Structural optimization of a CXCR2-directed antagonist that indirectly inhibits γ-secretase and reduces Aβ

Article abstract of DOI:10.1016/j.bmc.2009.09.051

Amyloid β (Aβ), a key molecule in the pathogenesis of Alzheimer's disease (AD), is derived from the amyloid precursor protein (APP) by sequential proteolysis via β- and γ-secretases. Because of their role in generation of Aβ, these enzymes have emerged as important therapeutic targets for AD. In the case of γ-secretase, progress has been made towards designing potent inhibitors with suitable pharmacological profiles. Direct γ-secretase inhibitors are being evaluated in clinical trials and new strategies are being explored to block γ-secretase activity indirectly as well. In this regard, we have previously reported an indirect regulation of γ-secretase through antagonism of CXCR2, a G-protein coupled receptor (GPCR). We demonstrated that N-(2-hydroxy-4-nitrophenyl)-N′-(2-bromophenyl)urea (SB225002), a selective inhibitor of CXCR2 also plays a role in an indirect inhibition of γ-secretase. Furthermore, we reported a ～5-fold difference in the selective inhibition of APP versus Notch processing via γ-secretase following treatment with SB225002. Herein we describe the synthesis and optimization of SB225002. By determination of the structure-activity relationship (SAR), we derived small molecules that inhibit Aβ40 production with IC₅₀ values in the sub-micromolar range in a cell-based assay and also validated the potential of CXCR2 as a new target for therapeutic intervention in AD.

Full text of DOI:10.1016/j.bmc.2009.09.051

Bioorganic & Medicinal Chemistry 17 (2009) 8102–8112
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural optimization of a CXCR2-directed antagonist that indirectly inhibits
c-secretase and reduces Ab
*
Pancham Bakshi , Chao Jin, Pierre Broutin, Beniam Berhane, Jon Reed, Michael Mullan
Roskamp Institute, 2040 Whitfield Avenue, Sarasota, FL 34243, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2009
Revised 17 September 2009
Accepted 30 September 2009
Available online 3 October 2009
Amyloid b (Ab), a key molecule in the pathogenesis of Alzheimer’s disease (AD), is derived from the amy-
loid precursor protein (APP) by sequential proteolysis via b- and
generation of Ab, these enzymes have emerged as important therapeutic targets for AD. In the case of
-secretase, progress has been made towards designing potent inhibitors with suitable pharmacological
profiles. Direct -secretase inhibitors are being evaluated in clinical trials and new strategies are being
explored to block -secretase activity indirectly as well. In this regard, we have previously reported an
indirect regulation of -secretase through antagonism of CXCR2, a G-protein coupled receptor (GPCR).
We demonstrated that N-(2-hydroxy-4-nitrophenyl)-N0-(2-bromophenyl)urea (SB225002), a selective
inhibitor of CXCR2 also plays a role in an indirect inhibition of -secretase. Furthermore, we reported
-secretase following
c-secretases. Because of their role in
c
c
c
Keywords:
Alzheimer’s disease
Secretase
CXCR2
Amyloid beta
c
c
a ꢀ5-fold difference in the selective inhibition of APP versus Notch processing via
c
treatment with SB225002. Herein we describe the synthesis and optimization of SB225002. By determi-
nation of the structure–activity relationship (SAR), we derived small molecules that inhibit Ab40 produc-
tion with IC50 values in the sub-micromolar range in a cell-based assay and also validated the potential of
CXCR2 as a new target for therapeutic intervention in AD.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
N-terminally truncated form of Ab called p3 (a non-amyloidogenic
fragment) and AICD.3
Alzheimer’s disease (AD) is a progressive dementia believed to
Because of the evident involvement of Ab in the pathogenesis of
AD, several drug discovery efforts are aimed toward inhibition of
Ab production, prevention of Ab aggregation, and enhancing Ab
clearance. Preventing Ab production has focused on targeting the
enzymes involved in APP processing.4,5 Detailed structural charac-
terization of b-secretase6–9 led to the discovery of many transition-
state based inhibitors with activity in the low nanomolar range,10
though their in vivo efficacy was found to be limited.10,11 The large
active site of b-secretase has made it difficult to develop small mol-
ecules with suitable pharmacological profiles.12 This problem has
recently been overcome, and these well-designed inhibitors await
further investigation of their potential as drug candidates.12–14 De-
spite significant progress towards the biochemical characterization
be caused by accumulation of Ab in the brain.1 Ab peptides are gen-
erated by the sequential action of b- and
c-secretases on the
amyloid precursor protein (APP), a type 1 integral membrane pro-
tein.2 In this amyloidogenic cleavage pathway, b-secretase (also re-
ferred to as beta-site APP cleaving enzyme, or BACE) generates the
NH2-terminus of Ab, cleaving APP at the cell-surface to produce a
soluble version of APP (sAPPb) and a 99-residue COOH-terminal
fragment (C99) that remains membrane bound.2 C99 is then
cleaved by
the 4-kD Ab and a cytoplasmic tail, APP-intracellular domain
(AICD).2 Proteolysis of C99 by
-secretase is heterogeneous and
c-secretase in the intramembrane region to produce
c
produces Ab peptides that vary in length from 37 to 43 amino
acids. The most common Ab species are a 40-residue peptide
(Ab40), and a 42-residue peptide (Ab42).3 In an alternative non-
of
stood.15 Biochemical evidence demonstrates that the full spectrum
of -secretase activity can be reconstituted by the co-expression of
c-secretase, its mechanism of action is still not fully under-
amyloidogenic pathway,
produce soluble version of APP (sAPP
membrane bound COOH-terminal fragment (C83).3 C83 subse-
quently also serves as a substrate for -secretase, producing an
a
-secretase cuts within the Ab region to
c
a)
and an 83-residue
four integral membrane proteins, Presenilin (PS), Nicastrin (NCT),
Aph-1, and Pen-2, which are the minimally required constituents
c
of physiologically active
ated
c
-secretase.16–18 This membrane associ-
c
-secretase complex has resisted purification efforts that
* Corresponding author. Tel.: +1 941 752 2949x318; fax: +1 941 752 2948.
E-mail addresses: pbakshi@rfdn.org (P. Bakshi), cjin@rfdn.org (C. Jin), pebroutin
@yahoo.fr (P. Broutin), beniambt@gmail.com (B. Berhane), jreed@rfdn.org (J. Reed),
mmullan@rfdn.org (M. Mullan).
would allow crystallization and detailed characterization.15
Recently, cryoelectron microscopy images provided the first avail-
able structure of the
c
-secretase complex at 12 Å resolution.19,20
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.051

P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
8103
NH2
N
Additionally, several structurally diverse low molecular-weight
-secretase inhibitors have been reported that have helped to
understand the nature and role of -secretase with regard to its
structure and regulation.21 Based on the mechanism of inhibition
of -secretase, these inhibitors have been classified as direct and
indirect inhibitors.21 These inhibitors have enhanced our under-
standing of the binding pocket of -secretase, as well as the biomo-
lecular pathways that interact with and regulate -secretase and
Ab production.22–26 Recently, numerous
-secretase substrates
(a)
C
O
R
R
c
c
1a
1b
c
H
H
N
N
H2N
N
(b)
C
c
O
R
R'
R
R'
+
O
c
c
1c
1d
1b
(>30) have been identified that raise concerns about targeting
c
c
-secretase for therapeutic intervention of AD.5,27 Although one
-secretase inhibitor (LY450139) has advanced into late-phase
Scheme 1. Reaction conditions (a) DCM, aqueous saturated NaHCO3, phosgene
solution (20% in toluene), 0 °C, 30 min; (b) THF, 50 °C, 12 h.
clinical trials,28,29 the in vivo toxicity observed with use of
c
-secre-
tase inhibitors that block Ab production and apparently proteolysis
of the Notch receptor (an important substrate of -secretase) is a
cause of concern.5,21,27 Therefore it is desirable to develop
-secre-
(20% in toluene; 2 equiv) into the settled CH2Cl2 layer of the mix-
ture at 0 °C. After 30 min of vigorous stirring at room temperature,
the organic phase was separated, dried over Na2SO4, filtered and
concentrated. The N,N0-diarylurea 1d (2–46) was then made by
coupling another substituted aniline 1c (1 equiv) with the phenyl-
isocyanate 1b in a minimal amount of THF at 50 °C for 12 h
(Scheme 1). The final compound 1d was purified using flash chro-
matography and crystallization to >95% purity which was deter-
mined using HPLC and elemental analysis. Compounds that met
these purity criteria were then tested in the cell-based assay de-
scribed below.
c
c
tase inhibitors that have the ability to selectively inhibit Ab pro-
duction without hindering Notch proteolysis.27 In this regard,
evidence is emerging that many biochemical pathways can impact
c
-secretase activity.30–34
Towards finding such pathways that can alter c-secretase activ-
ity, we showed that CXCR2, a GPCR which mediates inflammatory
signaling through binding with its ligands, blocks Ab production
through inhibition of
c
-secretase.31 We also demonstrated that
SB225002, which is a selective antagonist for the CXCR2 recep-
tor,35 is a potent inhibitor of Ab production.31 On exploring the
inhibitory mechanism of SB225002 on Ab production, we observed
3. Biological evaluation
and reported a dose-dependent indirect inhibition of
activity with no effect on
- and b-secretase activity.31 Thus, our
initial study suggested an important role of CXCR2 as a novel target
in regulation of -secretase activity. At the time of our report, two
other labs independently reported the effect of other GPCRs on the
c-secretase
Recent studies performed by our group identified SB225002 (2)
as an inhibitor of Ab production through the indirect blocking of
c
a
-secretase activity.31 The cellular and molecular biology data re-
c
vealed a pivotal role of an inflammatory receptor, CXCR2, in regu-
lation of
-secretase.31 SB225002 (2), reported to be a selective
inhibitor of the CXCR2 receptor,35 was also found to indirectly in-
c
regulation of
c-secretase activity, although the proposed mecha-
nisms of action of these GPCRs differ from one another.32,33
Herein we describe the synthesis and structural optimization of
SB225002 with the aims of improving activity and determining the
steric and electronic requirements for binding to CXCR2, which
hibit c-secretase activity without any effect on a- and b-secretase
activities.31 In this study we focused our effort to (1) improve
activity (2) determine the steric and electronic requirement of
SB225002 (2) pharmacophore needed for interaction with the
then results in indirect inhibition of
ent study also elucidates the importance of the phenolic 2-hydroxy
moiety necessary for -secretase inhibition. The acidic nature of
c-secretase activity. The pres-
CXCR2 binding site required for blocking
(3) elucidate the importance of the phenolic 2-hydroxy moiety of
the SB225002 (2) pharmacophore for -secretase inhibition. The
c-secretase activity, and
c
c
phenolic 2-hydroxy has been suggested as an important structural
feature in CXCR2 antagonism and inhibition of its associated
downstream pathways.36,37
acidic nature of the phenolic 2-hydroxy of SB225002 (2) has been
shown to be an important structural feature in antagonizing CXCR2
and its association with G-proteins.36–38
Our previous work demonstrated that the CXCR2 antagonists,
SB225002 (2) and Repertaxin (chemically dissimilar from
SB225002 (2)), inhibit Ab40 and 42 production with tight correla-
2. Results and discussion
2.1. Chemistry
tion between their IC50 values, suggesting a similar c-secretase is
responsible for Ab40 and 42 generation.31 Thus in the present
study, the SAR of synthesized compounds was derived by evaluat-
ing their effect on Ab40 production in Chinese Hamster Ovary
(CHO) cells stably transfected with human APP 751 (7w CHO cell
line (7w))26,39 as described previously. Briefly, stock concentrations
of analogues were made in DMSO and added to the media to a final
concentration of 1% DMSO; positive controls contained 1% DMSO
only. The compounds were incubated with cells for 18 h, media
was collected, centrifuged, and protein-normalized supernatant
was checked for secreted Ab40 by ELISA.
The optimized compounds derived through the SAR were fur-
ther validated for their inhibitory effect on agonist-induced calcium
mobilization assay using a previously described procedure35 with
slight modifications. Briefly, 7w cells in a 96-well plate were loaded
with Fura-4 NW (no wash required) using manufacturer’s instruc-
tions. The stock concentrations of analogues (in 1% DMSO, final con-
centration) were added at the indicated concentrations (negative
controls contained 1% DMSO only), followed 15 s later by addition
We divided SB225002 (2) into two domains (A and B) for SAR
optimization.
For the synthesis of methoxy substituted N,N0-diphenyl urea
analogues (11–16), we methylated the phenolic hydroxyl group
of commercially available substituted anilines. Briefly, the hydro-
xyl substituted aniline was dissolved in acetone with K2CO3
(1.2 equiv), and was subsequently alkylated by MeI (5 equiv). The
reaction was stirred at 50 °C for 15 h, then quenched with H2O,
and extracted with CH2Cl2. The methoxy-containing substituted
anilines was dried, concentrated, purified and subsequently cou-
pled with phenylisocyanate as described below to obtain com-
pounds 11–16.
The final compounds 2–46 were synthesized using a previously
described route.26 Briefly, to access the compounds 2–46, substi-
tuted phenylisocyanate 1b (Scheme 1), was made by stirring
substituted aniline 1a (1 equiv) in a mixture of CH2Cl2 and aqueous
saturated NaHCO3, followed by the addition of phosgene solution

8104
P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
of Gro
a
(33 nM final concentration). The maximal calcium concen-
and a bulky group like –Br, –Cl or even –OMe on the second phenyl
tration attained after Gro
a
stimulation was quantitated as de-
ring. Together, these data indicate a preference for (1) a phenolic –
OH (or at least a substituent that can be involved in H-bonding
(both –OH and –F containing molecules are active)) on one ring
(preferably at position 2) and (2) a bulky group substitution on
the second ring (although 4 contains both –OH and –F, it is less
active than 14 that contains a bulky group and –F). Changing the
position of –F on the phenyl ring of domain A from position 2 to
3 was found to be detrimental for activity (Table 1, compare 14
vs 15), which is consistent with our earlier results. Also, exchang-
ing the position of –NO2 and –OMe on the phenyl ring of domain B
while keeping –F at position 2 on the ring of domain A (16) reduced
activity (Table 1, compare 14 vs 16). This further confirms the
requirement of a substituent on one ring that can be either in-
volved in H-bonding or have an acidic proton and a bulky group
on the second ring.
After demonstrating the importance of phenolic –OH group in
domain B, we next focused on optimizing domain A keeping do-
main B unchanged. To optimize steric effects and further map the
binding domain of CXCR2, we substituted domain A with multiple
halogens while leaving domain B unchanged as in SB225002 (2).
We synthesised several di-halosubstituted compounds, and their
inhibitory effect on Ab40 production is illustrated inTable 2. Substi-
tution at positions 2 and 4 of domain A with –Br (17), –Cl (18) and
–F (19) exhibited inhibitory activity in order of –Cl (18) > –Br
(17) > –F (19) (Table 2). Because of the difficulty encountered in
synthesis of dibrominated analogues, we chose to synthesize di-
chloro-substituted analogues instead. On exploring the optimal po-
sition of the –Cl groups on the phenyl ring A, the order of activity
was found to be 2,4-dichloro (18) > 2,5-dichloro (20) > 3,4-dichloro
(21) (Table 2). However, the order of activity with difluoro substitu-
tion was found to be 3,4-difluoro (22) > 2,4-difluoro (19) (Table 2).
For comparison, we made several tri-substituted derivatives (23–
28) using commercially available –Cl and –F tri-substituted ani-
lines. However, the syntheses were sluggish and resulted in poor
yield of the final compounds. With Cl substitution, the order of
activity was found to be in order of 2,3,4-trichloro (23) = 2,4,5-tri-
chloro (24) (Table 2). Interestingly, for trifluoro-substituted ana-
logues , the order of activity was found to be 3,4,5-trifluoro
(25) = 2,3,4-trifluoro (27) > 2,4,5-trifluoro (28) > 2,4,6-trifluoro
(26) (Table 2). Importantly, trichloro-substituted analogues were
found to be less active than di-substituted and even mono-substi-
tuted analogues. In contrast, the trifluoro analogues inhibited
scribed previously.35
We initially focused on hydrogen and different halogen substi-
tutions at position 2 on the phenyl ring of domain A, keeping the
same position of –NO2 and –OH groups on the phenyl ring of do-
main B in SB225002 (2). Substituting the 2-Br from SB225002 (2)
with –H (3), –F (4) and –Cl (5) revealed that both –Br (2) and –Cl
(5) at position 2 (Table 1) are well-tolerated. However, both H
(3) and F (4) substitutions in position 2 reduced the potency by
approximately 20-fold compared to the lead (Table 1).
A significant reduction in activity was noticed with both re-
moval of phenolic –OH (6) (Table 1, compare 4 vs 6) or with ex-
change of positions between –OH and –NO2 group (7) (Table 1,
compare 4 vs 7) from domain B, suggesting the requirement for
the –OH group at position 20 of the phenyl ring for optimum activ-
ity. Removal of either the 40-NO2 group alone (Table 1, compare 8
vs 5 and 9 vs 2) or both 20-OH and 40-NO2 (10) from the phenyl ring
of domain B was found to be detrimental for inhibition of Ab40
production. Methylation of 20-OH on the phenyl ring of domain B
to 20-OMe (Table 1), with the domain A phenyl ring substituted
at position 2, either with –H or –Br or –Cl or –F resulted in com-
pounds 11, 12, 13 and 14, respectively. Compounds 11, 12 and
13 lacked activity even at 100 lM (Table 1, compare 11 vs 3; 12
vs 2; 13 vs 5), implicating the 20-OH group on the phenyl ring of
domain B as an important chemical feature necessary for blocking
c-secretase through CXCR2. However, compound 14 (Table 1) was
found to be more active than analogue 4 (Table 1), indicating the
requirement of a H-bonding group at position 2 of one phenyl ring
Table 1
Inhibitory effect of compounds on Ab40 production in 7w cells
A
H
H
B
N
N
R
R'
O
a
Compound
no.
Domain A
R
Domain B
R0
Ab40/IC50
M)
(
l
0
SB225002
R
2 = Br
R2 = OH,
0.5 0.03
0
(2)
R4 = NO2
0
3
R2 = R3 = R4 = R5 = R6 = H R2 = OH,
12 0.1
0
R4 = NO2
0
4
5
R2 = F
R2 = Cl
R2 = F
R2 = OH,
10 0.08
0.6 0.05
0
Table 2
R4 = NO2
0
Inhibitory effect of compounds on Ab40 production in 7w cells
R2 = OH,
0
R4 = NO2
OH
40
H
H
2
6
7
R
= NO2
30 0.2
NA
N
N
3
0
R
2 = F
R4 = OH,
R4
6
0
R2 = NO2
O
NO2
5
0
8
9
R2 = Cl
R
R2 = OH
NA
NA
NA
NA
0
2 = Br
R2 = Br
R2 = OH
a
Compound no.
R
Ab40/IC50 (lM)
10b
11
0
R2 = R3 = R4 = R5 = R6 = H R2 = OMe,
17
18
19
20b
21
22
23
24
25
26
27
28
R
R
R
2 = R4 = Br
2 = R4 = Cl
2 = R4 = F
0.8 0.08
0.3 0.02
15 0.1
0.5 0.1
0.7 0.2
0
R4 = NO2
0
12
R2 = Br
R2 = Cl
R2 = F
R3 = F
R2 = F
R2 = OMe,
NA
NA
0
R4 = NO2
R2 = R5 = Cl
0
R2 = OMe,
R
R
3 = R4 = Cl
3 = R4 = F
13
0
R4 = NO2
8
3
3
8
0.06
0.02
0.01
0.1
0
14b
15b
16
R2 = OMe,
5
0.1
R2 = R3 = R4 = Cl
0
R4 = NO2
R
R
R
R
R
2 = R4 = R5 = Cl
3 = R4 = R5 = F
2 = R4 = R6 = F
2 = R3 = R4 = F
2 = R4 = R5 = F
0
R2 = OMe,
12 0.2
10 0.1
0
R4 = NO2
15 0.1
0.1
10 0.3
0
R4 = OMe,
8
0
R2 = NO2
NA: not active at 100 lM.
NA: not active at 100
l
M.
a
a
Values are the mean of three separate experiments SEM.
Values are the mean of three separate experiments SEM.
b
b
Purchased from Sigma–Aldrich.
Purchased from Sigma–Aldrich.

P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
8105
Ab40 production with almost the same potency as mono and di-
fluoro analogues, suggesting stringent steric and spatial require-
ments of the CXCR2 interacting site.
logues with only 2,4-dimethyl and 3,5-dimethyl analogues com-
pared to 29. This is consistent with our earlier findings with di-
halosubstituted analogues, and suggests stringent steric require-
ments at position 2 of the phenyl ring of domain A.
To more precisely define the electronic versus steric require-
ments on ring A for inhibition of Ab production, we made a third
series of compounds (Table 3) using a more diverse set of substit-
uents. For this we purchased all commercially available mono-al-
kyl (methyl to butyl) substituted anilines. The syntheses became
more difficult with increase in bulk, and so we restricted the series
to no more than a butyl group. Substitution of position 2 on the
phenyl ring of domain A with methyl, ethyl and n-propyl groups
(2-n-butyl aniline is not commercially available) resulted in com-
pounds (29), (30) and (31), respectively (Table 3). The order of
activity was n-propyl (31) = n-ethyl (30) > n-methyl (29) (Table
3), suggesting that an increase in steric bulk at position 2 of the
phenyl ring of domain A correlates with an improved inhibitory
effect on Ab production. The ability to lower Ab40 production
was significantly reduced by changing the position of similar sub-
stituents on the phenyl ring (Table 3, compare 30 vs 33 and 34 and
31 vs 35 (3n-propyl aniline is not commercially available)). Also,
branching at position 2 of the phenyl ring of domain A and groups
larger than 3-carbon containing alkyl chains reduced inhibitory
activity (Table 3, compare 31 vs 36; 31 vs 32 and 37 and 32 vs
37). Next, in order to see if additional bulk at other positions of do-
main A would improve activity, we used dialkyl-substituted ani-
lines to make compounds 38–46 (Table 4). All dialkyl derivatives
showed less activity compared to monoalkyl-substituted ana-
Using the SAR, we were able to derive two compounds, 30 and
31, that reduced Ab40 production via inhibition of
c-secretase
activity beyond starting molecules 2 and 5 (Tables 1 and 3; Fig. 1).
Finally, to determine if compounds 30 and 31 (optimized for Ab
inhibition) were also functional CXCR2 antagonists, we monitored
their effects on intracellular calcium mobilization stimulated by
GRO
compounds 30, 31 and SB225002 (2) produced a concentration-
dependent inhibition of GRO -mediated calcium mobilization
with ꢀIC50 values of 100 nM, 300 nM and 1 M, respectively
(Fig. 2). The order of potency of these compounds towards inhibi-
tion of Ab production and inhibition of GRO -mediated calcium
a. For a positive control, we used SB225002 (2). In these cells,
a
l
a
mobilization is the same. Further, we found a tight correlation
((regression) r2 = 0.989) between the IC50 for Ab inhibition with
120
SB225002 (2)
5
30
100
31
80
60
40
20
0
Table 3
Inhibitory effect of compounds on Ab40 production in 7w cells
OH
2
H
H
N
N
3
R4
6
O
NO2
5
-20
0.001
0.01
0.1
1
10
100
a
Compound no.
R
Ab40/IC50
(lM)
[Compound] (μM)
29
30
31
32
33
34
35
36
37
R
R
R
R
R
2 = Me
2 = Et
2
0.03
0.09 0.05
0.1 0.08
1
4
3
4
Figure 1. Effects of designed inhibitors SB225002 (2), 5, 30 and 31 on Ab40
production. 7w cells stably transfected with human APP751 were treated with
various concentrations of the indicated compounds for 18 h, whereupon the media
was collected for Ab sandwich ELISA analysis. Values are means s.d. from three
separate experiments and are expressed relative to control values.
2 = n-Pr
2 = sec-Bu
3 = Et
0.01
0.02
0.06
0.02
R4 = Et
R
R
R
4 = n-Pr
2 = i-Pr
2 = t-Bu
0.3 0.01
15 0.05
120
NA: not active at 100
l
M.
SB225002 (2)
31
30
a
Values are the mean of three separate experiments SEM.
100
80
60
40
20
0
Table 4
Inhibitory effect of compounds on Ab40 production in 7w cells
OH
2
H
H
N
N
3
R4
6
O
NO2
5
a
Compound no.
R
Ab40/IC50
(
lM)
38
39
40
41
42
43
44
45
46
R
R
R
R
R
R
2 = R4 = Me
3
0.02
-20
2 = R5 = Me
10 0.02
15 0.1
3 = R4 = Me
-40
3 = R5 = Me
2
7
3
5
8
0.1
0.2
0.06
0.02
0.01
0.01
0.1
1
10
100
1000
2 = Et, R6 = Me
2 = Et, R6 = i-Pr
[Compound] (μM)
R2 = iPr, R6 = Me
2 = R6 = i-Pr
Figure 2. Effects of designed inhibitors SB225002 (2), 30 and 31 on Gro
calcium mobilization. 7w cells were pretreated for 15
concentrations of SB225002 (2), 30, 31 (10–100,000 nM) before the addition of
the agonist (Gro ). Each data point represents means s.d (n = 8) and the result is a
representative from three separate experiments.
a
-induced
R
R
2 = sec-Bu, R6 = Et
15 0.1
s
with the indicated
NA: not active at 100
l
M.
a
a
Values are the mean of three separate experiments SEM.

8106
P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
60000
50000
40000
30000
20000
10000
0
study led to the conclusion that the phenolic –OH, a prerequisite
for antagonism of the CXCR2 receptor,44 is also required to inhibit
Ab production through indirect inhibition of -secretase activity.
c
Further, we found that in addition to the acidic proton of the phe-
nolic-OH at position 2 of ring A, the ability of the substituents to
undergo H-bonding, has a positive effect on the interaction with
the CXCR2 binding domain. Thus, effects of systematic chemical
manipulation in the parent molecule on Ab production suggest that
there are stringent steric and electronic requirements at position 2.
Specifically, we found a preference for (1) a bulky group at position
2 of one of the phenyl rings and (2) a substituent at position 20 on
the other phenyl ring that can be involved in H-bonding or have
acidic proton. Using this SAR, we were able to derive two com-
pounds, 30 and 31, that reduced Ab40 production via inhibition
of c-secretase activity beyond starting molecules 2 and 5 (Tables
0
2000
4000
6000
8000
10000
12000
14000
16000
1 and 3; Fig. 1). Finally, we confirmed that compounds 30 and
31, which were optimized for Ab production inhibition, are also
Aβ 40 (IC50
)
functional CXCR2 antagonists, as both of them inhibited Groa-in-
Figure 3. Correlation between the IC50 for Ab inhibition with IC50 for inhibition of
Gro -induced intracellular calcium mobilization. Nonlinear regression was used to
derive the correlation between the IC50 values for Ab inhibition (x-axis) with IC50
values for Gro -induced intracellular calcium mobilization inhibition (y-axis) for
compounds SB225002 (2), 4, 5, 14, 20, 30, 31 and 36 (r = 0.9949; r2 = 0.9898).
a
duced intracellular calcium mobilization in a dose-dependent
manner (Fig. 2). Importantly, as demonstrated inFigure 3, we found
a tight correlation ((regression) r2 = 0.989) between the IC50 for Ab
a
and the IC50 for Groa-induced intracellular calcium mobilization
for compounds SB225002 (2), 4, 5, 14, 20, 30, 31 and 36. In conclu-
sion, we have identified compounds that maximize the inhibition
of CXCR2-mediated c-secretase activity, which in addition to their
IC50 for inhibition of Groa-induced intracellular calcium mobiliza-
tion for compounds SB225002 (2), 4, 5, 14, 20, 30, 31 and 36
(Fig. 3). This observation and our previously published studies lend
further support to the hypothesis that CXCR2 regulates c-secretase
Ab-lowering properties are likely to mitigate the CXCR2-mediated
inflammation which accompanies and exacerbates the AD.
The data in this manuscript and in recently published work
demonstrates that antagonism of CXCR2 with structurally unre-
activity and Ab production.
4. Conclusions
lated ligands31 reduces
c-secretase activity, suggesting an impact
on the pathways from the former to the latter. Furthermore, we
have also validated CXCR2 as a potential target in gamma-secre-
tase regulation in vivo (Bakshi et al., unpublished result). The dif-
ferential manipulation of downstream pathways (permissive
antagonism) of CXCR2 needs further exploration with these com-
Alzheimer’s disease (AD) is a progressive chronic disorder that
leads to cognitive decline.1 Recent advances in therapeutic inter-
vention in AD through blocking
direct and indirect inhibitors of
loose substrate specificity of -secretase, in vivo studies suggest
that inhibition of -secretase with compounds that equally prevent
processing of APP and apparently Notch can result in toxic-
ity.5,21,24,27 It has been demonstrated that
-secretase-mediated fi-
nal processing of APP can be regulated through different
biochemical pathways.30,32,33,21,24,40,41 For instance,
-secretase
has been shown to be modulated through another GPCR, GPR3,
that affects cell-surface localization of the mature -secretase
c
-secretase activity have identified
c
-secretase.21,24 Because of the
c
pounds and may lead to inhibition of c-secretase-mediated APP
processing and without inhibition of other substrates.
c
5. Experimental procedures
c
5.1. General techniques
c
All reactions requiring anhydrous conditions were conducted in
oven-dried glassware under an atmosphere of nitrogen. Prepara-
tive chromatographic separations were performed using a Combi-
flash Companion (Isco Inc., Lincoln, NE); reactions were followed
by TLC analysis using silica plates with a fluorescent indicator
(254 nm) and visualized with UV phosphomolybdic acid or 4-hy-
droxy-3-methoxybenzaldehyde. All commercially available re-
agents were purchased from Aldrich and Acros and were
typically used as supplied unless stated otherwise.
c
and thus impacts
c-secretase specificity towards APP and Notch
processing.33 In this regard, through studying the association of
chemokine receptors with altered APP processing and increased
production of Ab, we have demonstrated an important role of
CXCR2 in regulation of
c-secretase activity and in inhibition of
Ab40/42 production.31 Further, we reported a small molecule,
SB225002 (2), known to selectively antagonize CXCR2,35 which
blocked Ab40/42 production in a cell-based assay without affecting
a
- and b-secretases.31 SB225002 (2) was found to alter APP pro-
HPLC analyses were carried out on an Agilent 1100 series sys-
tem using an analytical HPLC column (Eclipse XDB-C18, 5 lm,
cessing through indirect regulation of
c-secretase activity via
CXCR2 antagonism.31 Specifically, we found that antagonism of
CXCR2 leads to reduction in PS1-CTF (a critical component of
4.6 mm i.d.) and a diode array as the detector. Compounds were
separated over a period of 40 min gradient of water and acetoni-
trile (0–80%) at a flow rate of 1 ml/min. 1H and 13C NMR spectra
were recorded in Fourier transform mode at field strengths speci-
fied on a Varian AS500 spectrometer. Chemical shifts are expressed
in parts per million (ppm) downfield of tetramethylsilane (singlet
at 0 ppm, TMS) as an internal standard for 1H resonances. Multi-
plicities in the 1H NMR spectra are described as: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, br = broad; cou-
pling constants (J) are reported in hertz (Hz). High resolution mass
spectra (HRMS) were measured on a LTQ-Orbitrap mass spectrom-
eter (calibrated immediately prior to analysis) utilizing an Advion
Bioscience Nanomate electrospray source. Full scan MS spectra
c-secretase, required for its activity) at the protein level that in
turn leads to degradation, and thus reduction of other -secretase
c
components.31 This finding is consistent with recent findings that
demonstrate that inhibition of basal activity of c-jun-NH2-terminal
kinase (JNK) (JNK, downstream of CXCR2) represses the expression
of FLPS42 and reduces the stabilization of PS1-CTF.34 Similarly, it is
plausible that CXCR2 might mediate stabilization of PS1-CTF (half
life ꢀ 24 h),43 an endoproteolysed product of FLPS43 via JNK.
In the present study, we sought to define the topological
requirements around SB225002 (2) by iterative modification of
substituents around the pharmacophore. This systematic SAR

P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
8107
were acquired in Fourier Transform (FTMS) mode at a resolution of
60,000 FWHM in centroid mode. Samples were dissolved in 50%
methanol, 0.1% formic acid. Reserpine was added to each sample
116.5 (CAr), 122.5 (CAr), 122.6 (CAr), 123.3 (CAr), 123.9 (CAr), 124.2
(CAr), 127.7 (CAr), 129.5 (CAr), 135.2 (CAr), 136.1 (CAr), 145.5 (CAr),
152.3 (CO). HRMS (ES) m/z 308 (M+H)+, m/z 308.0329 (calcd for
C13H11ClN3O4 (M+H)+: 308.036).
at 2 ng/l
l as an internal lockmass standard ([M+H+] = 609.2812).
Ion mass/charge (m/z) ratios are reported as values in atomic mass
units.
5.2.5. 1-(2-Fluorophenyl)-3-(4-nitrophenyl)urea (6)
Method A was utilized for synthesis. Brown solid (22% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 7.06–7.11 (1H,
m, CHAr), 7.16–7.22 (2H, m, CHAr), 8.03 (4H, A2B2, JAB = 9.2 Hz
CHAr), 8.29 (1H, br t, J = 8.0 Hz, CHAr), 9.12 (1H, br s, NH); 13C
NMR (acetone-d6, 125 MHz) d 99.6 (CAr), 119.7 (CAr), 122.5 (CAr),
122.6 (CAr), 126.0 (CAr), 128.2 (CAr), 129.4 (CAr), 129.8 (CAr),
132.2 (CAr), 147.0 (CAr), 150.9 (CAr), 156.5 (CAr), 158.5 (CO). HRMS
(ES) m/z 276 (M+H)+; m/z 276.0707 (calcd for C13H11FN3O3
(M+H)+: 276.0706).
5.2. General method of synthesis
5.2.1. Method A: 1-(2-bromophenyl)-3-(2-hydroxy-4-
nitrophenyl)urea SB225002 (2)
2-Bromophenylisocyanate was made by stirring 2-bromoani-
line (0.57 mL; 5 mmol) in a mixture of CH2Cl2 and aqueous satu-
rated NaHCO3, followed by the addition of phosgene solution
(20% in toluene; 5.18 mL, 10 mmol) into the settled CH2Cl2 layer
of the mixture. After 30 min of vigorous stirring, the organic
phase was separated, dried over Na2SO4, filtered and concen-
trated. The 2-hydroxy-4-nitroaniline (154.1 mg, 1 mmol) was
then coupled with bromophenylisocyanate in a minimal amount
of THF at 50 °C for 12 h. The reaction was concentrated and puri-
fied by flash chromatography over silica gel (EtOAc/hexanes 1:1).
The solid obtained after evaporation was crystallized from ace-
tone/hexane to >98% purity. Yellow solid (34% yld); 1H NMR (ace-
tone-d6, 500 MHz) d 7.06 (1H, td, J = 1.3, 7.9 Hz, CHAr), 7.41 (1H,
td, J = 1.3, 7.9 Hz, CHAr), 7.63 (1H, dd, J = 1.3, 7.9 Hz, CHAr), 7.77
(1H, d, J = 2.5 Hz, CHAr), 7.84 (1H, dd, J = 2.5, 9.0 Hz, CHAr), 8.19
(1H, dd, J = 1.3, 7.9 Hz, CHAr), 8.47 (1H, d, J = 9.0 Hz, CHAr), 8.57
(1H, br s, NH), 8.95 (1H, br s, NH), 9.85 (1H, br s, OH); 13C
NMR (acetone-d6, 125 MHz) d 109.5 (CHAr), 114.1 (CAr), 116.5
(CHAr), 118.2 (CHAr), 123.7 (CHAr), 125.0 (CHAr), 128.2 (CHAr),
132.8 (CHAr), 135.2 (CAr), 137.2 (CAr), 142.2 (CAr), 145.6 (CAr),
152.4 (CO). HRMS (ES) m/z 352 (M+H)+; m/z 351.9858 (calcd for
C13H11BrN3O4 (M+H)+: 351.9855).
5.2.6. 1-(2-Fluorophenyl)-3-(4-hydroxy-2-nitrophenyl)urea (7)
Method A was utilized for synthesis. Brown solid (69% yld; >98%
purity); 1H NMR (acetone-d6, 500 MHz) d 7.04–7.09 (1H, m, CHAr),
7.13–7.19 (2H, m, CHAr), 7.26 (1H, dd, J = 2.6, 9.1 Hz, CHAr), 7.57
(1H, dd, J = 2.6 Hz, CHAr), 8.22 (1H, t, J = 7.9 Hz, CHAr), 8.30 (1H, d,
J = 9.1 Hz, CHAr), 8.94 (2H, br s, NH), 9.47 (1H, br s, OH); 13C NMR
(acetone-d6, 125 MHz) d 115.0 (CAr), 119.7 (CAr), 126.7 (CAr),
127.6 (CAr), 128.1 (CAr), 129.1 (CAr), 130.0 (CAr), 123.2 (CAr), 132.3
(CAr), 143.5 (CAr), 156.9 (CAr), 157.2 (CAr), 158.6 (CO). HRMS (ES)
m/z 292 (M+H)+; m/z 292.0658 (calcd for C13H11FN3O4 (M+H)+:
292.0655).
5.2.7. 1-(2-Chlorophenyl)-3-(2-hydroxyphenyl)urea (8)
Method A was utilized for synthesis. White solid (80% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 6.76 (t, J = 7.5 Hz, 1H,
CHAr), 6.87–6.81 (m, 2H, CHAr), 7.03 (t, J = 7.5 Hz, 1H, CHAr), 7.29
(t, J = 7.5 Hz, 1H, CHAr), 7.45 (d, J = 7.5 Hz, 1H, CHAr), 8.00 (d,
J = 8.0 Hz, 1H, CHAr), 8.10 (d, J = 7.5 Hz, 1H, CHAr), 8.95 (s, 1H,
NH), 8.97 (br s, 1H, NH), 9.89 (s, 1H, OH); 13C NMR (DMSO-d6,
125 MHz) d 115.3 (CHAr), 119.8 (CHAr), 120.2 (CHAr), 122.9 (CHAr),
123.0 (CHAr), 123.2 (CHAr), 124.0 (CHAr), 128.1 (CHAr), 128.2 (CAr),
130.0 (CAr), 136.9 (CAr), 146.9 (CAr), 153.2 (CO). HRMS (ES) m/z
263 (M+H)+ ; m/z 263.0585 (calcd for C13H12ClN2O2 (M+H)+:
263.0587).
5.2.2. 1-(2-Hydroxy-4-nitrophenyl)-3-phenylurea (3)
Method A was utilized for synthesis. Yellow solid (49% yld; >98%
purity); 1H NMR (acetone-d6, 500 MHz) d 7.06 (1H, t, J = 7.4 Hz,
CHAr), 7.33 (2H, t, J = 7.4 Hz, CHAr), 7.59 (2H, d, J = 8.0 Hz, CHAr),
7.76 (1H, d, J = 2.0 Hz, CHAr), 7.83 (1H, dd, J = 2, 9.0 Hz, CHAr), 8.33
(1H, br s, NH), 8.49 (1H, d, J = 9.0 Hz, CHAr), 8.92 (1H, br s, NH),
9.89 (1H, br s, OH); 13C NMR (acetone-d6, 125 MHz) d 109.3 (CAr),
116.6 (CAr), 117.5 (CAr), 118.8 (CAr), 118.9 (CAr), 122.9 (CAr), 129.0
(CAr), 135.5 (CAr), 139.5 (CAr), 139.7 (CAr), 141.8 (CAr), 146.1 (CAr),
152.2 (CO). HRMS MS (ES) m/z 274 (M+H)+; m/z 274.0841 (calcd
for C13H12N3O4 (M+H)+: 274.084).
5.2.8. 1-(2-Bromophenyl)-3-(2-hydroxyphenyl)urea (9)
Method A was utilized for synthesis. White solid (62% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 6.75 (t, J = 7.5 Hz, 1H,
CHAr), 6.87–6.81 (m, 2H, CHAr), 6.98 (t, J = 7.5 Hz, 1H, CHAr), 7.33
(t, J = 7.5 Hz, 1H, CHAr), 7.61 (d, J = 7.5 Hz, 1H, CHAr), 7.96 (d,
J = 8.5 Hz, 1H, CHAr), 7.99 (d, J = 8.0 Hz, 1H, CHAr), 8.80 (s, 1H,
NH), 8.95 (s, 1H,NH), 9.89 (s, 1H, OH); 13C NMR (DMSO-d6,
125 MHz) d114.5 (CHAr), 115.3 (CHAr), 119.8 (CHAr), 120.2 (CHAr),
122.9 (CHAr), 124.2 (CHAr), 124.9 (CHAr), 128.2 (CHAr), 128.6 (CAr),
133.2 (CAr), 138.1 (CAr), 146.9 (CAr), 153.3 (CO). HRMS (ES) m/z
307 (M+H)+; m/z 307.0081 (calcd for C13H12BrN2O2 (M+H)+:
307.0082).
5.2.3. 1-(2-Fluorophenyl)-3-(2-hydroxy-4-nitrophenyl)urea (4)
Method A was utilized for synthesis. Yellow solid (82% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 7.07–7.10 (1H, m,
HAr), 7.16–7.21 (2H, m, HAr), 7.77 (1H, d, J = 2.6 Hz, HAr), 7.84
(1H, d, J = 2.6, 9.0 Hz, HAr), 8.34 (1H, t, J = 9.0 Hz, HAr), 8.51 (1H,
d, J = 9.0 Hz, HAr), 8.78 (1H, br s, NH), 8.90 (1H, br s, NH), 9.90
(1H, br s, OH); 13C NMR (acetone-d6, 125 MHz) d 109.3 (CAr),
115.0 (CAr), 116.5 (CAr), 117.8 (CAr), 121.5 (CAr), 123.4 (CAr), 124.7
(CAr), 127.6 (CAr), 135.3 (CAr), 142.0 (CAr), 145.4 (CAr), 152.2 (CAr),
153.8 (CO). HRMS (ES) m/z 292 (M+H)+; m/z 292.0659 (calcd for
C13H11FN3O4 (M+H)+: 292.0655).
5.2.9. Method B: 1-(2-methoxy-4-nitrophenyl)-3-phenylurea (11)
To a solution of 2-amino-5-nitrophenol (52.4 mg, 0.34 mmol,
1 equiv) in acetone (3 mL) was added K2CO3 (97 mg, 0.41 mmol,
1.2 equiv) followed by MeI (0.11 mL, 1.71 mmol, 5 equiv). The
reaction was stirred at 50 °C for 15 h, quenched with H2O and ex-
tracted with DCM. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The crude product was
purified by chromatography over silica gel. The 2-methoxy-4-
nitroaniline (168.15 mg, 1 mmol) was then coupled with the 1-
phenylisocyanate (synthesized as described in method A) in a
minimal amount of THF at 50 °C for 12 h. The reaction was con-
centrated and purified by chromatography over silica gel. Yellow
5.2.4. 1-(2-Chlorophenyl)-3-(2-hydroxy-4-nitrophenyl)urea (5)
Method A was utilized for synthesis. Yellow solid (26% yld; >98%
purity); 1H NMR (acetone-d6, 500 MHz) d 7.11 (1H, td, J = 1.4,
7.8 Hz, CHAr), 7.38 (1H, td, J = 1.4, 8.3 Hz, CHAr), 7.45 (1H, dd,
J = 1.4, 8.0 Hz, CHAr), 7.77 (1H, d, J = 2.6 Hz, CHAr), 7.84 (1H, dd,
J = 2.6, 9.1 Hz, CHAr), 8.30 (1H, dd, J = 1.4, 8.3 Hz, CHAr), 8.49 (1H,
d, J = 9.1 Hz, CHAr), 8.72 (1H, br s, NH), 8.93 (1H, br s, NH), 9.85
(1H, br s, OH); 13C NMR (acetone-d6, 125 MHz) d 109.4 (CAr),

8108
P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
solid (46% yld; >98% purity); 1H NMR (acetone-d6, 500 MHz) d
4.05 (3H, s, CH3), 7.05 (1H, br t, J = 8.3 Hz, CHAr), 7.32 (2H, td,
J = 0.9, 8.3 Hz, CHAr), 7.57 (2H, dd, J = 0.9, 8.3 Hz, CHAr), 7.79
(1H, d, J = 2.5 Hz, CHAr), 7.91 (1H, dd, J = 2.5, 9.1 Hz, CHAr), 8.38
(1H, br s, NH), 8.57 (1H, d, J = 9.1 Hz, CHAr), 8.86 (1H, br s, NH);
5.2.14. 1-(2,4-Dichlorophenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (18)
Method A was utilized for synthesis. Yellow solid (35% yld; >98%
purity); 1H NMR (acetone-d6, 500 MHz) d 7.40 (1H, dd, J = 2.4,
8.9 Hz, CHAr), 7.52 (1H, dd, J = 2.4 Hz, CHAr), 7.76 (1H, d, J = 2.5 Hz,
CHAr), 7.84 (1H, dd, J = 9, 11.5 Hz, CHAr), 8.34 (1H, d, J = 9.0 Hz,
CHAr), 8.48 (1H, d, J = 9.0 Hz, CHAr), 8.80 (1H, br s, NH), 8.94 (1H,
br s, NH), 9.94 (1H, br s, OH). HRMS (ES) m/z 342 (M+H)+, m/z
342.0031 (calcd for C13H10Cl2N3O4 (M+H)+: 342.0040).
13C NMR (acetone-d6, 125 MHz)
d 56.3 (CH3), 105.5 (CHAr),
116.7 (CHAr), 117.6 (CHAr), 118.9 (CHAr), 122.9 (CHAr), 129.0
(CAr), 136.2 (CAr), 139.6 (CAr), 147.5 (CAr), 152.0 (CO). HRMS (ES)
m/z 288 (M+H)+; m/z 288.0909 (calcd for C14H14N3O4 (M+H)+:
288.0906).
5.2.15. 1-(2,4-Difluorophenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (19)
5.2.10. 1-(2-Bromophenyl)-3-(2-methoxy-4-nitrophenyl)urea
(12)
Method A was utilized for synthesis. Yellow solid (43% yld; >98%
purity); 1H NMR (acetone-d6, 500 MHz) d 7.01–7.06 (1H, m, CHAr),
7.11 (1H, ddd, J = 2.9, 8.7, 11.4 Hz, CHAr), 7.76 (1H, d, J = 2.6 Hz,
CHAr), 7.83 (1H, dd, J = 2.6, 9.1 Hz, CHAr), 8.29 (1H, td, J = 6.1,
9.1 Hz, CHAr), 8.49 (1H, d, J = 9.1 Hz, CHAr), 8.73 (1H, br s, NH),
8.86 (1H, br s, NH), 9.93 (1H, br s, OH); 13C NMR (acetone-d6,
125 MHz) d 103.6 (CHAr), 109.3 (CHAr), 111.0 (CAr), 111.2 (CAr),
116.5 (CHAr), 117.7 (CHAr), 122.8 (CAr), 122.8 (CAr), 135.2 (CHAr),
142.1 (CAr), 145.3 (CHAr), 152.2 (CAr), 159.0 (CO). HRMS (ES) m/z
310 (M+H)+, m/z 310.0565 (calcd for C13H10F2N3O4 (M+H)+:
310.0561).
Method B was utilized for synthesis. Yellow solid (14% yld; >98%
purity); 1H NMR (acetone-d6, 500 MHz) d 4.06 (3H, s, CH3), 7.05 (1H,
td, J = 7.9, 1.4 Hz, CHAr), 7.40 (1H, t, J = 8.2 Hz, CHAr), 7.63 (1H, d,
J = 7.0 Hz, CHAr), 7.82 (1H, d, J = 2.3 Hz, CHAr), 7.93 (1H, dd, J = 2.3,
9.1 Hz, CHAr), 8.19 (1H, d, J = 8.3 Hz, CHAr), 8.46 (1H, br s, NH), 9.03
(1H, d, J = 8.2 Hz, CHAr), 9.05 (1H, br s, NH); 13C NMR (acetone-d6,
125 MHz) d 56.3 (CH3), 105.6 (CHAr), 114.0 (CAr), 117.4 (CHAr),
117.4 (CHAr), 123.6 (CHAr), 125.0 (CHAr), 128.2 (CHAr), 132.8 (CHAr),
136.0 (CAr), 137.2 (CAr), 142.2 (CAr), 147.9 (CAr), 152.1 (CO). HRMS
(ES) m/z 366 (M+H)+; m/z 366.0031 (calcd for C14H13BrN3O4
(M+H)+: 366.0011).
5.2.16. 1-(3,4-Dichlorophenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (21)
5.2.11. 1-(2-Chlorophenyl)-3-(2-methoxy-4-nitrophenyl)urea
(13)
Method A was utilized for synthesis. Yellow solid (31% yld; 95%
purity); 1H NMR (DMSO-d6, 500 MHz,) d 7.30 (1H, dd, J = 2.5, 9.0 Hz,
CHAr), 7.56 (1H, d, J = 9.0 Hz, CHAr), 7.68 (1H, d, J = 2.5 Hz, CHAr),
7.77 (1H, dd, J = 2.5, 9.0 Hz, CHAr), 7.91 (1H, d, J = 2.5 Hz, CHAr),
8.35 (1H, d, J = 9.0 Hz, CHAr), 8.79 (1H, s, NH), 9.87 (1H, s, NH),
11.20 (1H, br s, OH); 13C NMR (DMSO-d6, 125 MHz) d 109.2 (CAr),
116.6 (CAr), 117.4 (CAr), 119.0 (CAr), 120.0 (CAr), 124.4 (CAr), 131.5
(CAr), 131.9 (CAr), 135.5 (CAr), 140.1(CAr), 141.7 (CAr), 146.1 (CAr),
152.4 (CO); HRMS (ES) 342 (M+H)+; m/z 342.1 (calcd for
C13H10Cl2N3O4 (M+H)+: 341.997).
Method B was utilized for synthesis. Yellow solid (26% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 4.06 (3H, s,
CH3), 7.10 (1H, td, J = 1.4, 8.0 Hz, CHAr), 7.35 (1H, td, J = 1.4,
8.0 Hz, CHAr), 7.44 (1H, dd, J = 1.4, 8.0 Hz, CHAr), 7.81 (1H, d,
J = 2.5 Hz, CHAr), 7.93 (1H, dd, J = 2.5, 9.1 Hz, CHAr), 8.29 (1H, dd,
J = 1.4, 8.0 Hz, CHAr), 8.58 (1H, d, J = 9.1 Hz, CHAr), 8.60 (1H, br s,
NH), 9.03 (1H, br s, NH); 13C NMR (acetone-d6, 125 MHz) d 26.3
(CH3), 105.6 (CHAr), 117.3 (CHAr), 117.4 (CHAr), 122.5 (CHAr),
123.2 (CAr), 124.2 (CHAr), 127.7 (CHAr), 129.5 (CHAr), 136.0 (CAr),
136.0 (CAr), 142.2 (CAr), 147.9 (CAr), 152.0 (CO). HRMS (ES) m/z
322 (M+H)+; m/z 322.0519 (calcd for C14H13ClN3O4 (M+H)+:
322.0516).
5.2.17. 1-(3,4-Difluorophenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (22)
Method A was utilized for synthesis. Yellow solid (52% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 7.19–7.21 (1H, m,
CHAr), 7.27 (1H, q, J = 9.4 Hz, CHAr), 7.75–7.87 (3H, m, CHAr), 8.33
(1H, br s, CHAr), 8.47 (1H, d, J = 9.0 Hz, NH), 9.09 (1H, br s, NH),
9.92 (1H, br s, OH); 13C NMR (acetone-d6, 125 MHz) d 113.1 (CAr),
114.4 (CHAr), 119.9 (CHAr), 121.8 (CHAr), 122.7 (CHAr), 140.3 (CHAr),
141.9 (CAr), 147.2 (CAr), 150.1 (CAr), 150.4 (CHAr), 152.1 (CAr), 154.4
(CAr), 156.3 (CO). HRMS (ES) m/z 310 (M+H)+; m/z 310.0568 (calcd
for C13H10F2N3O4 (M+H)+: 310.0561).
5.2.12. 1-(2-Fluorophenyl)-3-(4-methoxy-2-nitrophenyl)urea
(16)
Method B was utilized for synthesis. Yellow solid (40% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 3.92 (3H, s, CHAr),
7.05–7.11 (1H, m, CHAr), 7.15–7.21 (2H, m, CHAr), 7.36 (1H, dd,
J = 3.1, 9.3 Hz, CHAr), 7.64 (1H, d, J = 3.1 Hz, CHAr), 8.23 (1H, td,
J = 1.5, 8.1 Hz, CHAr), 8.40 (1H, d, J = 9.3 Hz, CHAr), 9.00 (1H, br s,
NH), 9.56 (1H, br s, NH); 13C NMR (acetone-d6, 125 MHz) d 55.7
(CH3), 108.4 (CHAr), 115.0 (CHAr), 115.2 (CHAr), 122.2 (CHAr),
122.5 (CAr), 123.6 (CHAr), 123.7 (CAr), 124.6 (CAr), 124.6 (CHAr),
125.2 (CHAr), 129.0 (CAr), 152.3 (CAr), 154.8 (CO). HRMS (ES) m/z
306 (M+H)+; m/z 306.0814 (calcd for C14H13FN3O4 (M+H)+:
306.0812).
5.2.18. 1-(2-Hydroxy-4-nitrophenyl)-3-(2,3,4-trichlorophenyl)
urea (23)
Method A was utilized for synthesis. Yellow solid (28% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 7.56 (1H, d,
J = 9.2 Hz, CHAr), 7.75 (1H, d, J = 2.5 Hz, CHAr), 7.82 (1H, dd, J = 2.5,
9.1 Hz, CHAr), 8.30 (1H, d, J = 9.2 Hz, CHAr), 8.47 (1H, d, J = 8.1 Hz,
CHAr), 8.89 (1H, br s, NH), 8.98 (1H, br s, NH), 9.89 (1H, br s, OH);
13C NMR (acetone-d6, 125 MHz) d 109.3 (CHAr), 116.4 (CHAr),
118.2 (CAr), 120.8 (CAr), 123.0 (CHAr), 126.8 (CAr), 128.6 (CAr),
130.9 (CHAr), 134.8 (CHAr), 137.0 (CAr), 142.3 (CAr), 145.6 (CAr),
152.0 (CO). HRMS (ES) m/z 376 (M+H)+; m/z 375.9561 (calcd for
C13H9Cl3N3O4 (M+H)+: 375.958).
5.2.13. 1-(2,4-Dibromophenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (17)
Method A was utilized for synthesis. Yellow solid (27% yld; >98%
purity); 1H NMR (acetone-d6, 500 MHz) d 7.58 (1H, dd, J = 2.2,
8.9 Hz, CHAr), 7.76 (1H, d, J = 2.5 Hz, CHAr), 7.81 (1H, d, J = 2.2 Hz,
CHAr), 7.84 (1H, dd, J = 2.5, 9.1 Hz, CHAr), 8.19 (1H, d, J = 8.9 Hz,
CHAr), 8.48 (1H, d, J = 9.1 Hz, CHAr), 8.65 (1H, br s, NH), 8.99 (1H,
br s, NH), 9.91 (1H, br s, OH); 13C NMR (acetone-d6, 125 MHz) d
109.4 (CHAr), 114.4 (CAr), 115.6 (CHAr), 116.4 (CAr), 118.2 (CHAr),
124.5 (CHAr), 131.2 (CHAr), 134.7 (CHAr), 135.0 (CAr), 136.9 (CAr),
142.3 (CAr), 145.6 (CAr), 152.2 (CO). HRMS (ES) m/z 431 (M+H)+,
m/z 430.9191 (calcd for C13H10Br2N3O4 (M+H)+: 430.9186).
5.2.19. 1-(2-Hydroxy-4-nitrophenyl)-3-(2,4,5-trichlorophenyl)
urea (24)
Method A was utilized for synthesis. Yellow solid (44% yld;
165 mg; 0.44 mmol; >98% purity); 1H NMR (acetone-d6,

P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
8109
500 MHz) d 7.68 (1H, s, CHAr), 7.76 (1H, d, J = 2.5 Hz, CHAr), 7.84
(1H, dd, J = 2.5, 9.1 Hz, CHAr), 8.50 (1H, d, J = 9.1 Hz, CHAr), 8.64
(1H, s, CHAr), 8.89 (1H, br s, NH), 9.00 (1H, br s, NH), 9.92 (1H, br
s, OH); 13C NMR (acetone-d6, 125 MHz) d 109.3 (CHAr), 116.4
(CHAr), 118.2 (CHAr), 121.8 (CAr), 122.4 (CHAr), 125.5 (CAr), 130.3
(CHAr), 131.0 (CAr), 134.7 (CAr), 136.3 (CAr), 142.4 (CAr), 145.7
(CAr), 151.9 (CO). HRMS (ES) m/z 376 (M+H)+; m/z 375.954 (calcd
for C13H9Cl3N3O4 (M+H)+: 375.958).
125 MHz) d 18.7 (CH), 109.2 (CHAr), 116.6 (CHAr), 117.3 (CHAr),
122.5 (CHAr), 124.0 (CHAr), 126.8 (CHAr), 129.1 (CHAr), 130.9 (CAr),
136.3 (CAr), 137.5 (CAr), 141.2 (CAr), 146.0 (CAr), 153.0 (CO). HRMS
(ES) m/z 288 (M+H)+; m/z 288.0981 (calcd for C14H14N3O4
(M+H)+: 288.0984).
5.2.25. 1-(2-Hydroxy-4-nitrophenyl)-3-(2-ethylphenyl)urea (30)
Method A was utilized for synthesis. Yellow solid (53% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 1.26 (t, J = 7.0 Hz,
3H, CH3), 2.54 (t, J = 7.5 Hz, 2H, CH2), 6.94 (s, 1H, CHAr), 7.08 (d,
J = 7.0 Hz, 1H, CHAr), 7.14 (d, J = 7.0 Hz, 1H, CHAr), 7.62 (s, 1H, CHAr),
7.75 (d, J = 9.0 Hz, 1H, CHAr), 7.80 (d, J = 7.5 Hz, 1H, CHAr), 8.41 (d,
J = 9.0 Hz, 1H, CHAr), 8.84 (s, 1H, NH), 9.18 (s, 1H, NH), 11.10 (br
s, 1H, OH); 13C NMR (DMSO-d6, 125 MHz) d 15.6 (CH), 21.5 (CH),
109.2 (CHAr), 116.6 (CHAr), 117.2 (CHAr), 123.6 (CHAr), 124.0 (CHAr),
126.8 (CHAr), 129.4 (CHAr), 130.6 (CAr), 136.3 (CAr), 136.9 (CAr),
141.2 (CAr), 146.0 (CAr), 153.0 (CO). HRMS (ES) m/z 301 (M+H)+;
m/z 301.1065 (calcd for C15H16N3O4 (M+H)+: 301.1063).
5.2.20. 1-(2-Hydroxy-4-nitrophenyl)-3-(3,4,5-trifluorophenyl)
urea (25)
Method A was utilized for synthesis. Yellow solid (36% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 7.42 (2H, m, CHAr),
7.76 (1H, d, J = 2.6 Hz, CHAr), 7.85 (1H, dd, J = 2.6, 9.1 Hz, CHAr), 8.36
(1H, br s, NH), 8.46 (1H, d, J = 9.1 Hz, CHAr), 9.22 (1H, br s, NH),
10.04 (1H, br s, OH); 13C NMR (acetone-d6, 125 MHz) d 104.2
(CHAr), 109 (CHAr), 113.5(CHAr), 118.7 (CHAr), 132.7 (CAr), 134.6
(CAr), 145.3 (CAr), 146 (CAr), 147.2 (CAr), 148.9 (CAr), 152 (CO). HRMS
(ES) m/z 328 (M+H)+; m/z 328.0465 (calcd for C13H9F3N3O4 (M+H)+:
328.0467).
5.2.26.1-(2-Hydroxy-4-nitrophenyl)-3-(2-propylphenyl)urea (31)
Method A was utilized for synthesis. Yellow solid (36% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 0.93 (t, J = 7.0 Hz, 3H, CH3),
1.56 (m, 2H, CH2), 2.59 (t, J = 7.5 Hz, 2H, CH2), 7.03 (td, J = 7.5,
1.5 Hz, 1H, CHAr), 7.14–7.19 (m, 2H, CHAr), 7.66–7.69 (m, 2H, CHAr),
7.73 (dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.36 (d, J = 9.0, 1H, CHAr), 8.76 (s,
1H, NH), 9.11 (s, 1H, NH), 11.06 (br s, 1H, OH); 13C NMR (DMSO-d6,
125 MHz) d 14.5 (CH), 23.3 (CH), 33.3 (CH), 109.2 (CHAr), 116.6
(CHAr), 117.4 (CHAr), 124.1 (CHAr) , 124.5 (CHAr), 126.7 (CHAr),
130.1 (CHAr), 134.1 (CAr), 136.3 (CAr), 136.7 (CAr), 141.2 (CAr),
146.0 (CAr), 153.3 (CO). HRMS (ES) m/z 316 (M+H)+; m/z 316.1296
(calcd for C16H18N3O4 (M+H)+: 316.1297).
5.2.21. 1-(2-Hydroxy-4-nitrophenyl)-3-(2,4,6-trifluorophenyl)
urea (26)
Method A was utilized for synthesis. Yellow solid (21% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 7.07 (1H, dd,
J = 8.9, 7.7 Hz, CHAr), 7.76 (1H, d, J = 2.5 Hz, CHAr), 7.79 (1H, dd,
J = 2.5, 9.0 Hz, CHAr), 8.39 (1H, d, J = 9.0 Hz, CHAr), 8.47 (1H, br s,
NH), 8.61 (1H, br s, NH); 13C NMR (acetone-d6, 125 MHz) d 100.4
(CAr), 100.4 (CAr), 100.6 (CHAr), 100.8 (CAr), 100.2 (CAr), 116.3 (CHAr),
117.4 (CHAr), 135.2 (CAr), 142.1 (CAr), 145.5 (CAr), 152.5 (CO). HRMS
(ES) m/z 328 (M+H)+, m/z 328.0468 (calcd for C13H9F3N3O4 (M+H)+:
328.0467).
5.2.27. 1-(2-sec-Butylphenyl)-3-(2-hydroxy-4-nitrophenyl)urea
(32)
5.2.22. 1-(2-Hydroxy-4-nitrophenyl)-3-(2,3,4-trifluorophenyl)
urea (27)
Method A was utilized for synthesis. Yellow solid (30% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 0.78 (t, J = 7.5 Hz, 3H, CH3),
1.16 (d, J = 7.0 Hz, 3H, CH3), 1.56 (m, 2H, CH2), 2.97 (m, 1H, CH),
7.09–7.16 (m, 2H, CHAr), 7.24 (dd, J = 7.5, 2.0 Hz, 1H, CHAr), 7.56
(dd, J = 7.5, 2.0 Hz, 1H, CHAr), 7.66 (d, J = 2.5 Hz, 1H, CHAr), 7.73
(dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.35 (d, J = 9.0 Hz, 1H, CHAr), 8.78 (s,
1H, NH), 9.05 (s, 1H, NH), 11.06 (br s, 1H, OH); 13C NMR (DMSO-
d6, 125 MHz) d 12.6 (CH), 21.9 (CH), 30.4 (CH), 34.2 (CH), 109.1
(CHAr), 116.6 (CHAr), 117.2 (CHAr), 125.3 (CHAr), 125.4 (CHAr),
126.3 (CHAr), 126.5 (CHAr), 136.0 (CAr), 136.4 (CAr), 140.1 (CAr),
141.1 (CAr), 145.9 (CAr), 153.3 (CO). HRMS (ES) m/z 330 (M+H)+;
m/z 330.1453 (calcd for C17H20N3O4 (M+H)+: 330.1454).
Method A was utilized for synthesis. Yellow solid (41% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 7.21 (1H, dd,
J = 2.4, 10.2, 18.3 Hz, CHAr), 7.77 (1H, d, J = 2.5 Hz, CHAr), 7.84
(1H, dd, J = 2.5, 9.0 Hz, CHAr), 8.06–8.12 (1H, m, CHAr), 8.49 (1H,
d, J = 9.0 Hz, CHAr), 8.74 (1H, br s, NH), 8.98 (1H, br s, NH), 9.92
(1H, br s, OH); 13C NMR (acetone-d6, 125 MHz) d 109.3 (CHAr),
111.6 (CHAr), 111.6 (CAr), 111.8 (CHAr), 111.8 (CAr), 115.8 (CAr),
115.9 (CAr), 116.5 (CHAr), 117.8 (CHAr), 136.0 (CAr), 142.2 (CAr),
145.4 (CAr), 152.1 (CO). HRMS (ES) m/z 328 (M+H)+; m/z
328.0474 (calcd for C13H9F3N3O4 (M+H)+: 328.0467).
5.2.23. 1-(2-Hydroxy-4-nitrophenyl)-3-(2,4,5-trifluorophenyl)
urea (28)
5.2.28. 1-(3-Ethylphenyl)-3-(2-hydroxy-4-nitrophenyl)urea (33)
Method A was utilized for synthesis. Yellow solid (42% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 1.18 (t, J = 7.5 Hz,
3H, CH3), 2.58 (q, J = 7.5 Hz, 2H, CH2), 6.85 (d, J = 7.5 Hz, 1H, CHAr),
7.19 (t, J = 7.5 Hz, 1H, CHAr), 7.25 (d, J = 8.5 Hz, 1H, CHAr), 7.33 (s,
1H, CHAr), 7.65 (d, J = 2.5 Hz, 1H, CHAr), 7.74 (dd, J = 9.0, 2.5 Hz,
1H, CHAr), 8.36 (d, J = 9.5 Hz, 1H, CHAr), 8.71 (s, 1H, NH), 9.53 (s,
1H, NH), 11.09 (s, 1H, OH); 13C NMR (DMSO-d6, 125 MHz) d 16.2
(CH), 28.9 (CH), 109.1 (CHAr), 116.3 (CHAr), 116.7 (CHAr), 117.1
(CHAr), 118.2 (CHAr), 122.5 (CHAr), 129.5 (CHAr), 136.1 (CAr), 139.9
(CAr), 141.2 (CAr), 145.1(CAr), 145.8 (CAr), 152.6 (CO). HRMS (ES)
m/z 302 (M+H)+; m/z 302.1138 (calcd for C15H16N3O4 (M+H)+:
302.1141).
Method A was utilized for synthesis. Yellow solid (59% yld;
>98% purity); 1H NMR (acetone-d6, 500 MHz) d 7.35 (1H, td,
J = 7.35, 10.6 Hz, CHAr), 7.76 (1H, d, J = 2.5 Hz, CHAr), 7.85 (1H, dd,
J = 2.5, 9.1 Hz, CHAr), 7.36 (1H, m, CHAr), 8.46 (1H, d, J = 9.1 Hz,
CHAr), 8.79 (1H, br s, NH), 9.05 (1H, br s, NH); 13C NMR (acetone-
d6, 125 MHz) d 105.0 (CHAr), 105.2 (CHAr), 105.2 (CAr), 105.4 (CAr),
109.2 (CHAr), 109.4 (CAr), 109.4 (CAr), 116.4 (CHAr), 117.8 (CHAr),
134.9 (CAr), 142.2 (CAr), 145.4 (CAr), 152.0 (CO). HRMS (ES) m/z
328 (M+H)+, m/z 328.0463 (calcd for C13H9F3N3O4 (M+H)+:
328.0467).
5.2.24. 1-(2-Hydroxy-4-nitrophenyl)-3-o-tolylurea (29)
Method A was utilized for synthesis. Yellow solid (48% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 2.26 (s, 3H, CH3),
6.99 (s, 1H, CHAr), 7.16 (d, J = 7.0 Hz, 1H, CHAr), 7.19 (d, J = 7.0 Hz,
1H, CHAr), 7.67 (s, 1H, CHAr), 7.75 (d, J = 9.0 Hz, 1H, CHAr), 7.78 (d,
J = 7.5 Hz, 1H, CHAr), 8.38 (d, J = 9.0 Hz, 1H, CHAr), 8.82 (s, 1H,
NH), 9.16 (s, 1H, NH), 11.07 (br s, 1H, OH); 13C NMR (DMSO-d6,
5.2.29. 1-(4-Ethylphenyl)-3-(2-hydroxy-4-nitrophenyl)urea (34)
Method A was utilized for synthesis. Yellow solid (53% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 1.18 (t, J = 8.0 Hz, 3H, CH3),
2.57 (q, J = 7.5 Hz, 2H, CH2), 7.15 (d, J = 8.5 Hz, 1H, CHAr), 7.38(d,
J = 8.5 Hz, 1H, CHAr), 7.67 (d, J = 2.5 Hz, 1H, CHAr), 7.78 (dd, J = 9.0,

8110
P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
2.5 Hz, 1H, CHAr), 8.37 (d, J = 9.0 Hz, 1H, CHAr), 8.71 (s, 1H, NH), 9.51
(s, 1H, NH), 11.13 (br s, 1H, OH); 13C NMR (DMSO-d6, 125 MHz) d
16.5 (CH), 28.2 (CH), 109.1 (CHAr), 116.7 (CHAr), 117.0 (CHAr),
119.1 (CHAr), 128.8 (CHAr), 136.2 (CAr), 137.6 (CAr), 138.4 (CAr),
141.2 (CAr), 145.9 (CAr), 152.6 (CO). HRMS (ES) m/z 302 (M+H)+;
m/z 302.1138 (calcd for C15H16N3O4 (M+H)+: 302.1144).
(dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.39 (d, J = 9.0 Hz, 1H, CHAr), 8.77 (s,
1H, NH), 9.17 (s, 1H, NH), 11.07 (br s, 1H, OH); 13C NMR (DMSO-
d6, 125 MHz) d 18.3 (CH), 21.6 (CH), 109.2 (CHAr), 116.7 (CHAr),
117.4 (CHAr), 123.1 (CHAr), 124.7 (CHAr), 125.9 (CHAr), 130.8 (CAr),
135.8 (CAr), 136.4 (CAr), 137.3 (CAr), 141.2 (CAr), 146.1 (CAr), 153.0
;
(CO). HRMS (ES) m/z 302 (M+H)+ m/z 302.1138 (calcd for
C15H16N3O4 (M+H)+: 302.1141).
5.2.30.1-(2-Hydroxy-4-nitrophenyl)-3-(4-propylphenyl)urea (35)
Method A was utilized for synthesis. Yellow solid (48% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 0.90 (t, J = 7.5 Hz, 3H, CH3),
1.58 (sextet, J = 7.5 Hz, 2H, CH2), 2.52 (t, J = 7.5 Hz, 2H, CH2), 7.13 (d,
J = 8.0 Hz, 1H, CHAr), 7.38(d, J = 8.5 Hz, 1H, CHAr), 7.67 (d, J = 2.0 Hz,
1H, CHAr), 7.77 (dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.38 (d, J = 9.0 Hz, 1H,
CHAr), 8.72 (s, 1H, NH), 9.51 (s, 1H, NH), 11.11 (s, 1H, OH); 13C NMR
(DMSO-d6, 125 MHz) d 14.3 (CH), 24.9 (CH), 37.3 (CH), 109.1 (CHAr),
116.8 (CHAr), 117.1 (CHAr), 119.0 (CHAr), 129.4 (CHAr), 136.2 (CAr),
136.8 (CAr), 137.6 (CAr), 141.2 (CAr), 145.9 (CAr), 152.6 (CO). HRMS
(ES) m/z 316 (M+H)+; m/z 316.1294 (calcd for C16H18N3O4
(M+H)+: 316.1297).
5.2.35. 1-(3,4-Dimethylphenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (40)
Method A was utilized for synthesis. Yellow solid (53% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 2.18 (s, 3H, CH3),
2.21 (s, 3H, CH3), 7.06 (d, J = 8.5 Hz, 1H, CHAr), 7.19 (d, J = 8.0 Hz,
1H, CHAr), 7.25(s, 1H, CHAr), 7.67 (d, J = 2.5 Hz, 1H, CHAr), 7.76
(dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.37 (d, J = 9.0 Hz, 1H, CHAr), 8.70 (s,
1H, NH), 9.43 (s, 1H, NH), 11.07 (br s, 1H, OH); 13C NMR (DMSO-
d6, 125 MHz) d 19.4 (CH), 20.4 (CH), 109.1 (CHAr), 116.5 (CHAr),
116.8 (CHAr), 117.0 (CHAr), 120.3 (CHAr), 130.5 (CHAr), 130.8 (CAr),
136.2 (CAr), 137.3 (CAr), 137.6 (CAr), 141.2 (CAr), 145.9 (CAr), 152.6
(CO). HRMS (ES) m/z 302 (M+H)+; m/z 302.1139 (calcd for
C15H16N3O4 (M+H)+: 302.1141).
5.2.31. 1-(2-Hydroxy-4-nitrophenyl)-3-(2-isopropylphenyl)urea
(36)
Method A was utilized for synthesis. Yellow solid (46% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 1.19 (d, J = 7.0 Hz,
6H, CH3), 3.20 (m, 1H, CH), 7.09–7.17 (m, 2H, CHAr), 7.29 (dd,
J = 7.5, 1.5 Hz, 1H, CHAr), 7.59 (dd, J = 8.0, 1.5 Hz, 1H, CHAr), 7.66
(d, J = 3.0 Hz, 1H, CHAr), 7.73 (dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.36
(d, J = 9.5, 1H, CHAr), 8.81 (s, 1H, NH), 9.08 (s, 1H, NH), 11.06 (br
s, 1H, OH); 13C NMR (DMSO-d6, 125 MHz) d 23.9 (CH), 27.4 (CH),
109.1 (CHAr), 116.6 (CHAr), 117.2 (CHAr), 125.0 (CHAr), 125.2 (CHAr),
126.1 (CHAr), 126.4 (CHAr), 135.5 (CAr), 136.4 (CAr), 141.0 (CAr),
141.1(CAr), 145.9 (CAr), 153.5 (CO). HRMS (ES) m/z 316 (M+H)+;
m/z 316.129 (calcd for C16H18N3O4 (M+H)+: 316.1297).
5.2.36. 1-(3,5-Dimethylphenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (41)
Method A was utilized for synthesis. Yellow solid (36% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 2.26 (s, 6H, CH3), 6.67 (s,
1H, CHAr), 7.10 (s, 1H, CHAr), 7.67(d, J = 2.0 Hz, 1H, CHAr), 7.76 (dd,
J = 9.0, 2.0 Hz, 1H, CHAr), 8.37 (d, J = 8.0 Hz, 1H, CHAr), 8.73 (s, 1H,
NH), 9.45 (s, 1H, NH), 11.08 (br s, 1H, OH); 13C NMR (DMSO-d6,
125 MHz) d 21.8 (CH), 109.1 (CHAr), 116.6 (CHAr), 117.1 (CHAr),
124.7 (CHAr), 136.2 (CAr), 138.6 (CAr), 139.8 (CAr), 141.2 (CAr),
146.0 (CAr), 152.6 (CO). HRMS (ES) m/z 302 (M+H)+; m/z 302.1138
(calcd for C15H16N3O4 (M+H)+: 302.1141).
5.2.32. 1-(2-tert-Butylphenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (37)
5.2.37. 1-(2-Ethyl-6-methylphenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (42)
Method A was utilized for synthesis. Yellow solid (25% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 1.38 (s, 9H, CH3), 7.22–7.19
(m, 3H, CHAr), 7.40 (m, 1H, CHAr), 7.68 (d, J = 2.5 Hz, 1H, CHAr), 7.73
(dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.35 (d, J = 9.0 Hz, 1H, NH), 11.04 (br s,
1H, OH); 13C NMR (DMSO-d6, 125 MHz) d 31.3 (CH), 35.4 (C), 109.2
(CHAr), 116.7 (CHAr), 117.2 (CHAr), 127.0 (CHAr), 127.1 (CHAr), 131.9
(CHAr), 136.1 (CAr), 136.7 (CAr), 141.0 (CAr), 145.9 (CAr), 146.2 (CAr),
154.1 (CO). HRMS (ES) m/z 330 (M+H)+; m/z 330.1453 (calcd for
C17H20N3O4 (M+H)+: 330.1454).
Method A was utilized for synthesis. Yellow solid (42% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 1.14 (t, J = 7.5 Hz,
3H, CH3), 2.21 (s, 3H, CH3), 2.59 (q, J = 7.5 Hz, 2H, CH2), 7.20–7.10
(m, 3H, CHAr), 7.68 (s, 1H, CHAr), 7.74 (dd, J = 9.0, 2.5 Hz, 1H, CHAr),
8.65 (s, 1H, NH), 8.87 (s, 1H, NH), 11.09 (br s, 1H, OH); 13C NMR
(DMSO-d6, 125 MHz) d 15.5 (CH), 18.9 (CH), 25.2 (CH), 109.2
(CHAr), 116.7 (CHAr), 116.8 (CHAr), 126.9 (CHAr), 127.2 (CHAr),
128.5 (CHAr), 134.8 (CAr), 136.6 (CAr), 141.0 (CAr), 141.9 (CAr),
145.7 (CAr), 153.5 (CO). HRMS (ES) m/z 316 (M+H)+; m/z
316.1295 (calcd for C16H18N3O4 (M+H)+: 316.1297).
5.2.33. 1-(2,4-Dimethylphenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (38)
5.2.38. 1-(2-Ethyl-6-isopropylphenyl)-3-(2-hydroxy-4-nitro-
phenyl)urea (43)
Method A was utilized for synthesis. Yellow solid (39% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 2.23 (s, 3H, CH3),
2.25 (s, 3H, CH3), 6.98 (d, J = 8.0 Hz, 1H, CHAr), 7.02 (s, 1H, CHAr),
7.62 (d, J = 8.0 Hz, 1H, CHAr), 7.68 (d, J = 2.5 Hz, 1H, CHAr), 7.75
(dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.38 (d, J = 9.0 Hz, 1H, CHAr), 8.76 (s,
1H, NH), 9.09 (s, 1H, NH), 11.07 (br s, 1H, OH); 13C NMR (DMSO-
d6, 125 MHz) d 18.7 (CH), 21.1 (CH), 109.2 (CHAr), 116.7 (CHAr),
117.3 (CHAr), 122.9 (CHAr), 127.3 (CHAr), 129.3 (CHAr), 131.5 (CAr),
133.1 (CAr), 134.9 (CAr), 136.4 (CAr), 141.1 (CAr), 145.9 (CAr), 153.1
(CO). HRMS (ES) m/z 302 (M+H)+; m/z 302.1139 (calcd for
C15H16N3O4 (M+H)+: 302.1141).
Method A was utilized for synthesis. Yellow solid (23% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 1.22–1.12 (m, 9H, CH3), 2.56
(q, J = 7.5 Hz, 2H, CH2), 3.15 (septet, J = 7.0 Hz, 1H, CH), 7.12 (d,
J = 7.0 Hz, 1H, CHAr), 7.25–7.18 (m, 2H, CHAr), 7.68 (d, J = 2.5 Hz,
2H, CHAr), 7.74 (dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.32 (d, J = 9.0 Hz, 1H,
CHAr), 8.60 (s, 1H, NH), 8.87 (s, 1H, NH), 11.08 (br s, 1H, OH); 13C
NMR (DMSO-d6, 125 MHz) d 15.4 (CH), 24.7 (CH), 25.3 (CH), 28.6
(CH), 109.2 (CHAr), 116.8 (CHAr), 123.9 (CHAr), 126.6 (CHAr), 127.9
(CHAr), 133.3 (CAr), 136.7 (CAr), 140.9 (CAr), 142.5 (CAr), 145.6 (CAr),
147.1 (CAr), 154.2 (CO). HRMS (ES) m/z 344 (M+H)+; m/z
344.16108 (calcd for C18H22N3O4 (M+H)+: 344.161).
5.2.34. 1-(2,5-Dimethylphenyl)-3-(2-hydroxy-4-nitrophenyl)
urea (39)
Method A was utilized for synthesis. Yellow solid (47% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 2.22 (s, 3H, CH3),
2.27 (s, 3H, CH3), 6.82 (d, J = 8.0 Hz, 1H, CHAr), 7.07 (d, J = 7.5 Hz,
1H, CHAr), 7.64(s, 1H, CHAr), 7.68 (d, J = 2.5 Hz, 1H, CHAr), 7.75
5.2.39. 1-(2-Hydroxy-4-nitrophenyl)-3-(2-isopropyl-6-
methylphenyl)urea (44)
Method A was utilized for synthesis. Yellow solid (31% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 1.18 (d, J = 6.5 Hz, 6H, CH3),
2.22 (s, 3H, CH3), 3.19 (m, 1H, CH), 7.18–7.11 (m, 3H, CHAr), 7.69 (s,

P. Bakshi et al. / Bioorg. Med. Chem. 17 (2009) 8102–8112
8111
1H, CHAr), 7.74 (d, J = 8.5 Hz, 1H, CHAr), 8.34 (d, J = 8.5 Hz, 1H, CHAr),
8.65 (s, 1H, NH), 8.89 (s, 1H, NH), 11.06 (br s, 1H, OH); 13C NMR
(DMSO-d6, 125 MHz) d 19.1 (CH), 24.4 (CH), 28.6 (CH), 109.2 (CHAr),
116.9 (CHAr), 123.9 (CHAr), 127.6 (CHAr), 128.3 (CHAr), 134.0 (CAr),
136.8 (CAr), 141.0 (CAr), 145.7 (CAr), 146.5 (CAr), 153.8 (CO). HRMS
(ES) m/z 330 (M+H)+; m/z 330.1453 (calcd for C17H20N3O4 (M+H)+:
330.1454).
els secreted in conditioned media were determined using a quan-
titative human Ab40 sandwich ELISA kit as described in
manufacturer’s instructions.
5.6. Calcium mobilization assay
Compounds were tested in 7w cells. Cells were grown to ꢀ100%
confluency (30,000–40,000 cells) in 96-well plates. After removing
5.2.40. 1-(2,6-Diisopropylphenyl)-3-(2-hydroxy-4-
the growth medium from the adherent 7w cell culture, 100 ll of
nitrophenyl)urea (45)
the dye loading solution (made according to manufacturer’s
instructions) was carefully added to each well and the plate was
incubated at 37 °C for an hour. Following incubation, the dye solu-
Method A was utilized for synthesis. Yellow solid (28% yld;
>98% purity); 1H NMR (DMSO-d6, 500 MHz) d 0.76 (d, J = 6.5 Hz,
12H, CH3), 3.14 (septet, 7.0 Hz, 2H, CH), 7.17 (s, 1H, CHAr), 7.18
(s, 1H, CHAr), 7.28(t, J = 7.5 Hz, 1H, CHAr), 7.68 (d, J = 2.5 Hz, 1H,
CHAr), 7.74 (dd, J = 9.0, 2.5 Hz, 1H, CHAr), 8.32 (d, J = 9.5 Hz, 1H,
CHAr), 8.59 (s, 1H, NH), 8.86 (s, 1H, NH), 11.08 (br s, 1H, OH); 13C
NMR (DMSO-d6, 125 MHz) d 23.7 (CH), 24.7 (CH), 28.8 (CH),
109.2 (CHAr), 116.8 (CHAr), 116.9 (CHAr), 123.7 (CHAr), 128.2 (CHAr),
132.5 (CAr), 136.7 (CAr), 140.9 (CAr), 145.6 (CAr), 147.1 (CAr), 154.4
(CO); HRMS (ES) m/z 358 (M+H)+; m/z 358.1765 (calcd for
C19H24N3O4 (M+H)+: 358.1767).
tion was replaced with 100 ll of 1X HBSS buffer with component C
from the calcium assay kit. Stock concentrations of the compounds
in DMSO were added to 1X Hank’s balanced salt solution (HBSS) to
reach a final concentration of 1% DMSO. A minimum of nine differ-
ent concentrations of each compound were tested (n = 8 for each
concentration), and the experiment was repeated in triplicate.
Negative controls contained 1% DMSO alone. The plate was read
at Ex/Em: 494/516 in a Biotek Synergy (with automated injector)
plate reader, followed 15 s later by addition 50
Gro (final [Gro ] = 33 nM). The maximal calcium concentration
stimulation was quantitated as described
ll of 100 nM of
a
a
5.2.41. 1-(2-sec-Butyl-6-ethylphenyl)-3-(2-hydroxy-4-
nitrophenyl)urea (46)
attained after Gro
a
previously.35
Method A was utilized for synthesis. Yellow solid (25% yld; >98%
purity); 1H NMR (DMSO-d6, 500 MHz) d 0.76 (t, J = 7.5 Hz, 3H, CH3),
1.16–1.13 (m, 6H, CH3), 1.52 (br s, 2H, CH2), 2.52 (q, J = 7.0 Hz, 2H,
CH2), 2.90 (m, 1H, CH), 7.14–7.10 (m, 2H, CHAr), 7.23 (t, J = 7.5 Hz,
1H, CHAr), 7.68 (d, J = 2.0 Hz, 1H, CHAr), 7.74 (dd, J = 9.0, 2.5 Hz,
1H, CHAr), 8.32 (d, J = 9.0 Hz, 1H, CHAr), 8.57 (s, 1H, NH), 8.87 (s,
1H, NH), 11.08 (br s, 1H, OH); 13C NMR (DMSO-d6, 125 MHz) d
12.9 (CH), 15.3 (CH), 21.8 (CH), 25.4 (CH), 31.1 (CH), 35.6 (CH),
109.1 (CHAr), 116.8 (CHAr), 124.3 (CHAr), 126.5 (CHAr), 127.9 (CHAr),
133.9 (CHAr), 136.8 (CAr), 141.0 (CAr), 142.6 (CAr), 145.6 (CAr), 146.0
(CAr), 154.1 (CO); HRMS (ES) m/z 358 (M+H)+, m/z 358.1765 (calcd
for C19H24N3O4 (M+H)+: 358.1767).
5.7. Statistical analysis
All dose–response graphs were plotted using Sigma plot 8.0 and
axꢁx
the standard sigmoid 4-parameter equation y ¼ yo þ
was
o
ꢁ
b
used to fit the dose–response data. The scaling of x-axi1sþies in log.
Acknowledgments
We thank Dr. Michael Wolfe (Center for Neurologic Diseases,
Brigham and Women’s Hospital, Harvard Medical School, Boston,
Massachusetts, USA) for providing the 7w CHO cells used in this
study. The work was supported by the Robert and Diane Roskamp
Foundation.
5.3. Reagents for in vitro assays
All cell culture reagents were from Invitrogen, unless otherwise
noted. The BCA protein assay reagent kit was purchased from
Pierce Biotechnology, Inc. (Rockford, IL). The human Ab40 colori-
metric ELISA kit were purchased from BioSource International Inc
(Carlsbad, California). The Fluo-4 NW calcium assay kit was pur-
chased from Molecular Probes, Invirogen (Carlsbad, California).
All reagents used for synthesis were purchased from Aldrich and
Acros and were typically used as supplied unless otherwise stated.
Supplementary data
Supplementary data (HPLC data and elemental analysis of the
discussed compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2009.09.051.
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