Design and synthesis of procollagen C-proteinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.10.067

Non-peptidic inhibitors of procollagen C-proteinase (PCP) were designed from substrate leads. Compounds were optimized for potency and selectivity, with N-substituted aryl sulfonamide hydroxamates having the best combination of these properties. Compounds

Full text of DOI:10.1016/j.bmcl.2012.10.067

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7397–7401
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of procollagen C-proteinase inhibitors
Eric Turtle, Nicholas Chow, Charles Yang, Sergio Sosa, Udo Bauer, Mitch Brenner, David Solow-Cordero,
⇑
Wen-Bin Ho
FibroGen Inc., 409 Illinois St, SF, CA 94158, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Non-peptidic inhibitors of procollagen C-proteinase (PCP) were designed from substrate leads. Com-
pounds were optimized for potency and selectivity, with N-substituted aryl sulfonamide hydroxamates
having the best combination of these properties. Compounds 89 and 60 have IC50 values of 10 and
80 nM, respectively, against PCP; excellent selectivity over MMP’s 1, 2, and 9; and activity in cell-based
collagen deposition assays.
Received 20 June 2012
Revised 11 October 2012
Accepted 15 October 2012
Available online 24 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Procollagen C-proteinase
Fibrosis
Hydroxamate
Fibrosis is a pathological condition in which the normal wound
healing process goes awry, culminating in the excessive production
and deposition of collagen, the major component of scar tissue, in
the extracellular matrix.1,2 The disregulated formation of scar tis-
sue may occur in any organ, hindering function and potentially
leading to organ failure. Post operative and dermal scarring may
result in a variety of complications including physical restriction
or disfigurement. Currently, there are no adequate therapies for fi-
brotic conditions; new anti-fibrotics are needed for both systemic
and topical applications.
substrates, we devised alternative scaffolds which were elaborated
through iterative modifications into promising leads.
Design of initial peptidic inhibitors was guided by studies of
truncated PCP substrates. PCP cleavage sequences in known en-
zyme substrates types I, II, and III collagen indicate a substrate
preference for P3: Met or Tyr; P2: Arg or Tyr; P1: Ala or Gly; P10:
Asp, P20: Asp, Glu, or Gln; P30: Ala or Pro. A series of short peptides
derived from procollagen I–III sequences were examined as possi-
ble PCP substrates, measuring relative rates of cleavage to identify
preferred peptide structures (Table 1). Of those tested, an octapep-
Procollagen C-proteinase (PCP, also known as bone morphoge-
netic protein-1) is a zinc metallo endopeptidase of the astacin fam-
ily that cleaves both pro-collagen and non-collagen substrates
involved in forming functional collagen fibrils. As such it is an
attractive anti-fibrotic target.3–5 Types I–III collagens are initially
excreted as pro-collagen peptides which contain solubilizing N
and C terminal sequences. Cleavage of the globular C-terminal se-
quence is necessary to convert soluble pro-collagens into fully
formed, insoluble, collagen fibrils. In addition, PCP processes pro-
lysyl oxidase to its active form, which catalyzes the crosslinking
of collagens, contributing to the structural stability of collagen fi-
brils. Inhibition of PCP is expected to disrupt fibril formation and
stability, preventing the excess collagen deposition associated with
fibrosis.6,7
tide derived from the a1 strand of type III collagen, 1, demon-
strated the highest rate of cleavage. Limited truncation of 1 at
either the C or N termini retained activity, though further deletions
lead to substantially reduced cleavage by PCP. In addition, an ala-
nine scan of peptide 1 confirmed that the P10 aspartic acid was crit-
ical for recognition in this sequence, as illustrated by the complete
lack of activity against peptide 9.
Replacing the scissile bond in analogs of peptide 1 with zinc
binding groups such as thiols, hydroxamic acids, and carboxylic
acids provided initial inhibitors (Fig. 1). The hydroxamate capped
aspartate–glutamate mimetic 10, a mixture of 2 diastereomers
with an IC50 of 40
itor.8 Other zinc chelating functionalities were less potent: a car-
boxylic acid analog 11, IC50: 1500 M, was substantially lower in
activity while thiol 12 showed only moderate activity with an
IC50 of 85 M. Incorporation of a hydroxamate at the carboxy
lM, was the most effective peptide-based inhib-
l
Herein we describe the evolution and SAR of a series of potent,
selective, non-peptidic PCP inhibitors as part of our anti-fibrosis
programs. Starting from peptide-based PCP inhibitors and
l
terminus of the scissile bond of peptide 1 was also effective: the
tetra-peptide hydroxamate 13 inhibited PCP with an IC50 of
42 lM. Concurrent with this work others reported hydroxamate
⇑
dipeptides mimicking P10 and P20 side chains, which were more
effective then those derived from a N-terminal sequence.9
Corresponding author. Tel.: +1 415 978 1403; fax: 1 415 978 1901.
E-mail address: who@fibrogen.com (W.-B. Ho).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.067

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E. Turtle et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7397–7401
Table 1
10
Relative order of PCP activity on pro-collagen peptides
Substrate
Peptide sequence
Collagen chain
PCP activitya
OMe
1
2
3
4
5
6
7
8
9
Ac-PYYG-DEPM-NH2
Ac-PYYG-DEP-NH2
Ac-FYRA-DQPR-NH2
Ac-YYG-DEPM-NH2
Ac-PYYG-DE-NH2
Ac-YYRA-DDAN-NH2
Ac-FAPYYG-DEPMDF-NH2
Ac-PYYG-D-NH2
a
a
a
a
a
a
a
a
1(III)
1(III)
2(I)
1(III)
1(III)
1(I)
++++
+++
++
++
++
+
O
H
N
HO
N
H
S
O
O
HO2C
14 (IC50 = 10 µM)
1(III)
1(III)
+
ꢀ
Ac-PYYG-AEPM-NH2
ꢀ
ꢀ
CO2H
a
Relative rates of peptide cleavage by PCP.
OMe
OMe
O
O
HO
N
HO
N
N
H
S
N
H
S
O
O
O
O
CO2H
CO2H
O
HO2C
O
H
N
15 (IC50 = 345 µM)
H
N
H
17 (IC50 = 2.8 µM)
N
HS
N
H
HO
N
H
O
O
O
CO2H
CO2H
12 (IC50 = 85 µM)
10 (IC50 = 40 µM)
OMe
O
CO2H
HO
N
N
H
S
O
O
O
O
O
H
H
OH
N
N
N
H
Ac-Pro-Tyr-Tyr-Gly
HO
N
H
16 (IC50 = 16 µM)
Figure 2. Preliminary sulfonamide based leads.
O
CO2H
13 (IC50 = 42 µM)
11 (IC50 = 1500 µM)
amination using sodium triacetoxyborohydride or sodium cyano-
borohydride as the hydride source. In the second route, Michael
addition of a primary amine 30 into an acrylate ester provided a
N-propanoate ester functionalized amine 28. In both routes, the
resulting secondary amine, 28, was next coupled with the appro-
priate sulfonyl chloride in the presence of a base, typically triethyl-
amine, to form the elaborated tertiary sulfonamide. Alternatively,
in the third method, the secondary sulfonamide 32 is formed first
by reacting amine 30 and sulfonyl chloride 31 in the presence of
base, followed by alkylation of the sulfonamide nitrogen with an
alkylbromide ester. In all three routes, the esters of the penulti-
mate compounds were converted to the desired hydroxamic acids
29 by displacement of the alkoxy substituent using excess hydro-
xyl amine in methanol at room temperature.
Figure 1. Peptidic PCP inhibitors derived from peptide 1.
Other core scaffolds were explored using hydroxamates as the
key zinc chelating group. Modifying dipeptides such as 10 by
replacing the central amide with a sulfonamide was envisioned
0
0
to display potential P1 , P2 mimicking side chains and zinc binding
functionality from an alternative scaffold. Surprisingly, our initial
survey of N-substituted sulfonamide hydroxamates revealed that
the diacid functionalities preferred in substrates were not neces-
sary for potent inhibitory activity. Similar findings have been re-
ported by others where hydroxamate bearing scaffolds with
hydrophobic substituents have demonstrated potent binding.10–14
A hydroxamate inhibitor utilizing a N-arylsulfonamide aspartic
Optimization of the sulfonamide substituent R1 was undertaken
to identify substituent preference at this position (Table 3). Varying
the R1 group in 2 series, which contained either methylene or eth-
ylene linked hydroxamic acids (b = 1,2), showed that the ethylene
spacer was preferred. Mid micromolar potency inhibitors could
be obtained with methylene linked hydroxamates, but examples
such as 41, showed a much higher potency against MMP’s thought
to be critical to wound healing such as MMP 2 and 9 where it had
acid core, 14, IC50: 10 lM, showed greater potency then our initial
peptidyl Asp-Glu mimetic 10 (Fig. 2). Examples with acidic func-
tionality displayed from the sulfonamide nitrogen resulted lower
affinity, 15: IC50 of 345
benzyl group retained comparable binding, 16: IC50 of 16
Compounds containing both a carboxylic acid at the C- position
and a N-benzyl functionality further increased inhibitory potency,
17: IC50 of 2.8 M, but had liabilities of decreased activity in cell-
l
M, but replacing this acidic group with a
l
M.
a
l
IC50 values of 0.14 and 0.05
lM respectively, with moderate PCP
based assays and potential stability concerns due to observed
intramolecular cyclization to a less active species. Therefore, 16
was chosen as an starting point for optimization.
activity, IC50 = 19 M. In the ethylene linked hydroxamic acids ser-
l
ies, a variety of alkyl and aryl functionality retained micromolar
potency for this class, with the most potent of these compounds
bearing a phenethyl, 24, or para-methoxy phenethyl, 48, at the
R1 position. Retaining these preferred substrates at R1, optimiza-
tion of the aryl sulfonamide portion of the molecule, R, was exam-
ined next.
While a variety of R groups retain micromolar activity, appro-
priate substitution at the para position of phenylsulfonyl groups
provided several sub-micromolar inhibitors (Table 4). The para-
phenylsulfonyl hydroxyl imidamide analog, 60, IC50 = 80 nM,
showed excellent potency and selectivity versus MMP 1, 2, and 9.
To determine optimal placement of aromatic and zinc chelating
groups, a homologous series of sulfonamides was synthesized
around compound 16. Hydrocarbon spacers of varying lengths
were incorporated between the sulfonamide nitrogen and both
the hydroxamate functionality and the phenyl substituent. Of
those tested, ethyl spacers were identified as preferable for both
substituents. Compound 22, a = 2, b = 2, with an IC50 of 900 nM,
displayed a substantial improvement over the initial peptidyl
hydroxamate inhibitors (Table 2).
The compounds in Tables 2–4 were prepared via three general
synthetic methods. (Fig. 3, schemes I–III) In the first route, an ami-
no ester 26 and an aldehyde 27 were condensed via a reductive
Similarly, a para-methyl sulfonyl group, 65: IC50 = 0.29
lM, was
moderately potent and displayed excellent selectivity when

E. Turtle et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7397–7401
7399
Table 2
Table 4
Optimization of spacers for sulfonamide series
Optimization of sulfonamide substituent R
X
S
OMe
O
a
HO
N
N
S
b
H
O2
O
HO
N
R
O
N
Compound
number
a: (CH2)x spacer
length
b: (CH2)x spacer
length
IC50
M)
b
H
O
(
l
18
16
19
20
21
22
23
24
25
0
1
2
0
1
2
0
1
2
1
1
1
2
2
2
3
3
3
69
16
18
3
4.5
0.9
28
10
4.9
Compound
X
b: (CH2)x
R
IC50 (lM)
55
56
57
58
59
60
61
62
63
64
65
66
67
OMe
OMe
H
2
1
2
2
2
2
2
2
2
2
2
2
2
n-Bu
n-Bu
Ph
4-CO2H-Ph
2-CO2H-Ph
4-(C(@NOH)NH2)-Ph
3-(C(@NOH)NH2)-Ph
2-(C(@NOH)NH2)-Ph
4-(Ph-C(@O)NH)-Ph
4-(Ph-SO2-NH)-Ph
4-SO2Me-Ph
6.7
55
2.2
H
1.0
a
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
0.08
0.40
7.1
0.41
0.47
0.29
9.1
Table 3
Optimization of spacers for sulfonamide series
2-SO2Me-Ph
4-tBu-Ph
17
a
32% inhibition at 10 lM.
OMe
O
R1
N
HO
N
S
with either allyl bromide or 4-bromo-1-butene. The resulting al-
kenes were then epoxidized with mCPBA to give 75. The epoxide
rings were opened with thioacetic acid, and oxidized with PCC to
H
O
O
Compound
b: (CH2)x spacer
R1
IC50 (lM)
15
33
34
35
36
37
38
39
40
41
42
24
43
44
45
46
47
48
49
50
51
52
53
54
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
CH2CO2H
Adamantyl
sec-Butyl
CH2(4-F-Ph)
CH2CO2n-Bu
4-MeO-Ph
CH2CH2(4-MeO)Ph
CH2(4-MeO-Ph)
CH2(4-CF3-Ph)
CH2(4-Cl-Ph)
345
produce the acetate protected
a-keto thiols. The acetate was re-
a
moved with hydrazine, which minimized competing b-elimination
of the sulfonamide in the case of 69, to produce compounds 68 and
69.
112
34
167
73
18
29
The synthesis of N-hydroxy urea 70 was accomplished as shown
in Figure 4-II. The sulfonamide 73 was alkylated with bromoacet-
aldehyde diethyl acetal. The protected aldehyde was unmasked
under acidic conditions and condensed with hydroxylamine to give
the oxime 75. The oxime was reduced to the hydroxylamine and
N-acylated with TMS-isocyanate, which upon TMS exchange
furnished the desired N-hydroxy urea 70. Thiols 71 and 72 were
prepared as depicted in Figure 4-III. In the case of 71, N-alkylation
of 73 with 1-chloro-3-iodopropane and subsequent conversion
of the chloropropane substituent into a mercaptopropane via dis-
placement with thioacetic acid and hydrolysis to afford the desired
propanethiol 71. In the case of 72, a hydroxyethyl substituted
sulfonamide was synthesized by mesylation of 2-(4-methoxy-
phenyl)ethanol, N-alkylation of ethanol amine, and reaction of
the secondary amine with 4-(methylsulfonyl)benzene sulfonyl
chloride to give the ethanol substituted sulfonamide 76. The
alcohol was then mesylated, displaced with thiolacetic acid, and
hydrolyzed to give the desired ethanethiol 72.
67
19
CH(Ph)2
CH2CH2Ph
102
0.9
81
1.7
1.0
8.1
1.7
1.0
CH2CH2-N-morpholinyl
sec-Butyl
CH2-cyclhexane
CH2CH2(2-pyridyl)
4-MeO-Ph
CH2CH2(4-MeO-Ph)
CH2CH2(3-MeO-Ph)
CH2CH2(2-MeO-Ph)
CH2CH2(4-NO2-Ph)
CH2CH2(4- NH2SO2-Ph)
CH2CH2(3,4-diMeO-Ph)
H
2.3
b
11
23
42
166
a
9–21% Inhibition at 10
45% Inhibition at 10
l
M.
M.
b
l
As shown in Table 5, replacement of the hydroxamate function-
ality of 65 with other zinc chelating functionalities, including thi-
ols,
a-keto thiols, hydroxy-ureas, and carboxylic acids
counter screened against MMP 2 & 9 (IC50 >6 lM for both MMPs).
substantially decreased inhibitory potency. Variation in linker
length produced only minor changes in potency. While thiol con-
taining compounds 68 and 72 retained micromolar activity, poten-
tial limitations of thiols as drug-like functionality prioritized
continued optimization of our hydroxamates leads.
In the next iterative optimization, we fixed 4-methoxypheneth-
yl as the R1 group and ethyl-hydroxamic acid as the zinc binding
functionality while the para position of the aryl sulfonamide was
diversified further, (Table 4) since compounds with hydroxyl imi-
damide, amide, and sulfonamide moieties provided sub-micromo-
lar inhibition and improved selectivity. For example, compound 60
showed excellent potency; selectivity versus select MMPs, where
The scaffold of 65 was utilized to screen alternative zinc chelating
functionalities.
Hydroxamate containing compounds have potential liabilities
as systemic drugs: rapid metabolism or excretion, hydrolysis to re-
lease hydroxyl amine, and promiscuity for other zinc utilizing en-
zymes.15–18 Therefore, alternative zinc binding functionalities were
incorporated into this scaffold to determine effects on potency (Ta-
ble 5). The syntheses of several non-hydroxamic acids analogs of
65 are summarized in Figure 4. The synthesis of the a-keto thiols
68 and 69 are shown in the first scheme, 4-I. The secondary sulfon-
amide 74, synthesized by condensing 4-methoxyphenethylamine
with 4-(methylsulphonyl)benzene-sulfonyl chloride, was alkylated

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E. Turtle et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7397–7401
I
I
OMe
OMe
b
a
R1
b
a
a
R1
RO
OR
H2N
a
HN
S
N
H2N
+
OHC
27
HOHN
H
O
+
O
O
O
O
O
S
Me
Cl
26
b, c
28
S
Me
O
O
a
b
S
O
73
N
R1
O
O O
S
OMe
Ar
O
n
N
n
29
Br
O
O
S
O
II
b,c
O
Me
a
R1
a
R1
S
d
+
RO2C
RO
N
H2N
O
O
H
74
30
b, c
28
OMe
O
n
d,e,f
a
R1
HS
N
S
68: n=1
69: n=2
HOHN
N
O O
O
S
O
Ar
O
Me
29
S
O
O
III
a
e
II
R1
Cl
O
Ar
a
R1
HN
S
O
OMe
S
+
H2N
O
O
g,h,i
HON
Ar
N
73
30
31
HOHN
32
O
S
O
f, c
a
R1
b
Me
S
N
S
O
O
O
75
O O
Ar
OMe
OH
N
j,k,l
29
H2N
N
S
Figure 3. General synthesis of hydroxamates 15–67 and conditions: R = Et or Me;
(a) NaHB(OAc)3, TEA, DCM, rt, or NaBH3CN, AcOH, MeOH, rt; (b) ClSO2Ar, TEA, DCM,
rt; (c) NH2OH, MeOH, rt; (d) EtOH, reflux; (e) TEA, DCM, rt; (f) Br(CH2)bCO2R, NaH,
NaI, DMF, 90 °C.
O
O
O
Me
S
O
O
70
III
Table 5
OMe
Comparison of zinc chelating groups incorporated into compound 65 scaffold
m,n,o
HS
N
S
73
O
O
OMe
Me
S
O
O
71
O
S
O
OMe
OMe
X
N
p,q,r
HO
S
N
n
O
O
O
S
HO
O
a
Compound Number
n
X
IC50 (lM)
Me
S
76
65
68
69
70
71
72
2
1
2
2
3
2
–CONHOH
–COCH2SH
–COCH2SH
–N(OH)CONH2
–SH
0.29
4.7
13
>100
14
9.4
O
O
OMe
HS
N
s,t,u
O
S
–SH
O
Me
S
O
O
72
IC50’s for MMP 1, 2, and 9 were all greater than 25
cytotoxicity, as measured against proliferation of 3T3-L1 cells, with
an IC50 of 150 M. Therefore we examined polar linking groups for
lM; and low
Figure 4. Synthesis of non-hydroxamate inhibitors. n = 1 or 2; R = CH2CH2(4-
MeOPh); Conditions (yields, n = 1 and n = 2): (a) TEA, DCM, rt (93%); (b) NaH, DMF,
rt (n = 1); NaH, DMF, TBAI, 60 °C (n = 2), (50%); (c) mCPBA, DCM, rt (26% two steps,
75%); (d) AcSH, rt to 40 °C (69%, 65%); (e) PCC, celite, DCM, rt, (88%, 96%); (f)
Hydrazine, THF/DMF, ꢀ10 to ꢀ18 °C (91%, 90%); (g) NaH, BrCH2(OEt)2, TBAI, DMF,
80 °C (50%); (h) TsOH, Acetone, reflux (76%); (i) NH2OH, NaHCO3, EtOH/THF, rt,
(97%); (j) PyrBH3, THF 0 °C (90%); (k) TMS-NCO, dioxane, rt (quant); (l) H2O, (40%);
(m) NaH, ICH2CH2Cl, DMF, rt; (n) AcSH, K2CO3, DMF, 60 °C (40% for 2 steps); (o)
LiOH, THF/H2O, rt (42%); (p) MsCl, TEA, DCM, 0 °C to rt (70%); (q) ethanolamine, TEA,
THF, rt (92%); (r) ClSO2Ph(4-SO2Me), TEA, DMF, rt (85%); (s) MsCl, TEA, DCM, 0 °C to
rt (94%); (t) AcSH, DMF, Cs2CO3, rt (92%); (u) LiOH, THF/H2O/EtOH, rt, (72%).
l
the attachment of diversifying R2 and R3 groups at this position.
Utilizing a urea to display hydrophobic substituents from the 4-
postion of the phenylsufonamide, 80, resulted in several promising
compounds. Syntheses of sulfonamide–ureas were accomplished
by two general methods (Fig. 5). First, the isocyante of 4-isocya-
nate-phenylsulfonylchloride, 77, was chemoselectively coupled to
an amine to produce the appropriate di or tri substituted urea–sul-
fonyl chloride 78, which was then elaborated via our standard

E. Turtle et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7397–7401
7401
I
reduction of a 4-nitro precursor. Reaction of the amine with vari-
ous isocyanates gave disubstituted ureas. The final compounds
were, again, produced by conversion of the esters to hydroxamic
acids 80 with hydroxyl amine.
The ureas gave substantial improvement over the previous
para-substituted analogs, with IC50 values in the low to mid nano-
molar range (Table 6). Trisubstituted ureas bearing a benzyl or
phenyl substituent at R3 were less potent then the corresponding
disubstituted analogs, as seen in compounds 85 versus 89 and 82
versus 86.
O
O
S
O
O
S
a
Cl
O
Cl
R2
N
H
N
R3
NCO
77
78
OMe
OMe
H2N
b
O
The urea 89 was identified as a potent inhibitor of PCP with an
IC50 of 10 nM. When tested in a cellular assay, measuring inhibition
of collagen deposition in HFF cell cultures,19 89 displayed an IC50 of
+
MeO
N
H
O
79
MeO
1
l
M. The compound showed excellent selectivity versus several
matrix metalloproteinases (MMP-1 IC50 >25 M; MMP-2 IC50
>25 M; and MMP-9 IC50 >25 M). Compounds 89 and 60 possess
OMe
O
l
HO
l
l
N
N
S
O
H
c, d
O
promising properties for the development of topically applied anti-
scarring therapeutics. The additional optimization of this class of
inhibitors to improve the DMPK properties may also provide new
systemic anti-fibrotic agents.
O
R2
N
N
H
R3
80
II
OMe
O
References and notes
e,f
79
MeO
N
1. Hinz, B.; Gabbiani, G. F1000 Biol. Report 2010, 2, 78.
2. Zavoico, G. B. Drug Market Dev. 1999, 10, 2.
O
S
O
3. Lapiere, C. M.; Lenarers, A.; Kohn, I. D. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 3054.
4. Li, S.; Sieron, A. L.; Fertala, A.; Hojima, Y.; Arnold, W. V. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 5127.
5. Uzel, M. I.; Scott, I. C.; Babakhanlou-Chase, H.; Palamakumbura, A. H.; Pappano,
W. N.; Hong, H.-H.; Greenspan, D. S.; Trackman, P. C. J. Biol. Chem. 2001, 276,
22537.
NH2
81
OMe
O
HO
6. Prockop, D. J.; Sieron, A. L.; Li, S. Matrix Biol. 1998, 16, 399.
7. Turtle, E.; Ho, W.-B. Expert Opin. Ther. Pat. 2004, 14, 1185.
8. IC50 determination for PCP and MMP’s: A fluorogenic peptide containing a
consensus PCP cleavage site was used to assess the inhibitory activity of
compounds on PCP. In a similar assay, a fluorogenic peptide with the sequence
Mca-P-L-G-L-Dap(Dnp)-A-R-NH2 (kex = 360 nm, kem = 465 nm; Bachem catalog
no. M-1895) was used to measure the inhibitory activity of compounds on
MMP-1, MMP-2 and MMP-9. The latter enzymes were activated according to
the manufacturer’s instructions using p-amino phenylmercuric acetate. BMP-1,
and activated MMP-1, -2, and -9 were incubated for 1 h at 37 °C in the presence
of inhibitors and 50 mM of the appropriate fluorogenic peptide substrate, and
the increase in fluorescence was used to determine initial rates.
9. Ovens, A.; Joule, J. A.; Kadler, K. E. J. Peptide Sci. 2000, 6, 489.
10. Dankwardt, S. M.; Martin, R. L.; Chan, C. S.; VanWart, H. E.; Walker, K. A. M.;
Delae, N. G.; Robinson, L. A. Bioorg. Med. Chem. Lett. 2001, 11, 2085.
11. Robinson, L. A.; Wilson, D. M.; Delaet, N. G. J.; Bradley, E. K.; Dankwardt, S. M.;
Campbell, J. A.; Martin, R. L.; Van Wart, H. E.; Walker, K. A. M.; Sullivan, R. W.
Bioorg. Med. Chem. Lett. 2003, 13, 2381.
12. Delaet, N. G. J.; Robinson, L. A.; Wilson, D. M.; Sullivan, R. W.; Bradley, E. K.;
Dankwardt, S. M.; Martin, R. L.; Van Wart, H. E.; Walker, K. A. M. Bioorg. Med.
Chem. Lett. 2003, 13, 2101.
13. Fish, P. V.; Allan, G. A.; Bailey, S.; Blagg, J.; Butt, R.; Collis, M. G.; Greiling, D.;
James, K.; Kendall, J.; McElroy, A.; McCleverty, D.; Reed, C.; Webster, R.;
Whitlock, G. A. J. Med. Chem. 2007, 50, 3442.
14. Bailey, S.; Fish, P. V.; Billotte, S.; Bordner, J.; Greiling, D.; James, K.; McElroy, A.;
Mills, J. E.; Reed, C.; Webster, R. Bioorg. Med. Chem. Lett. 2008, 18, 6562.
15. Hajduk, P. J.; Shuker, S. B.; Nettesheim, D. G.; Craig, R.; Augeri, D. J.; Albert, D.
H.; Guo, Y.; Meadows, R. P.; Xu, L.; Michaelides, M.; Davisen, K.; Fesik, S. W. J.
Med. Chem. 2002, 45, 5628.
16. Singh, J.; Conzentino, P.; Cundy, K.; Gainor, J. A.; Gilliam, C. L.; Gordon, T. D.;
Johnson, J. A.; Morgan, B. A.; Schneider, E. D.; Wahi, R. C.; Whipple, D. A. Bioorg.
Med. Chem. Lett. 1995, 5, 337.
g,c
N
H
N
S
O
O
O
R2
N
H
N
R3
80
Figure 5. Synthesis of ureas. Conditions: (a) NHR2R3; THF, 0 °C; (b) EtOH, reflux; (c)
78, TEA, rt; (d) NH2OH, MeOH, rt; (e) ClSO2Ph-NO2, TEA, DCM, rt; (f) HCO2NH4, Pd/C,
MeOH/EtOAc, reflux; (g) R-NCO, DCM, reflux.
Table 6
Optimization of spacers for sulfonamide series 81
OMe
O
HO
N
N
S
H
O
O
O
R2
N
H
N
R3
Compound
R2
Ph
R3
IC50 (lM)
82
83
84
85
86
87
88
89
90
91
H
H
H
0.093
0.35
0.042
0.38
0.78
0.073
0.30
0.010
0.030
0.060
4-MeO-Ph
4-CF3-Ph
Bn
Bn
Ph
H
17. Flipo, M.; Charton, J.; Hocine, A.; Dassonneville, S.; Deprez, B.; Deprez-Poulain,
R. J. Med. Chem. 2009, 52, 6790.
Ph
4-Ph-Ph
4-Cl-Ph
Bn
CH2CH2Ph
Me
18. Becker, D. P.; Barta, T. E.; Bedell, L. J.; Boehm, T. L.; Bond, B. R.; Carroll, J.;
Carron, C. P.; Decrescenzo, G. A.; Easton, A. M.; Freskos, J. N.; Funckes-Shippy,
C. L.; Heron, M.; Hockerman, S.; Howard, C. P.; Kiefer, J. R.; Li, M. H.; Mathis,
K. J.; McDonald, J. J.; Mehta, P. P.; Munie, G. E.; Sunyer, T.; Swearingen, C. A.;
Villamil, C. I.; Welsch, D.; William, J. M.; Yu, Y.; Yao, J. J. Med. Chem. 2010, 53,
6653.
H
H
H
H
19. Franklin, T. J.; Morris, W. P.; Edwards, P. N.; Large, M. S.; Stephenson, R.
Biochem. J. 2001, 353, 333.
reaction sequences to hydroxamates 80. In the second method, an
4-aminophenylsulphone intermediate 81 was prepared from