentage mannitol penetrating did
not exceed 3% of the total applied dose after 24 h. The flux across
skin at steady state (µg/cm2/h) was calculated by taking the gradient
of the linear portion of a plot of the cumulative amount of
compound against time (15-24 h) and dividing by the exposed
surface area of the skin (0.63 cm2). The TED flux values are the
mean of 6-10 experiments. The flux ratio was then calculated by
dividing the flux of the test compound by the flux of the reference
(109) over the same time period. Experiments with propylene
glycol/citrate buffer/ethanol (80:10:10 gelled with 2.5% Carbopol)
as the TED formulation were performed in a similar matter.
Solubility in Phosphate Buffered Saline (PBS). The test
compound (ca. 1-5 mg) was weighed into a 2 mL eppendorf tube
in triplicate and then phosphate buffered saline (400 µL, 0.01M;
pH adjusted to pH 6.5) was added. These samples were shaken at
room temperature overnight and were then centrifuged at 15 000
rpm for 1 h. The supernatant was transferred to a fresh eppendorf
tube and centrifuged again at 15 000 rpm for 1 h. The pH of the
final supernatant was recorded and then diluted appropriately in
20/80 acetonitrile/water. Samples were analyzed by HPLC (Luna
(Phenomenex) C18, 150 × 4.6 mm, 5 µ pore size; gradient elution
20-90% 0.1% TFA MeCN/0.1% TFA water; flow rate ) 1 mL/
min; detection λ ) 220 nm).
Solubility in TED Formulation. Sufficient test compound was
weighed into 20 mL glass centrifuge tubes to ensure saturated
solubility. Aqueous propylene glycol (2 mL, 50% v/v) was added
and the tubes rotated on a blood tube rotator (model SB1, Stuart
Scientific, U.K.) for 72 h to ensure equilibration. The tubes were
inspected after 24 h to ensure an excess of test compound was
present. Tubes were centrifuged twice at 4000 rpm for 60 min,
and the supernatant was retained for use in penetration experiments.
A sample of the supernatant was taken, diluted using acetonitrile-
Milli-Q water (20:80) at ratios of 1 in 10, 1 in 100, and 1 in 1000
and then analyzed by HPLC. Experiments with propylene glycol/
citrate buffer/ethanol (80:10:10 gelled with 2.5% Carbopol) as the
TED formulation were performed in a similar matter.
Chemistry. General Details. Melting points (mp) were deter-
mined using open glass capillary tubes and either Gallenkamp or
Electrothermal melting point apparatus and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) data were obtained using a
Varian Unity 300 or a Varian Inova 400. Low-resolution mass
spectral (LRMS) data were recorded on a Fisons Instruments Trio
1000 (thermaspray) or a Finnigan Mat. TSQ 7000 (APCI). The
calculated and observed ions quoted refer to the isotopic composi-
tion of lowest mass. Elemental combustion analyses (Anal.) were
determined by Exeter Analytical U.K. Ltd. Column chromatography
was performed using Merck silica gel 60 (0.040-0.063 mm). Log
D7.4 determinations were similar to those described by Stopher and
McClean.26 pKas were determined by Sirius Analytical Instruments
Ltd (U.K.). When it was not possible to measure ionization
constants, values were calculated using Advanced Chemical
Development, Inc. (Toronto) software (ACD/pKa DB).
General Methods for the Synthesis of PCP Inhibitors 7-71.
Preparation of N-Hydroxy Amidines 73. (Method A): To a
solution of nitrile 72 (1.0 equiv) in methanol cooled to 0 °C were
added hydroxylamine hydrochloride (1.0 equiv) and triethylamine
(1.0 equiv), and the mixture was stirred at room temperature (or
heated to reflux) overnight. The mixture was cooled in an ice-bath
and the solid was collected by filtration, washed with methanol,
and dried to give the N-hydroxy amidine product 73.
Preparation of N-Acyloxy Amidines 75. (Method B): To a
solution of (2R)-2-(2-tert-butoxy-2-oxoethyl)-5-cyclohexylpentanoic
acid 74 (1.0 equiv) in dichloromethane (DCM) was added CDI (1.0
equiv), and the reaction was stirred at room temperature overnight.
The corresponding N-hydroxy amidine 73 (1.0 equiv) was added,
and the mixture was stirred at room temperature for 72 h (or heated
at reflux for 2 to 8 h). The solvent was removed in vacuo and the
residue was dissolved in ethyl acetate and washed with water. The
organic layer was dried (Na2SO4) and the solvent was evaporated.
The residue was taken crude onto the next stage.
Preparation of Oxadiazoles 76. (Method C): A solution of of
N-acyloxy amidine 75 (1.0 equiv) in xylene was heated to reflux
overnight. The reaction mixture was cooled to room temperature,
the solvent was removed in vacuo, and the residue was purified
by flash chromatography on silica gel to afford the oxadiazole pro-
duct 76.
Preparation of Succinic Acid 77. (Method D): To a solution
of succinate t-butyl ester 76 (1 equiv) in DCM was added TFA in
a 1:1 ratio, and the resulting solution was stirred at room temperature
for 2 h. The solvent and excess TFA were removed in vacuo, and
the residue was azeotroped with toluene to give a solid that was
triturated with diisopropyl ether (DIPE). The solid was collected
by filtration and dried under high vacuum to give the succinic acid
product 77.
Preparation of Hydroxamic Acid with iso-Butylchlorofor-
mate. (Method E): To a solution of succinic acid 77 (1 equiv) in
THF was added triethylamine (2 equiv). The reaction was cooled
to 0 °C in an ice bath, then iso-butylchloroformate (1 equiv) was
added and a precipitate began to form immediately. The mixture
was stirred for 1 h, then O-trimethylsilyl hydroxylamine (3.2 equiv)
was added, and the reaction was warmed to room temperature and
stirred overnight. Methanol was added, stirring was continued for
1.5 h, and then the solvent was removed in vacuo. The residue
was dissolved in ethyl acetate and washed with water. The organic
layer was dried (MgSO4) and the solvent was evaporated in vacuo.
The residue was purified by chromatography on silica gel to give
the hydroxamic acid product.
Preparation of Hydroxamic Acid with 1,1-Carbonyldiimida-
zole. (Method F): To a solution of succinic acid 77 (1 equiv) in
THF was added CDI (1.2 equiv). The mixture was stirred for 1 h,
then O-trimethylsilyl hydroxylamine (3.2 equiv) was added and the
reaction was stirred at room temperature overnight. Methanol was
added, stirring was continued for 1 h, and then the solvent was
removed in vacuo. The residue was purified by chromatography
on silica gel to give the hydroxamic acid product.
Hydrolytic Stability Measurement. The autoclave solution
stability of test compounds was determined between the pH range
of 1 to 12.6 in 0.2 M aqueous buffers. The test compound was
dissolved at 50 µg/mL in a number of aqueous buffers and
autoclaved at 121 °C for 15 min. HPLC analysis was used to
examine compound solution stability by comparing the percent main
band of test compound remaining with a control sample (T0, stored
at 4 °C).
Complete experimental procedures, along with spectroscopic and
analytical data, for the preparation of all intermediates and target

Inhibitors of Procollagen C-Proteinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3453
compounds for the PCP inhibitors 7-71 can be found in the
Supporting Information.
(1H, m), 8.05 (1H, s), 10.9-11.4 (2H, br s); LRMS m/z 414
(MNa+); Anal. (C18H24N5O5‚H2O‚0.1CH2Cl2‚0.1AcOH) C, H, N.
2-(5-{(1R)-4-Cyclohexyl-1-[2-(hydroxyamino)-2-oxoethyl]bu-
tyl}-1,2,4-oxadiazol-3-yl)isonicotinic Acid (26). Ethyl 2-[Amino-
(hydroxyimino)methyl]isonicotinate (84). Compound 84 was
obtained as a pale yellow solid (2.8 g) from ethyl 2-cyanoisoni-
cotinate by general method A: 1H NMR (DMSO-d6) δ 1.28 (3H,
t), 4.34 (2H, q), 5.87 (2H, s), 7.78 (1H, d), 8.25 (1H, s), 8.70 (1H,
d), 10.03 (1H, s); LRMS m/z 210 (MH+).
Ethyl 2-{5-[(1R)-1-(2-tert-Butoxy-2-oxoethyl)-4-cyclohexyl-
butyl]-1,2,4-oxadiazol-3-yl}isonicotinate (85). Compound 85 was
obtained as a colorless oil (18.9 g) from compounds 74 and 84 by
general methods B then C: 1H NMR (CDCl3) δ 0.80-0.92 (2H,
m), 1.08-1.48 (20H, m), 1.57-1.71 (5H, m), 1.71-1.94 (2H, m),
2.69 (1H, dd), 2.92 (1H, dd), 3.53-3.61 (1H, m), 4.46 (2H, q)
7.95 (1H, d), 8.64 (1H, s), 9.92 (1H, d); LRMS m/z 494 (MNa+),
472 (MH+); Anal. (C26H37N3O5‚EtOAc) C, H, N.
(3R)-6-Cyclohexyl-3-{3-[4-(ethoxycarbonyl)-2-pyridinyl]-1,2,4-
oxadiazol-5-yl}hexanoic Acid (77: R ) 4-Ethoxycarbonyl-2-
pyridine). (3R)-6-Cyclohexyl-3-{3-[4-(ethoxycarbonyl)-2-pyridinyl]-
1,2,4-oxadiazol-5-yl}hexanoic acid was obtained as a pale yellow
oil (4.6 g) by general method D: 1H NMR (CDCl3) δ 0.74-0.87
(2H, m), 1.04-1.38 (8H, m), 1.41 (3H, t), 1.55-1.70 (5H, m),
1.70-1.91 (2H, m), 2.83 (1H, dd), 3.06 (1H, dd), 3.53-3.62 (1H,
m), 4.43 (2H, q), 8.04 (1H, d), 8.67 (1H, s), 8.90 (1H, d); LRMS
m/z 416 (MH+).
Ethyl 2-(5-{(1R)-4-Cyclohexyl-1-[2-(hydroxyamino)-2-oxoet-
hyl]butyl}-1,2,4-oxadiazol-3-yl)isonicotinate. Ethyl 2-(5-{(1R)-
4-cyclohexyl-1-[2-(hydroxyamino)-2-oxoethyl]butyl}-1,2,4-oxadiazol-
3-yl)isonicotinate was obtained as a colorless oil (0.44 g) by general
method E: 1H NMR (CDCl3) δ 0.75-0.89 (2H, m), 1.11-1.40
(8H, m), 1.45 (3H, t), 1.53-1.97 (7H, m), 2.68 (1H, dd), 2.84 (1H,
dd), 3.67-3.79 (1H, m), 4.48 (2H, q), 7.98 (1H, d), 8.61 (1H, s),
8.90 (1H, d); LRMS m/z 453 (MNa+), 431 (MH+).
2-(5-{(1R)-4-Cyclohexyl-1-[2-(hydroxyamino)-2-oxoethyl]bu-
tyl}-1,2,4-oxadiazol-3-yl)isonicotinic acid (26). To a solution of
ethyl 2-(5-{(1R)-4-cyclohexyl-1-[2-(hydroxyamino)-2-oxoethyl]bu-
tyl}-1,2,4-oxadiazol-3-yl)isonicotinate (0.44 g, 1.0 mmol) in MeOH
(20 mL) was added a solution of LiOH (0.09 g, 2.0 mmol) in water
(7 mL), and the reaction was stirred at room temperature for 2 h.
The solvent was removed by evaporation in vacuo, and the residue
was partioned between water (20 mL) and DCM (20 mL). The
aqueous layer was separated, acidified to pH 4 with aqueous HCl
(2 M), and then extracted with DCM (2 × 20 mL). The combined
organic extracts were dried (MgSO4), and the solvent was removed
in vacuo to give 26 as a white solid (0.04 g): mp 117-122 °C; 1H
NMR (CD3OD) δ 0.80-0.93 (2H, m), 1.06-1.47 (8H, m), 1.53-
1.75 (5H, m), 1.75-1.93 (2H, m), 2.62 (1H, dd), 2.74 (1H, dd),
3.62-3.73 (1H, m), 8.07 (1H, d), 8.62 (1H, s), 8.89 (1H, d); LRMS
m/z 403 (MH+).
(3R)-6-Cyclohexyl-N-hydroxy-3-(3-{[(2-hydroxy-1,1-dimeth-
ylethyl)amino]methyl}-1,2,4-oxadiazol-5-yl)hexanamide (47). tert-
Butyl (3R)-6-Cyclohexyl-3-(3-{[(2-hydroxy-1,1-dimethylethyl)-
amino]methyl}-1,2,4-oxadiazol-5-yl)hexanoate(76: R)CH2NHC-
(Me)2CH2OH). A solution of tosylate 99 (3.71 g, 7.32 mmol) and
2-amino-2-methyl-1-propanol (2.10 mL, 22.0 mmol) in THF (8 mL)
was heated at 40 °C in a sealed vessel for 2 h. The reaction mixture
was cooled to room temperature and partioned between EtOAc (100
mL) and saturated aqueous NaHCO3 (100 mL). The organic layer
was washed with saturated brine (50 mL) and dried (MgSO4), and
the solvents were evaporated in vacuo to afford 76 (R ) CH2NHC-
(Me)2CH2OH) as a colorless oil (2.52 g, 81%): 1H NMR (CDCl3)
δ 0.84 (2H, m), 1.10 (6H, s), 1.15-1.35 (9H, m), 1.40 (9H, s),
1.60-1.80 (6H, m), 2.60 (1H, dd), 2.70 (1H, br s), 2.79 (1H, dd),
3.29 (2H, s), 3.42 (1H, m), 3.83 (2H, s); LRMS m/z 424 (MH+).
(3R)-6-Cyclohexyl-3-(3-{[(2-hydroxy-1,1-dimethylethyl)ami-
no]methyl}-1,2,4-oxadiazol-5-yl)hexanoicAcid(77: R)CH2NHC-
(Me)2CH2OH). (3R)-6-Cyclohexyl-3-(3-{[(2-hydroxy-1,1-dimeth-
ylethyl)amino]methyl}-1,2,4-oxadiazol-5-yl)hexanoic acid was
obtained as a colorless oil (2.95 g) by general method D: 1H NMR
(CDCl3) δ 0.82 (2H, m), 1.10-1.30 (8H, m), 1.39 (6H, d), 1.60-
Preparation of PCP Inhibitors 1, 25, 26, 47, 56, 66, and 67.
5-{(1R)-4-Cyclohexyl-1-[2-(hydroxyamino)-2-oxoethyl]butyl}-
1,2,4-oxadiazole-3-carboxamide (1). The preparation of 1 has been
1
reported previously:10 mp 136-138 °C; H NMR (DMSO-d6) δ
0.80 (2H, m), 1.27-0.99 (8H, m), 1.73-1.46 (7H, m), 2.56-2.39
(2H, m), 3.47 (1H, m), 8.05 (1H, br s), 8.26 (1H, br s), 8.81 (1H,
br s), 10.50 (1H, br s); LRMS m/z 323 (MH+); Anal. (C15H24N4O4‚
H2O) C, H, N.
(3R)-6-Cyclohexyl-3-[3-(2,4-dioxo-1,2,3,4-tetrahydro-5-pyri-
midinyl)-1,2,4-oxadiazol-5-yl]-N-hydroxyhexanamide (25). N′-
Hydroxy-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinecarboximi-
damide (73: R ) 5-Uracil). A mixture of 5-cyanouracil (2.0 g,
14.6 mmol), hydroxylamine hydrochloride (1.4 g, 20.0 mmol), and
triethylamine (2.8 mL, 20.0 mmol) in MeOH (50 mL) was heated
at reflux for 18 h. The solid was collected by filtration, washed
with MeOH, and dried to give the N-hydroxy amidine as a pale
yellow solid (1.8 g, 72%). 1H NMR (DMSO-d6) δ 5.71 (2H, br s),
7.62 (1H, s), 11.0-11.4 (1H, br s); LRMS m/z 193 (MNa+), 171
(MH+).
tert-Butyl (3R)-6-Cyclohexyl-3-[3-(2,4-dioxo-1,2,3,4-tetrahy-
dro-5-pyrimidinyl)-1,2,4-oxadiazol-5-yl]hexanoate (76: R )
5-Uracil). To a solution of 74 (0.45 g, 1.5 mmol) in DCM (20
mL) was added 1-hydroxybenzotriazole (0.245 g, 1.8 mmol), 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.34 g,
1.8 mmol), followed by N-hydroxy amidine 73 (R ) 5-uracil; 0.305
g, 1.8 mmol). Dimethylformamide (10 mL) was added to aid
solubility, and the mixture was stirred at room temperature for 18
h. The solvents were removed by evaporation in vacuo, and the
residue was dissolved in water (100 mL) and extracted with ethyl
acetate (2 × 100 mL). The combined organic extracts were dried
(MgSO4) and the solvent was removed by evaporation in vacuo to
afford 75 (R ) 5-uracil). Compound 75 (R ) 5-uracil) was
suspended in xylene (20 mL) and heated to reflux for 4 h. The
reaction mixture was cooled to room temperature, the solvent was
removed by evaporation in vacuo, and the residue was purified by
chromatography on silica gel using DCM-methanol (95:5) as eluent
to afford 76 (R ) 5-uracil) as a pale yellow solid (0.60 g, 92%):
1H NMR (DMSO-d6) δ 0.73-0.95 (2H, m), 1.03-1.20 (17H, m),
1.52-1.73 (7H, m), 2.64-2.74 (2H, m), 8.00 (1H, s), 11.0-11.7
(2H, br s); LRMS m/z 455 (MNa+).
(3R)-6-Cyclohexyl-3-[3-(2,4-dioxo-1,2,3,4-tetrahydro-5-pyri-
midinyl)-1,2,4-oxadiazol-5-yl]hexanoic Acid (77: R ) 5-Uracil).
To a solution of 76 (R ) 5-uracil; 0.58 g, 1.34 mmol) in DCM (20
mL) was added TFA (10 mL), and the solution was stirred at room
temperature for 2 h. The solvents were removed by evaporation in
vacuo, and the residue was azeotroped with toluene (×3) to afford
a brown solid that was triturated with DIPE. The solid was collected
by filtration and dried to give 77 (R ) 5-uracil) as a white solid
1
(0.40 g, 79%): mp 240-244 °C; H NMR (DMSO-d6) δ 0.72-
0.89 (2H, m), 1.03-1.17 (8H, m), 1.53-1.72 (7H, m), 2.66-2.81
(2H, m), 3.36-3.47 (1H, m), 8.01 (1H, s), 11.32 (1H, br s), 11.38
(1H, br s), 12.05-12.30 (1H, br s); LRMS m/z 375 (M - H).
(3R)-6-Cyclohexyl-3-[3-(2,4-dioxo-1,2,3,4-tetrahydro-5-pyri-
midinyl)-1,2,4-oxadiazol-5-yl]-N-hydroxyhexanamide (25). iso-
Butylchloroformate (0.11 mL, 0.9 mmol) was added to a stirred
solution of 77 (R ) 5-uracil; 0.30 g, 0.8 mmol) and triethylamine
(0.225 mL, 1.6 mmol) in THF (10 mL) at 0 °C, and the mixture
was stirred for 1 h. O-Trimethylsilyl hydroxylamine (0.32 mL, 2.6
mmol) was added, and the mixture was warmed to room temper-
ature and stirred for 2 h. MeOH (10 mL) was then added and the
reaction was stirred for 1 h. The solvents were evaporated in vacuo,
and the residue was partitioned between aqueous HCl (20 mL, 2
M) and EtOAc (100 mL). The aqueous layer was extracted with
EtOAc (2 × 100 mL), and the combined organic extracts were
dried (MgSO4) and evaporated in vacuo. The residue was purified
by chromatography on silica gel with DCM-methanol-acetic acid
(90:10:0.5) as eluent to give 25 as a white solid (0.19 g, 61%):
1
mp 210 °C; H NMR (DMSO-d6) δ 0.74-0.89 (2H, m), 1.06-
1.24 (8H, m), 1.53-1.69 (7H, m), 2.66-2.81 (2H, m), 3.39-3.47

3454 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Fish et al.
1.80 (7H, m), 2.78 (1H, dd), 2.92 (1H, dd), 3.42 (1H, m), 3.63
(2H, s), 4.24 (2H, m); LRMS m/z 368 (MH+).
and dried to afford acid 77 (R ) CH2NHSO2Me) as an off-white
solid (7.06 g, 87%): 1H NMR (CD3OD) δ 0.83 (2H, m), 1.10-
1.35 (8H, m), 1.60-1.80 (7H, m), 2.75 (1H, dd), 2.85 (1H, dd),
2.97 (3H, s), 3.48 (1H, m), 4.39 (2H, s); LRMS m/z 396 (MNa+);
Anal. (C16H27N3O5S) C, H, N.
(3R)-6-Cyclohexyl-N-hydroxy-3-(3-{[(2-hydroxy-1,1-dimeth-
ylethyl)amino]methyl}-1,2,4-oxadiazol-5-yl)hexanamide (47). Com-
pound 47 was obtained as a white foam (980 mg, 52%) by general
1
method E: mp 49-51 °C; H NMR (DMSO-d6) δ 0.80 (2H, m),
(3R)-6-Cyclohexyl-N-hydroxy-3-(3-{[(methylsulfonyl)amino]-
methyl}-1,2,4-oxadiazol-5-yl)hexanamide (56). A solution of 77
(R ) CH2NHSO2Me; 6.80 g, 18.2 mmol) and 2,6-lutidine (2.3 mL,
20 mmol) in THF (100 mL) at 0 °C under a nitrogen atmosphere
was treated with iso-butyl chloroformate (2.6 mL, 20 mmol) and
stirred at 0 °C for 1.5 h. O-(Trimethylsilyl)hydroxylamine (4.9 mL,
40.0 mmol) was added, and the reaction mixture was stirred for
18 h, warming to room temperature over this period of time. MeOH
was then added and the reaction mixture was stirred for an additional
2 h. The solvent was removed under reduced pressure, and the
residue was dissolved in EtOAc (400 mL). The organic solution
was washed with dilute HCl (350 mL, 1 M) and brine (200 mL),
dried (MgSO4), and then evaporated in vacuo. The solid residue
was recrystallized from hot EtOAc to afford hydroxamic acid 56
0.98 (6H, s), 1.05-1.25 (8H, m), 1.50-1.70 (7H, m), 2.40 (2H,
m), 3.20 (2H, m), 3.40 (1H, m), 3.73 (2H, s), 4.43 (1H, br s), 8.62
(1H, br s), 10.38 (1H, br s); LRMS m/z 383 (MH+); Anal.
(C19H34N4O4‚0.05H2O) C, H, N.
(3R)-6-Cyclohexyl-N-hydroxy-3-(3-{[(methylsulfonyl)amino]-
methyl}-1,2,4-oxadiazol-5-yl)hexanamide (56). (1Z)-N′-Hydroxy-
2-[(methylsulfonyl)amino]ethanimidamide(73: R)CH2NHSO2Me).
A solution of N-(cyanomethyl)methanesulfonamide (10.85 g, 81.0
mmol) in EtOH (370 mL) was treated with hydroxylamine
hydrochloride (5.63 g, 81.0 mmol) followed by a solution of NaOH
(3.24 g, 81.0 mmol) in water (125 mL), and the mixture was stirred
at room temperature for 20 h. All volatile solvents were removed
by evaporation under reduced pressure. The residue was extracted
with hot EtOH (×3) and decanted from the solid residues (NaCl).
The combined organic extracts were cooled, which gave a
precipitate. The solid was collected by filtration, washed with Et2O,
and dried to afford the N-hydroxy amidine as white crystals (9.77
g, 72%): 1H NMR (DMSO-d6) δ 2.87 (3H, s), 3.49 (2H, s), 5.22
(2H, br s), 7.09 (1H, br s), 9.00 (1H, s); LRMS m/z 190 (MNa+);
Anal. (C3H9N3O3S‚0.1H2O) C, H, N.
tert-Butyl (3R)-6-Cyclohexyl-3-(3-{[(methylsulfonyl)amino]-
methyl}-1,2,4-oxadiazol-5-yl)hexanoate (76: R ) CH2NHSO2Me).
Sodium (2R)-2-(2-tert-butoxy-2-oxoethyl)-5-cyclohexylpentanoate
(i.e., 74 sodium salt; 18.68 g, 58.4 mmol) was partitioned between
10% aqueous citric acid solution (190 mL) and EtOAc (190 mL).
The organic layer was washed with brine, dried (MgSO4), and
filtered, and the solvents were removed under reduced pressure to
afford the acid 74 (quant) as a colorless oil. A solution of 74 in
DCM (185 mL) was treated CDI (9.46 g, 58.4 mmol), and the
reaction mixture was stirred at room temperature for 1.5 h.
N-Hydroxy amidine 73 (R ) CH2NHSO2Me; 9.75 g, 58.4 mmol)
was added portionwise, but required the addition of DMF (50 mL)
to dissolve the solid completely. The reaction mixture was stirred
at room temperature for 18 h. The solvents were removed by
evaporation under reduced pressure. The residue was dissolved on
EtOAc (500 mL), washed with water (2 × 500 mL) and brine (200
mL), and dried (MgSO4), and the solvent was removed by
evaporation under reduced pressure to afford compound 75 (R )
CH2NHSO2Me) as a white solid (26.3 g, ∼100%): 1H NMR
(DMSO-d6) δ 0.81 (2H, m), 1.00-1.30 (9H, m), 1.37 (9H, s), 1.40-
1.65 (6H, m), 2.39 (1H, dd), 2.55 (1H, dd), 2.79 (1H, m), 2.94
(3H, s), 3.62 (2H, s), 6.19 (2H, br s), 7.30 (1H, br s); LRMS: m/z
470 (MNa+). Compound 75 (R ) CH2NHSO2Me; 26.0 g, 58.2
mmol) was dissolved in xylene (500 mL) and heated at 130 °C
under a nitrogen atmosphere for 20 h. The solvents were evaporated
in vacuo, and the crude reaction mixture was purified by column
chromatography on silica gel using pentane then a gradient of
DCM-MeOH (100:0 to 90:10) as eluent. Appropriate fractions
were combined, and the solvent was removed under reduced
pressure to give a brown residue that was then triturated with
pentane to afford the oxadiazole 76 (R ) CH2NHSO2Me) as a white
solid (9.43 g, 38%): 1H NMR (CD3OD) δ 0.83 (2H, m), 1.10-
1.30 (9H, m), 1.38 (9H, s), 1.60-1.75 (7H, m), 2.67 (1H, dd), 2.79
(1H, dd), 2.98 (3H, s), 3.42 (1H, m), 4.39 (2H, s); LRMS m/z 452
(MNa+).
as a white solid (6.19 g, 88%): [R]25 +18.5 (c 2.0, MeOH); mp
D
1
115-116 °C; H NMR (DMSO-d6) δ 0.80 (2H, m), 1.00-1.15
(8H, m), 1.50-1.70 (8H, m), 2.50-2.55 (2H, obs), 2.89 (3H, s),
3.41 (1H, m), 4.24 (2H, d), 7.63 (1H, br s), 8.62 (1H, br s), 10.38
(1H, br s); LRMS m/z 411 (MNa+); Anal. (C16H28N4O5S) C, H, N.
(3R)-6-Cyclohexyl-N-hydroxy-3-{3-[1-(methylsulfonyl)-4-pi-
peridinyl]-1,2,4-oxadiazol-5-yl}hexanamide (66). 1-(Methylsul-
fonyl)-4-piperidinecarbonitrile (72: R ) 1-(Methylsulfonyl)-4-
piperidinyl). 1-(Methylsulfonyl)-4-piperidinecarbonitrile was ob-
tained as a white solid (2.48 g, 42%; Et2O): 1H NMR (CD3OD) δ
1.85 (2H, m), 2.01 (2H, m), 2.81 (3H, s), 2.95 (1H, m), 3.18 (2H,
m), 3.41 (2H, m); LRMS m/z 211 (MNa+); Anal. (C7H12N2O2S)
C, H, N.
N′-Hydroxy-1-(methylsulfonyl)-4-piperidinecarboximida-
mide (73: R ) 1-(Methylsulfonyl)-4-piperidinyl). N′-Hydroxy-
1-(methylsulfonyl)-4-piperidinecarboximidamide was obtained as
a white solid by general method A and was used in the next step
without further purification.
tert-Butyl(3R)-3-{[({(Z)-amino[1-(methylsulfonyl)-4-piperidinyl]-
methylidene}amino)oxy]carbonyl}-6-cyclohexylhexanoate (75:
R ) 1-(Methylsulfonyl)-4-piperidinyl). tert-Butyl (3R)-3-{[({(Z)-
amino[1-(methylsulfonyl)-4-piperidinyl]methylidene}amino)oxy]-
carbonyl}-6-cyclohexylhexanoate (1.50 g, 1.83 mmol) was prepared
from 74 (0.55 g, 1.83 mmol) and 73 (R ) 1-(methylsulfonyl)-4-
piperidinyl); 1.20 g, 2.0 mmol) by general method B.
tert-Butyl (3R)-6-Cyclohexyl-3-{3-[1-(methylsulfonyl)-4-pip-
eridinyl]-1,2,4-oxadiazol-5-yl}hexanoate (76: R ) 1-(Methyl-
sulfonyl)-4-piperidinyl). A solution of 75 (R ) 1-(methylsulfonyl)-
4-piperidinyl; 1.50 g, 1.83 mmol) in toluene (100 mL) was treated
with pyridine (90 µL, 1.83 mmol) and anhydrous ZnCl2 (150 mg,
1.83 mmol), and the mixture was heated at reflux for 20 h. The
cooled reaction mixture was diluted with EtOAc (100 mL) and
washed with water (100 mL) and saturated brine (100 mL). The
organic layer was dried (Na2SO4) and the solvent was evaporated
in vacuo. The residue was purified by chromatography on silica
gel using cyclohexane-EtOAc (90:10 to 50:50) as eluent to give
76 (R ) 1-(methylsulfonyl)-4-piperidinyl) as an oil, which was
recrystallization from pentane as a white solid (320 mg, 36%): 1H
NMR (CDCl3) δ 0.83 (2H, m), 1.10-1.35 (8H, m), 1.40 (9H, s),
1.60-1.80 (7H, m), 1.99 (2H, m), 2.14 (2H, br d), 2.60 (1H, dd),
2.71-2.80 (4H, dd and s), 2.95 (3H, m), 3.41 (1H, m), 3.74 (2H,
m); LRMS m/z 506 (MNa+); Anal. (C24H41N3O5S) C, H, N.
(3R)-6-Cyclohexyl-3-(3-{[(methylsulfonyl)amino]methyl}-1,2,4-
oxadiazol-5-yl)hexanoic Acid (77: R ) CH2NHSO2Me). A
solution of 76 (R ) CH2NHSO2Me; 9.36 g, 21.8 mmol) in toluene
(100 mL) was treated with TFA (50 mL) and stirred under a
nitrogen atmosphere at room temperature for 20 h. The solvent was
removed under reduced pressure and azeotroped with EtOAc. The
residue was dissolved in EtOAc (250 mL) and washed with H2O
followed by brine. The organic layer was dried (MgSO4) and the
solvent was removed under reduced pressure. The residue was
triturated with Et2O to yield a solid that was collected by filtration
(3R)-6-Cyclohexyl-3-{3-[1-(methylsulfonyl)-4-piperidinyl]-
1,2,4-oxadiazol-5-yl}hexanoic Acid (77: R ) 1-(Methylsulfonyl)-
4-piperidinyl). (3R)-6-Cyclohexyl-3-{3-[1-(methylsulfonyl)-4-
piperidinyl]-1,2,4-oxadiazol-5-yl}hexanoic acid was obtained as a
white solid (245 mg, 89%; triturated with pentane) by general
method D: 1H NMR (CDCl3) δ 0.81 (2H, m), 1.05-1.35 (8H, m),
1.55-1.85 (7H, m), 1.97 (2H, m), 2.10 (2H, m), 2.70-2.80 (4H,
dd and s), 2.93 (4H, m), 3.45 (1H, m), 3.72 (2H, m); LRMS m/z
426 (M - H); Anal. (C20H33N3O5S) C, H, N.

Inhibitors of Procollagen C-Proteinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3455
(3R)-6-Cyclohexyl-N-hydroxy-3-{3-[1-(methylsulfonyl)-4-pi-
peridinyl]-1,2,4-oxadiazol-5-yl}hexanamide (66). (3R)-6-Cyclo-
hexyl-N-hydroxy-3-{3-[1-(methylsulfonyl)-4-piperidinyl]-1,2,4-
oxadiazol-5-yl}hexanamide was obtained as fluffy white solid (204
mg, 85%; triturated with DIPE) by general method E: 1H NMR
(CD3OD) δ 0.83 (2H, m), 1.05-1.35 (8H, m), 1.55-1.75 (7H, m),
1.88 (2H, m), 2.09 (2H, m), 2.48 (1H, dd), 2.60 (1H, dd), 2.82
(3H, s), 2.98 (3H, m), 3.52 (1H, m), 3.70 (2H, m); LRMS m/z 465
(MNa+); Anal. (C20H34N4O5S) C, H, N.
(3R)-6-Cyclohexyl-N-hydroxy-3-(3-{[(phenylsulfonyl)amino]-
carbonyl}-1,2,4-oxadiazol-5-yl)hexanamide (67). tert-Butyl (3R)-
6-Cyclohexyl-3-(3-{[(phenylsulfonyl)amino]carbonyl}-1,2,4-ox-
adiazol-5-yl)hexanoate (107: R ) H). To a suspension of sodium
hydride (0.65 g of 60% dispersion in mineral oil, 16.4 mmol) in
THF (80 mL) at 0 °C was added dropwise a solution of 106 (3.0
g, 8.2 mmol) and freshly distilled benzenesulfonyl chloride (1.15
mL, 9.0 mmol) in THF (20 mL). The reaction was stirred at 0 °C
for 3 h then quenched with aqueous HCl (2 M) and warmed to
room temperature. The mixture was diluted with water (50 mL)
and then extracted with ethyl acetate (3 × 100 mL). The combined
organic extracts were dried (Na2SO4), and the solvent was removed
in vacuo. The residue was purified by chromatography on a Biotage
405 cartridge with DCM-methanol (100:0 then 95:5) as eluent to
give 107 (R ) H) as a clear oil (2.8 g, 67%): 1H NMR (CDCl3)
δ 0.78-0.91 (2H, m), 1.11-1.55 (18H, m), 1.55-1.79 (6H, m),
2.65 (1H, dd), 2.80 (1H, dd), 3.44-3.52 (1H, m), 7.56 (2H, dd),
7.66 (1H, dd), 8.17 (2H, d); LRMS m/z 504 (M - H); Anal.
(C25H35N3O6S‚0.5H2O) C, H, N.
Pereira, Simon Planken, Michelle Ronald, and John Wardale.
We thank Professor Efrat Kessler for kindly providing H-
procollagen. We also thank members of the Physical Sciences
group for spectroscopic and analytical services.
3
Supporting Information Available: Complete experimental
details, along with spectroscopic and analytical data, for the
preparation of all intermediates and target compounds for the PCP
inhibitors 7-71. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Trojanowska, M.; LeRoy, E. C.; Eckes, B.; Krieg, T. Pathogenesis
of Fibrosis: Type I Collagen and the Skin. J. Mol. Med. 1998, 76,
266-274.
(2) Border, W. A.; Noble, N. A. Transforming Growth Factor-â in Tissue
Fibrosis. N. Engl. J. Med. 1994, 331 (10), 1286-1292.
(3) Lee, S.; Solow-Corero, D. E.; Kessler, E.; Takahara, K.; Greenspan,
D. S. Transforming Growth Factor-â Regulation of Bone Morpho-
genetic Protein-1/Procollagen C-Proteinase and Related Proteins in
Fibrogenic Cell and Keratinocytes. J. Biol. Chem. 1997, 272 (30),
19059-19066.
(4) Kessler, E. Procollagen C-Proteinase. The Handbook of Proteolytic
Enzymes; Academic Press: New York, 1998; pp 1-5.
(5) Wozney, J. M.; Rosen, V.; Celeste, A. J.; Mitsock, L. M.; Whitters,
M. J.; Kriz, R. W.; Hewick, R. M.; Wang, E. A. Novel Regulators
of Bone Formation: Molecular Clones and Activities. Science 1988,
242, 1528-1531.
(6) Kessler, E.; Takahara, T.; Biniamov, L.; Brusel, M.; Greenspan, D.
S. Bone Morphogenetic Protein-1: The Type-1 Procollagen C-
Proteinase. Science 1996, 271, 360-362.
(7) Panchenko, M. V.; Stetler-Stevenson, W. G.; Trubetskoy, O. V.;
Gacheru, S. N.; Kagan, H. M. Metalloproteinase Activity Secreted
by Fibrogenic Cells in the Processing of Prolyl Oxidase. J. Biol.
Chem. 1996, 271, 7113-7119.
(8) Lijnen, H. R.; Collen, D. Role of Plasminogen and MMP Systems
in Wound Healing. Plasminogen 2003, 189-200.
(9) For a recent review, see: Turtle, E. D.; Ho, W.-B. Inhibition of
Procollagen C-Proteinase: Fibrosis and Beyond. Expert Opin. Ther.
Pat. 2004, 14, 1185-1197.
(10) (a) Bailey, S.; Billotte, S.; Derrick, A. M.; Fish, P. V.; James, K.;
Thomson, N. M. Preparation of Oxazolyl- and Oxadiazolyl-Contain-
ing Hydroxamic Acids Useful as Procollagen C-Proteinase Inhibitors.
WO 2001047901, 2001. (b) Bailey, S.; Fish, P. V.; James, K.;
McElroy, A. Discovery of UK-383,367, A Potent and Selective
Nonpeptidic Inhibitor of Procollagen C-Proteinase for the Treatment
of Dermal Scarring. Abstracts of Papers, 227th ACS National
Meeting, Anaheim, CA, March 28-April 1, 2004; American Chemi-
cal Society: Washington, DC, 2004. (c) Allan, G. A.; Gedge, J. I.;
Nedderman, A. N. R.; Roffey, S. J.; Small, H. F.; Webster, R.
Pharmacokinetics and Metabolism of UK-383,367 in Rats and Dogss
A Rationale for Long-Lived Plasma Radioactivity. Xenobiotica 2006,
36, 399-418.
(11) Fray, M. J.; Dickinson, R. P. Discovery of Potent and Selective
Succinyl Hydroxamate Inhibitors of Matrix Metalloprotease-3 (Stromel-
ysin-1). Bioorg. Med. Chem. Lett. 2001, 11, 571-574.
(12) (a) Barry, B. W. Dermatological Formulations, in Drugs and the
Pharmaceutical Sciences. Marcel Dekker, Inc.: New York, 1983;
pp 127-233. (b) Degim, I. T. Understanding Skin Penetration:
Computer Aided Modeling and Data Interpretation. Curr. Comp.-
Aided Drug Des. 2005, 1, 11-19.
(13) Roberts, M. S. In Structure-Permeability Considerations in Percu-
taneous Absorption in Prediction of Percutaneous Penetrations
Methods, Measurements, and Modelling; Scott, R. C., Guy, R. H.,
Hadgraft, J., Bodde, H. E., Eds.; IBC Technical Services: United
Kingdom, 1991; Vol. 2, pp 210-228.
(14) Kasting, G. B.; Smith, R. L.; Cooper, E. R. Effect of Lipid Solubility
and Molecular Size on Percutaneous Absorption. Pharmacol. Skin;
Karger: Basel, 1987; Vol. 1, pp 138-153.
(15) Hadgraft, J.; Valenta, C. pH, pKa, and Dermal Delivery. Int. J. Pharm.
2000, 200, 243-247.
(3R)-6-Cyclohexyl-3-(3-{[(phenylsulfonyl)amino]carbonyl}-
1,2,4-oxadiazol-5-yl)hexanoic Acid (108: R ) H). A solution of
107 (R ) H; 2.65 g, 1.0 mmol) and TFA (6 mL) in DCM (30 mL)
was stirred at room temperature for 2 h. A further portion of TFA
(1 mL) was added and stirring was continued for an additional 1
h. The solvents were evaporated in vacuo, and the residue was
azeotroped with toluene (×3) to give 108 (R ) H) as an oil (2.28
g): 1H NMR (DMSO-d6) δ 0.72-0.85 (2H, m), 1.01-1.26 (8H,
m), 1.52-1.69 (7H, m), 2.67-2.81 (2H, m), 3.39-3.50 (1H, m),
7.56-7.68 (2H, m), 7.68-7.76 (1H, m), 7.97 (2H, d); LRMS m/z
448 (M - H).
(3R)-6-Cyclohexyl-N-hydroxy-3-(3-{[(phenylsulfonyl)amino]-
carbonyl}-1,2,4-oxadiazol-5-yl)hexanamide (67). To a solution
of 108 (R ) H; 2.24 g, 5.0 mmol) in THF (40 mL) was added
2,6-lutidine (1.07 g, 10.0 mmol), the solution was cooled to 0 °C,
and then iso-butylchloroformate (0.66 mL, 5.05 mmol) was added.
The reaction was stirred at 0 °C for 1 h, then O-trimethylsilyl
hydroxylamine (1.83 mL, 15.0 mmol) was added, and the reaction
was warmed to room temperature and stirred overnight. Methanol
(25 mL) was added, and stirring was continued for 30 min. The
solvent was evaporated in vacuo, and the residue was partitioned
between ethyl acetate (75 mL) and aqueous HCl (35 mL, 2 M).
The aqueous layer was extracted with ethyl acetate (2 × 60 mL),
and the combined organic extracts were then dried (Na2SO4) and
the solvent was removed in vacuo. The residue was purified by
chromatography on a Biotage CP-18 reverse phase 40S cartridge
with acetonitrile-water (30:70 to 50:50) as eluent to afford 67 as
a foam. Recrystallization from toluene gave 67 as a white crystalline
1
solid (1.0 g, 51%): mp 149 °C; H NMR (DMSO-d6) δ 0.74-
0.89 (2H, m), 1.06-1.24 (8H, m), 1.53-1.69 (7H, m), 2.66-2.81
(2H, m), 3.39-3.47 (1H, m), 8.05 (1H, s), 10.9-11.4 (2H, br s);
LRMS m/z 414 (MNa+); Anal. Calcd. for C18H24N5O5‚H2O‚0.1CH2-
Cl2‚0.1AcOH) C, H, N.
Acknowledgment. We would like to thank our following
colleagues for their expert and enthusiastic technical as-
sistance: Lyndsey Addison, Yvonne Ailwood, Cathy Allan,
Louise Angel, Stephane Billotte, Gerwyn Bish, Mike Closier,
Usa Datta, Vikki Dawe, Neil Flanagan, Samantha Gaboardi,
John Harvey, Carol Loosley, Debbie Lovering, Liz King, Kerry
Malloy, Kathryn McAllister, Emma Newstead, Jane Nightingale,
Rachel Oni-orison, Julie Owen, Christele Pasquinet, Clint
(16) Potts, R. O.; Guy, R. H. A Predictive Algorithm for Skin Perme-
ability: The Effects of Molecular Size and Hydrogen Bond Activity.
Pharm. Res. 1995, 12, 1628-1633.
(17) To establish a baseline level of acceptable flux in our in vitro TED
model, a set of compounds with commercial precedence of successful
transdermal delivery were evaluated, utilizing the same vehicle and
test conditions.

3456 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Fish et al.
(18) Korbonits, D.; Bako, E. M.; Horvath, K. A Novel Ring Transforma-
tion of 1,2,4-Oxadiazoles. A Convenient General Synthesis of 3-
Aminopyrazolines from Primary Amines. J. Chem. Res. 1979, (S),
64-65.
(19) (a) Clark, R. A. F.; McCoy, G. A.; Folkvord, J. M.; McPherson, J.
M. TGF-â1 Stimulates Cultured Human Fibroblasts to Proliferate
and Produce Tissue-Like Fibroplasia: A Fibronectin Matrix-De-
pendent Event. J. Cell. Physiol. 1997, 170, 69-80. (b) Newstead,
E. L.; Gaboardi, S.; Huggins, J. P.; Greiling, D. Features of keloid
tissue can be mimicked in an in Vitro fibroplasias model. J. Dermatol.
Sci. 1998, 16 (suppl. 1), S128.
(20) Hartigan, N.; Garrigue-Antar, L.; Kadler, K. E. Bone Morphogenetic
Protein-1 (BMP-1). Identification of the Minimal Domain Structure
for Procollagen C-Proteinase Activity. J. Biol. Chem. 2003, 278,
18045-18049.
(21) Beekman, R.; Drijfhout, J. W.; Bloemhoff, W.; Ronday, H. K.; Tak,
P. P.; te Koppele, J. M. Convenient Fluorometric Assay for Matrix
Metalloproteinase Activity and its Application in Biological Media.
FEBS Lett. 1996, 390, 221-225.
(22) Hojima, Y.; van der Rest, M.; Prockop, D. J. Type I Procollagen
Carboxy-Terminal Proteinase from Chick Embryo Tendons. J. Biol.
Chem. 1985, 260, 15996-16003.
(23) Purification performed by Centre for Applied Microbiology &
Research, Porton Down, U.K.
(24) Biolise Software was purchased from BGM Labtech GmbH, Hanns-
Martin-Schleyer-Str.10, 77656 Offenburg, Germany.
(25) Lee, S.; Solow-Cordero, D. E.; Kessler, E.; Takahara, K.; Greenspan,
D. S. Transforming Growth Factor-â Regulation of Bone Morpho-
genetic Protein-1/Procollagen C-Proteinase and Related Proteins in
Fibrogenic Cells and Keratinocytes. J. Biol. Chem. 1997, 272,
19059-19066.
(26) Stopher, D.; McClean, S. An Improved Method for the Determination
of Distribution Coefficient. J. Pharm. Pharmacol. 1990, 42, 144.
JM061010Z