Design, synthesis, and evaluation of novel 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and selective CXCR2 chemokine receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.08.014

We describe herein a novel series of 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and selective inhibitors against the CXCR2 chemokine receptor and IL-8-mediated chemotaxis of a CXCR2-expressing cell line. Furthermore, these alkyl-hydrazine series inhibitors such as 5b demonstrated acceptable metabolic stability when incubated in human and rat microsomes.

Full text of DOI:10.1016/j.bmcl.2009.08.014

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5741–5745
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of novel 3-amino-4-hydrazine-cyclobut-3-
ene-1,2-diones as potent and selective CXCR2 chemokine receptor antagonists
a
a
Shilan Liu a, Yinhui Liu a, Hongmei Wang a, YiLi Ding a, Hao Wu a, , Jingchao Dong , Angela Wong ,
*
Shu-Hui Chen a, , Ge Li , Manuel Chan b,c, Nicole Sawyer b,c, Francois G. Gervais b,c, Martin Henault b,c
,
a
*
Stacia Kargman b,c, Leanne L. Bedard b,c, Yongxin Han b,c, Rick Friesen b,c, Robert B. Lobell d, David M. Stout e
a WuXi PharmaTech Co. Ltd, 288 FuTe Zhong Road, No. 1 Building, WaiGaoQiao Free Trade Zone, Shanghai 200131, PR China
b Department of Medicinal Chemistry, Merck Frosst Center for Therapeutic Research, Merck-Frosst Canada Ltd, PO Box 1005, Pointe Claire-Dorval, Quebec, Canada H9R 4P8
c Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Merck-Frosst Canada Ltd, PO Box 1005, Pointe Claire-Dorval, Quebec,
Canada H9R 4P8
d Merck Research Laboratories, West Point, PA 19486, USA
e Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe herein a novel series of 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and
selective inhibitors against the CXCR2 chemokine receptor and IL-8-mediated chemotaxis of a CXCR2-
expressing cell line. Furthermore, these alkyl-hydrazine series inhibitors such as 5b demonstrated
acceptable metabolic stability when incubated in human and rat microsomes.
Ó 2009 Published by Elsevier Ltd.
Received 25 April 2009
Revised 31 July 2009
Accepted 1 August 2009
Available online 7 August 2009
Keywords:
3,4-Dioxocyclobut-1-enyl-hydrazine
Antagonists
CXCR2
Chemokine receptor
Owing to the relevance of IL-8 (CXCL8) and related chemokines
in a wide range of inflammatory diseases such as arthritis, asthma,
and COPD, the search for small-molecule antagonists for CXCR2
has attracted a lot of attention within the past two decades.1,2 As
a result of these efforts, many structurally diverse CXCR2 antago-
nists have been identified, which include the bis-aryl urea series
such as 1,3–6 the 3,4-diamino-cyclobut-3-ene-1,2-dione series such
as 2a and 2b,7–9 the thiazolopyrimidine series 3,10–12 and the 3,4-
diamino-substituted 1,2,5-thiadiazole series 413–15 as shown in
Figure 1. A careful analysis on SAR trends reported for Series 2
antagonists7–9 suggested the possibility of replacing the alkyl
amine moiety as installed for 2a or 2b with a hydrazine linkage
as seen for Series 5 (see Fig. 1). The rationale for incorporation of
hydrazine moiety in Series 5 antagonists was further supported
by the observation that such linkage was incorporated into HIV
protease inhibitors by Reddy et al.,16 Human Rhino Viruses (HRV)
3C protease inhibitors by Kati et al.,17 HCV protease inhibitors by
Bailey et al.,18 as well as SARS 3CL protease inhibitors by Anand
et al.19 Furthermore, a recently FDA approved HIV protease inhib-
itor Reyataz with hydrazine linkage incorporated as subunit fur-
ther validates our design concept for Series 5 antagonists20 (see
Fig. 2).
With the intention to validate our hypothesis, we designed a
number of N-alkyl, both electron rich and deficient N-aryl, and N-
acyl substituted hydrazines as the initial target compounds (5a–
j) as shown in Figure 3. In this communication, we describe the de-
sign, synthesis, and SAR trend observed with the novel hydrazine
linkage bearing cyclobutene diones 5 as CXCR2 antagonists. The
most promising compound identified within this series thus far,
5b showed good receptor binding potency, functional activity,
and excellent selectivity against CXCR1, and acceptable metabolic
stability, thus rendering itself as a new lead compound for further
optimization.
Chemical synthesis: The syntheses of all target compounds were
accomplished according to the chemistries depicted in Scheme 1
through 6. It is worthwhile to mention that many attempts to pre-
pare these seemingly related target analogs (5a–j) via a convergent
route such as that shown in Scheme 1 were not successful (e.g., for
5d–f). Consequently, multiple routes were exploited for the prepa-
ration of various hydrazine bearing cyclobutene diones derivatives
5.
The syntheses of compounds 5a and 5f were completed accord-
ing to Scheme 1. Towards this end, coupling of the known
* Corresponding authors. Tel.: +86 21 50463721; fax: +86 21 50463718.
E-mail address: chen_shuhui@wuxiapptec.com (S.-H. Chen).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.08.014

5742
S. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5741–5745
O
O
OH
HN
N
Cl
O
S
N
H
N
H2N
N
H
F
N
NH2
S
N
N
Cl
R
H
H
O
N
OH
O
S
OH
Cl
F
O
2a R = 2-Furyl (SCH 527123)
2b R = Et
1 (SB-332235)
3 (AZ Series)
O
O
S
N
N
N
N
Wherein R = H, alkyl, acyl,
aryl, heteroaryl, etc
N
H
N
H
N
R
N
N
H
H
OH
O
OH
5
O
4 (SP Series)
Figure 1. Representative CXCR2 antagonists.
O
O
O
O
H
H
H
N
Boc
N
N
Br
N
H
N
N
O
N
H
N
H
N
N
H
H
O
OH
O
O
O
O
C(O)NH
HIV Protease Inhibitor
2
HRV Protease Inhibitor
N
Reyataz (BMS)
HIV Protease Inhibitor
O
OH
H
O
H
N
N
N
O
O
N
H
N
H
O
O
H
O
N
CO H
2
BnO
O
O
O
O
O
O
H
H
H
H
N
AcHN
N
N
N
N
N
N
N
N
H
H
H
H
O
O
HN
N
O
O
O
N
CO H
2
CO H
2
O
H
O
NO
2
SARS 3CL Protease Inhibitor
HCV Protease Inhibitor
Figure 2. Selected examples of hydrazine bearing viral protease inhibitors.
O
O
O
H
R=:
N
N
N
H
F
R
H
N
5e
5c
5d
5a
5b
OH
O
5a-i
O
O
O
N
F
MeO
F
N
H
N
N
H2N
5i
5f
5g
5h
OH
O
5j
Figure 3. Detailed target list for series 5 antagonists.
substituted aniline 68 with diethyl squarate 7 in EtOH afforded the
expected adduct 8 (70%), which was converted to free hydrazine
bearing derivative 9 (94%) upon treatment with hydrazine-hy-
drate. Compound 9 was further converted to the mono-N-ethyl
analog 5a via a reductive amination reaction in low yield. Subse-
quent N-alkylation of 5a with para-fluorobenzyl bromide thus
yielded 5h in 30% yield.
As shown in Scheme 2, the synthesis of 5b was accomplished in
65% yield via condensation of the intermediate 8 (see Scheme 1)
with N,N-di-ethyl hydrazine 13, which was in turn prepared from
Boc-hydrazine via a two-step sequence consisting of N-alkylation
and Boc-deprotection.
Scheme 3 shows the synthetic route utilized for the preparation
of three N-aryl bearing analogs 5d, 5e, and 5f. In this event, various

S. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5741–5745
5743
arylamines 15d–f were firstly converted to their respective N-aryl-
N-alkyl amines 16d–f according to the protocol of Sajiki,21 which
were then transformed into their corresponding N-nitroso deriva-
tives 17d–f via an N-nitrosation reaction.22 17d–f were further
converted to the requisite N-aryl-N-ethyl hydrazines 18d–f upon
a Zn mediated reduction.23 Treatment of the intermediate 8 with
18d–f thus afforded the desired final products 5d–f in moderate
yields.
The synthesis of N-pyrido bearing analog 5g was achieved as
described in Scheme 4. Reaction of 2-fluoropyridine 19 with N-
ethyl hydrazine afforded the desired adduct 20, which was con-
densed with the already described intermediate 8 to yield the de-
sired N-pyrido derivative 5g in moderate yield.
point out that direct base mediated N-alkylation (e.g., NaH/EtI)
would produce N-di-alkylated compound as the main product.
Subsequent treatment of 23 with either of two acyl chlorides pro-
vided 24c (98%) and 24i (80%), which were further reacted with
HCl in ether to afford the N-ethyl-N-acyl hydrazines 25c and 25i
in excellent yields. Final condensation of 25c and 25i with 8 affor-
ded the desired analogs 5c and 5i, albeit in low yield.
As highlighted in Scheme 6, the synthesis of the mono-N-ethyl
derivative 5j began with coupling of two known intermediates,
namely N-ethyl-N0-Boc hydrazine 23 and 8. The expected product
26 was obtained in 70% yield, which was then treated with TFA
to provide the desired analog 5j in 85% yield.
After the compounds synthesized, their biological activities
were evaluated. The novel series of CXCR2 antagonists described
herein (5a–j) were evaluated for their binding affinity against the
CXCR2 receptor according to a literature protocol with minor mod-
ification.8,24 Promising compounds emerging from this evaluation
The preparation of two N-acylated analogs is depicted in
Scheme 5. In this case, N-Boc hydrazine 21 was converted to its
mono-alkylated product 23 via a reductive amination reaction
through imine intermediate 22 in good yield. It is worthwhile to
O
O
O
O
O
OH
iii
O
O
ii
i
NH2
+
N
N
H
NH
NH2
N
H
OEt
70%
94%
N
N
EtO
OEt
OH
OH
O
8
6
9
7
O
O
O
O
O
O
O
iv
15%
v
F
N
H
HN NH
5 a
N
H
HN N
N
H
HN
N
N
N
N
30%
OH
OH
OH
5h
O
O
O
10
Scheme 1. Reagent and conditions: (i) EtOH, rt,12 h; (ii) EtOH, hydrzine hydrate, rt, 8 h; (iii) acetaldehyde, MeOH, rt 4 h; (iv)BH3ꢀTHF, MeOH, 0 °C to rt; (v) DMF, K2CO3,
4-fluorobenzyl bromide, rt, 3 h.
O
O
iii
i
ii
BocHNNH2
BocHNN
H2NN
N
H
NH
N
95%
65%
N
30%
OH
11
12
5b
O
13
Scheme 2. Reagent and conditions: (i) NaBH3CN, CH3CHO, EtOH, rt, 5 h, (ii)HCl/Et2O, rt, 2 h; (iii) compound 8, EtOH, rt, 12 h.
O
O
ii
H
N
NO
N
NH2
N
iv
iii
i
NH2
Ar
15d-f
Ar
N
H
NH
N
85-94%
Ar
Ar
R
54-85%
37-75%
N
30-40%
16d-f
17d-f
18d-f
OH
O
5d, 5e, 5f
5d: R = Ph; 5e: R = 4-F-Ph; 5f: R = 4-MeO-Ph
Scheme 3. Reagent and conditions: (i) MeCN, Pd/C, MeOH, rt 16 h; (ii) HCl, ice, NaNO2 0–5 °C; (iii) Zn, AcOH, 5–10 °C; (iv) compound 8, EtOH, rt, 16 h.
O
O
ii
i
N
N
H
HN N
N
N
N
F
35%
44%
N
NH2
OH
19
20
5 g
O
Scheme 4. Reagent and conditions: (i) EtNHNH2, DIEA, 150 °C, microwave 30 min.; (ii) compound 8, EtOH, K2CO3, 40 °C, 12 h.

5744
S. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5741–5745
H
N
H
i
ii
NH2
N
Boc
NH2.HCl
O
iii
N
N
iv
BocHN
N
BocHN
N
H
Boc
95%
75%
R2
O
80%
95%
21
22
R2
23
24c or 24i
25c or 25i
O
O
O
N
v
R2
N
H
HN
10%-20%
N
OH
5 c
5i
or
O
Scheme 5. Reagent and conditions: (i) CH3CHO, toluene, 50 °C, 1 h; (ii) DIBAL-H, THF, ꢁ30 to 40 °C,4 h; (iii) AcCl or 4-fluorobenzoyl chloride, CH2Cl2, pyridine, 4 h; (iv)HCl/
Et2O, rt 1 h; (v) compound 8, EtOH, K2CO3, rt, 12 h.
O
O
N
O
O
N
H
N
i
ii
N
H
NHBoc
N
H
Boc
N
H
NH2
N
85%
70%
N
OH
23
26
OH
O
5 j
O
Scheme 6. Reagent and conditions: (i) compound 8, EtOH, K2CO3, rt 12 h; (ii)TFA, CH2CL2, rt,4 h.
were further evaluated for selectivity against the CXCR1 receptor
and for functional activity.8 Benchmark compound 2b was in-
cluded as a positive control. The literature reported data on 2a
(SCH527123) is also included for comparison.8 The testing results
are highlighted in Table 1.
Benchmark compound performance: As shown in Table 1, the po-
sitive control 2b showed CXCR2 inhibitory potency (IC50) of 15 nM
and 60-fold selectivity against CXCR1 according to Dwyer et al.8
The same compound exhibited CXCR2 binding affinity (Ki) of
16 nM and 90-fold selectivity versus CXCR1 in our assays [MK]. It
is worthwhile to point out that the furyl bearing version of 2b, ana-
log 2a (SCH527123) was reported to be fourfold more potent
(IC50 = 3.8 nM) against CXCR2 and yet less selective (sevenfold)
against CXCR1 in comparison to 2b.8
SAR trend observed in CXCR2 binding assay: When evaluated for
CXCR2 binding affinity, the newly designed diethyl-hydrazine
antagonist 5b (a close analog of 2b having one additional nitrogen
in place of a carbon) exhibited Ki value of 120 nM. Replacement of
one ethyl from 5b with an acetyl moiety led to 5c, which was found
to be threefold less potent than 5b (Ki = 320 nM) in the binding as-
say. When three closely related hydrazine bearing analogs were
tested in the CXCR2 binding inhibition assay, 5b was found to be
2- or threefold more potent than 5c or 5a (with one ethyl group re-
placed by a hydrogen), respectively.24 It should be pointed out
herein that 5j shown in Scheme 6 (the N-ethyl isomer of 5a) was
found totally inactive in the binding assay. This SAR trend found
within hydrazine series is in good agreement with the literature re-
port on the Schering CXCR2 antagonist series 2a and 2b.7–9
On the other hand, incorporation of a series of ethylaryl hydra-
zine moieties into 5b resulted in compounds 5d–g. It is noted
that 5d and 5g retained the similar binding affinity (Ki = 110–
130 nM) for CXCR2 as that found with 5b (Ki = 120 nM). Further-
more, since the pyridyl moiety presented in 5g should promote
aqueous solubility relative to the phenyl bearing antagonist 5d,
thus the physical chemical properties (e.g., solubility, membrane
permeability, and even tissue distribution, etc.) of 5d and 5g
could be quite different, which brings in added advantage of this
series compound.
Further inspection of the binding data obtained for 5e and 5f re-
veals that whilst installment of an electron donating group (OMe
for 5f) on the phenyl ring of 5d had minimal effect on CXCR2 bind-
ing (Ki = 180 nM), addition of an electron withdrawing group (F for
5e) on the same phenyl ring in 5d resulted in fourfold reduction in
CXCR2 binding affinity (Ki = 540 nM). Comparative evaluation of
CXCR2 binding affinity of p-F-Ph bearing analog 5e (Ki = 540 nM)
and its p-F-Bn counterpart 5h (Ki = 7.2 lM) indicated a sharp 13-
fold drop in binding potency for the latter. Surprisingly, replace-
ment of the benzyl linker in 5h with its corresponding benzoyl
moiety led to 5i, which displayed close to 30-fold enhanced bind-
ing potency (Ki = 260 nM) relative to 5h. Taken together all of the
SAR data obtained with the ethylaryl hydrazine series compounds,
compounds 5d and 5g were the most promising CXCR2 antagonists
synthesized thus far (Table 1).
SAR trend observed in CXCR-1 selectivity assay: Encouraged by
their CXCR2 binding affinities, promising novel hydrazine bearing
antagonists 5b and 5d–g were tested in a CXCR1 binding assay.
To our satisfaction, the diethyl hydrazine analog 5b demonstrated
80-fold selectivity against CXCR1. This level of selectivity is quite
comparable to that exhibited by 2b (60–90-fold) (Table 1).
As also shown in Table 1, when tested in the CXCR1 selectivity
assay, a series of ethylaryl hydrazine analogs 5d, 5f, and 5g showed
about 40-fold selectivity. In addition, whilst p-F-Ph analog 5e
showed >20-fold selectivity, its related analog 5i exhibited 2x im-
proved selectivity (>46-fold).
Table 1
Effects of 5a–i on CXCR2 and CXCR-1 binding inhibition and CXCR2 in CHO cells
Compd CXCR-2 Ki/[IC50
V)
]
CXCR-1 Ki/[IC50
M)
]
CXCR-2 [6] functional IC50
(lM)
(
l
(l
2a
2b
[0.0038] Ref. 8
[0.015] Ref. 8
0.016
[0.026] Ref. 8
[0.91] Ref. 8
1.5
//
//
0.012 (n = 5)
//
0.046 (n = 5)
//
5a
5b
[5.49] Ref. 24
0.12 (n = 4)
[1.86] Ref. 24
[3.29] Ref. 24
0.32 (n = 4)
0.11 (n = 3)
0.55 (n = 3)
0.18 (n = 3)
0.13 (n = 3)
7.2 (n = 3)
//
9.7 (n = 3)
//
//
>12 (n = 3)
4.1 (n = 3)
>12 (n = 3)
6.5 (n = 3)
5.2 (n = 3)
>12 (n = 3)
>12 (n = 3)
5c
//
1.25 (n = 3)
0.054 (n = 5)
0.84 (n = 4)
0.10 (n = 5)
0.075 (n = 5)
6.34 (n = 5)
0.84 (n = 4)
5d
5e
5f
5g
5h
5i
0.26 (n = 3)

S. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5741–5745
5745
3. Widdowson, K. L.; Elliott, J. D.; Veber, D. F.; Nie, H.; Rutledge, M. C.; McCleland,
B. W.; Xiang, J.-N.; Jurewicz, A. J.; Hertzberg, R. P.; Foley, J. J.; Griswold, D. E.;
Martin, L.; Lee, J. M.; White, J. R.; Sarau, H. M. J. Med. Chem. 2004, 47, 1319.
4. Nie, H.; Widdowson, K. L.; Palovich, M. R.; Fu, W.; Elliott, J. D.; Bryan, D. L.;
Burman, M.; Schmidt, D. B.; Foley, J. J.; Sarau, H. M.; Busch-Petersen, J. Bioorg.
Med. Chem. Lett. 2006, 16, 5513.
5. Wang, Y.; Busch-Petersen, J.; Wang, F.; Ma, L.; Fu, W.; Kerns, J. F.; Jin, J.;
Palovich, M. R.; Shen, J.-K.; Burman, M.; Foley, J. J.; Schmidt, D. B.; Hunsberger,
G. E.; Sarau, H. M.; Widdowson, K. L. Bioorg. Med. Chem. Lett. 2007, 17, 3864.
6. McCleland, B. W.; Davis, R. S.; Palovich, M. R.; Widdowson, K. L.; Werner, M. L.;
Burman, M.; Foley, J. J.; Schmidt, D. B.; Sarau, H. M.; Rogers, M.; Salyers, K. L.;
Gorychi, P. D.; Roethke, T. J.; Stelman, G. J.; Azzarano, L. M.; Ward, K. W.; Busch-
Petersen, J. Bioorg. Med. Chem. Lett. 2007, 17, 1713.
7. Merritt, J. R.; Rokosz, L. L.; Nelson, K. H., Jr.; Kaiser, B.; Wang, W.; Stauffer, T. M.;
Ozgur, L. E.; Schilling, A.; Li, G.; Baldwin, J. J.; Taveras, A. G.; Dwyer, M. P.; Chao,
J. Bioorg. Med. Chem. Lett. 2006, 16, 4107.
8. Dwyer, M. P.; Yu, Y.; Chao, J.-P.; Aki, C.; Chao, J.-H.; Biju, P.; Girijavallabhan, V.;
Rindgen, D.; Bond, R.; Mayer-Ezel, R.; Jakway, J.; Hipkin, R. W.; Fossetta, J.;
Gonsiorek, W.; Bian, H.; Fan, X.; Terminnelli, C.; Fine, J.; Lundell, D.; Merritt, J.
R.; Rokosz, L. L.; Kaiser, B.; Li, G.; Wang, W.; Stuffer, T.; Ozgur, L.; Baldwin, J. J.;
Taveras, A. G. J. Med. Chem. 2006, 49, 7603.
SAR trend observed in functional assay: When tested in a human
neutrophil chemotaxis assay, compound 5b exhibited functional
inhibitory activity with an IC50 value of 46 nM. The N-ethyl-N-Ac
incorporated analog of 5c showed much weaker potency with an
IC50 value close to 1.25 lM. The positive control 2b demonstrated
the good potency with IC50 value of 15 nM.
To our satisfaction, three ethylaryl hydrazine bearing analogs
5d, 5f, and 5g also exhibited impressive potencies (IC50) ranging
from 54 to 100 nM, which are almost equal potent to that demon-
strated by 5b. It is also worthwhile to mention that the functional
activities detected with these N-ethylaryl hydrazine analogs tracks
well with the receptor binding potencies obtained (Table 1).
In accordance with the reduced CXCR2 binding affinities, com-
pounds 5e and 5i exhibited about 10-fold weaker functional activ-
ity than 5g or 5f with IC50 value of 840 nM. The least potent analog
5 h displayed weakest functional activity (IC50 = 6.34 lM).
9. Chao, J.-H.; Taveras, A. G.; Chao, J.-P.; Aki, C.; Dwyer, M.; Yu, Y.; Purakkattle, B.;
Rindgen, D.; Jakway, J.; Hipkin, W.; Fosetta, J.; Fan, X.; Lundell, D.; Fine, J.;
Minnicozzi, M.; Phillips, J.; Merritt, J. R. Bioorg. Med. Chem. Lett. 2007, 17, 3778.
10. Baxter, A.; Cooper, A.; Kinchin, E.; Moakes, K.; Unitt, J.; Wallace, A. Bioorg. Med.
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11. PCT Patent: WO2006064228 (AZ).
12. PCT Patent: WO2001058906 (AZ).
13. PCT Patent: WO2005066147A1 (Jul. 21, 2005) (Schering).
In vitro microsomal stability evaluation—in light of the demon-
strated promising binding affinity for the CXCR2 receptor and good
potency in functional assay, three newly designed hydrazine con-
taining antagonists 5b, 5d, and 5g along with two positive controls
2a and 2b were tested for metabolic stability upon incubation with
human liver microsome at 37 °C for 30 min (with 1 lM final con-
centration for each compound) according to the protocol reported
by Merritt et al.7 All test compounds including 5b, 5d, and 5g exhib-
ited good stability with >50% of drug remaining after incubation at
37 °C for 120 min (T1/2 >2 h). Furthermore, compound 5b showed
good stability against rat microsome with a T1/2 value of 84 min.
In this communication, we have reported our preliminary data
on the discovery of a novel series of 3-amino-4-hydrazine-cyclo-
but-3-ene-1,2-dione containing CXCR2 antagonists including the
bis-N-ethyl bearing analog 5b. This compound was shown to pos-
sess potent CXCR2 binding affinity (Ki = 120 nM), adequate CXCR1
selectivity (80-fold), functional activity (IC50 = 46 nM) against IL-8-
mediated chemotaxis in a Chinese hamster ovary (CHO) cell line
(CXCR2 expressing line) as well as acceptable rat and human
microsomal stability. In addition, replacement of the bis-N-ethyl
moiety in 5b with N-ethyl-N-aryl hydrazines led to 5d and 5g, each
of which displayed good CXCR2 binding affinity (Ki = 110 or 130 nM)
and acceptable human microsomal stability (T1/2 >120 min). It is
conceivable that further modification of either bis-N-alkyl or
N-alkyl-N-aryl hydrazine moieties could yield more potent and
selective CXCR2 antagonists. The results of this research will be
reported in due time.
14. PCT Patent: 2005068460A1 (Jul. 28, 2005) (Schering).
15. Biju, P.; Yu, Y. Tetrahedron Lett. 2007, 48, 5279.
16. For hydrazine moiety incorporated HIV protease inhibitors, see: Reddy, G. S. K.
K.; Ali, A.; Nalam, M. N. L.; Anjum, S. G.; Cao, H.; Nathans, R. S.; Schiffer, C. A.;
Rana, T. M. J. Med. Chem. 2007, 50, 4316.
17. For hydrazine moiety incorporated HRV 3C protease inhibitors, see: Kati, W.
M.; Sham, H. L.; McCall, J. O.; Montgomery, D. A.; Wang, G. T.; Rosenbrook,
W.; Miesbauer, L.; Buke, A.; Norbeck, D. W. Arch. Biochem. Biophys. 1999, 362,
363.
18. For hydrazine bearing HCV protease inhibitor, see: Bailey, M. D.; Halmos, T.;
Goudreau, N.; Lescop, E.; Llinas-Brunet, M. J. Med. Chem. 2004, 47, 3788.
19. For hydrazine bearing SARS 3CL protease inhibitor, see: Anand, K.; Ziebuhr, J.;
Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R. Science 2003, 300, 1763.
20. For hydrazine moiety bearing approved drug (Reyataz), see: Bold, G.; Faessler,
A.; Capraro, H.-G.; Cozens, R.; Klimkait, T.; Lazdins, J.; Mestan, J.; Poncioni, B.;
Rosel, J.; Stover, D.; Tintelnot-Blomley, M.; Acemoglu, F.; Beck, W.; Boss, E.;
Eschbach, M.; Hurlimann, T.; Masso, E.; Roussel, S.; Ucci-Stoll, K.; Wyss, D.;
Lang, M. J. Med. Chem. 1998, 41, 3387.
21. Sajiki, H.; Ikawa, T.; Hirota, K. Org. Lett. 2004, 6, 4977.
22. Iranpoor, N.; Firouzabadi, H.; Pourali, A.-R. Synthesis 2003, 10, 1591.
23. Iffland, D. C.; Cerda, E. J. Org. Chem. 1963, 28, 2769.
24. WX’s Binding assay conditions:Reagent: wheatgerm-agglutinin coated SPA
beads and [
125I]IL-8 were from GE Healthcare (Piscataway, NJ), receptor
membranes CXCR2 and CXCR1 were from Chemicon (Temecula, CA). HEPES
was from Sigma (St Louis, MO). MgCl2 and KOH were from SCRC (Shanghai,
China). DMSO was from Sigma–Aldrich (Steinheim, Germany).Method: for each
200
(0.020
l
L reaction, a working mixture of receptor over-expressing membranes
g/ CXCR2 or 0.040 g/ CXCR1) and 2 mg/mL wheatgerm-
l
lL
l lL
agglutinin coated SPA beads was prepared in assay buffer. The assay buffer
was 25 mM HEPES, 3 mM MgCl2, pH 7.4. This mixture was incubated on ice for
5 min. A 0.040 nM 125I labeled IL-8 stock solution was prepared in the assay
buffer. Test compounds were serially diluted by half-log concentration in
DMSO. The above solutions were added to a 96 well plate (Perking Elmer) in
Acknowledgment
We are indebted to Kai Gu and Dr. Ning Zhao for analytic
support.
the following sequence: 45
100 L of membranes and SPA bead mixture and 50
stock solution. The assay plates were incubated for 2 h at room temperature,
keeping from light. Binding was detected using Perking Elmer-Wallace
lL assay buffer and 5
l
L test compound or DMSO,
l
lL [125I]IL-8 solution
References and notes
a
Microbeta 1450 liquid scintillation counter. The data was analyzed using
SigmaPlot (Systat Software Inc., San Jose, CA).
1. Review: Busch-Petersen, J. Curr. Top. Med. Chem 2006, 6, 1345.
2. Review: Moser, B.; Wolf, M.; Walz, A.; Loetscher, P. Trends Immunol. 2004, 25, 75.