Arylacetamide-derived fluorescent probes: Synthesis, biological evaluation, and direct fluorescent labeling of κ opioid receptors in mouse microglial cells

Article abstract of DOI:10.1021/jm950813b

Fluorescein isothiocyanate isomer I (FITC-I) conjugates of 2-(3,4- dichlorophenyl)-N-methyl-N-[1-(3- or 4-aminophenyl)-2-(1- pyrrolidinyl)ethyl]acetamide (10 and 14) were prepared either without or with an intervening mono-, di-, or tetraglycyl linker. The 3-substituted fluorescent probes (2-5) were found to retain potent agonist activity in smooth muscle preparations as well as high κ receptor affinity and selectivity in receptor binding assays. The 4-substituted series (6-9) were substantially less potent than the corresponding 3-substituted compounds. Flow cytometric analysis demonstrated high levels of direct κ-specific staining of mouse microglial cells by the fluorescent probe 5 containing a tetraglycyl linker, as indicated by a 41% decrease in percent cells positively labeled and a 61% decrease in mean fluorescence intensity in the presence of the κ-selective antagonist, norbinaltorphimine (norBNI). In similar studies, the probe 2 without a linker exhibited only nonspecific binding. This is the first report of direct, selective staining of κ opioid receptors by a fluorescent nonpeptide opioid ligand. The results of the present study illustrate the importance of introducing hydrophilic linkers to reduce nonspecific binding of fluorescent probes for opioid receptors.

Full text of DOI:10.1021/jm950813b

J . Med. Chem. 1996, 39, 1729-1735
1729
Ar yla ceta m id e-Der ived F lu or escen t P r obes: Syn th esis, Biologica l Eva lu a tion ,
a n d Dir ect F lu or escen t La belin g of K Op ioid Recep tor s in Mou se Micr oglia l
Cells
An-Chih Chang,† Chun C. Chao,‡ Akira E. Takemori,§ Genya Gekker,‡ Shuxian Hu,‡ Phillip K. Peterson,‡ and
Philip S. Portoghese*,†
Department of Medicinal Chemistry, College of Pharmacy, and Department of Pharmacology, Medical School,
University of Minnesota, and Minneapolis Medical Research Foundation, Minneapolis, Minnesota 55455
Received November 2, 1995X
Fluorescein isothiocyanate isomer I (FITC-I) conjugates of 2-(3,4-dichlorophenyl)-N-methyl-
N-[1-(3- or 4-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (10 and 14) were prepared either
without or with an intervening mono-, di-, or tetraglycyl linker. The 3-substituted fluorescent
probes (2-5) were found to retain potent agonist activity in smooth muscle preparations as
well as high κ receptor affinity and selectivity in receptor binding assays. The 4-substituted
series (6-9) were substantially less potent than the corresponding 3-substituted compounds.
Flow cytometric analysis demonstrated high levels of direct κ-specific staining of mouse
microglial cells by the fluorescent probe 5 containing a tetraglycyl linker, as indicated by a
41% decrease in percent cells positively labeled and a 61% decrease in mean fluorescence
intensity in the presence of the κ-selective antagonist, norbinaltorphimine (norBNI). In similar
studies, the probe 2 without a linker exhibited only nonspecific binding. This is the first report
of direct, selective staining of κ opioid receptors by a fluorescent nonpeptide opioid ligand. The
results of the present study illustrate the importance of introducing hydrophilic linkers to reduce
nonspecific binding of fluorescent probes for opioid receptors.
Among the three major types of opioid receptors (µ,
κ, and δ),1 κ receptors have attracted special attention
because they mediate analgesia with minimal depend-
ence and respiratory depression.2 In addition to anal-
gesia, other potential applications of κ-selective agonists
have been reported.3 Because κ receptors represent
important therapeutic targets, selective ligands can
serve as important pharmacologic tools in order to gain
a better understanding of their mechanisms of action.
Moreover, the need for additional selective ligands has
been heightened by the recent cloning of κ opioid
receptors.4-9
only in nonspecific binding. Here we report on the
development of κ-selective fluorescent probes 2-9 that
are derived from the potent κ-selective agonist, (S)-2-
(3,4-dichlorophenyl)-N-methyl-N-[1-phenyl-2-(1-pyr-
rolidinyl)ethyl]acetamide (1, ICI199,441).20 Members of
this series retain κ agonist activity and selectivity, and
one of its members (5) has been found to label κ opioid
receptors. This is the first report of direct κ receptor
selective staining by a nonpeptide fluorescent opioid
ligand.
Design Ra tion a le a n d Ch em istr y
Fluorescent ligands can be used to label receptors in
cell cultures or tissue preparations. The fluorescent
labeling can be studied by fluorescence microscopy,10,11
confocal laser microscopy,12 or flow cytometry.13 Fur-
thermore, the high sensitivity of fluorescent labeling
may also allow the detection of receptors in heteroge-
neous populations of immune cells where radioligand
binding studies have failed to do so.13 Although immu-
nofluorescent methods have been useful in identifying
opioid receptors,14 pharmacologically active fluorescent
opioid ligands would have a potential advantage over
biologically inactive immunofluorescent probes because
they can in principle be used to correlate the staining
of opioid receptors with function. In spite of reports on
the synthesis of fluorescent opioid ligands,15-19 no
successful applications of such ligands for the direct
visualization of opioid receptors have been published.
Recently, a fluorescein-conjugated arylacetamide ligand
was successfully employed in the indirect immuno-
fluorescent detection of κ opioid receptors in the mouse
immune system,13 but direct staining with this resulted
Because structure-activity relationship studies have
indicated that the central phenyl group of 1 can tolerate
substitutions with minimal effect on affinity and agonist
potency,20 we chose to attach a fluorescein moiety to the
meta and para positions. The required m- and p-NH2
intermediates, 10 and 14, were prepared as previously
reported.21 Intermediate 10 was obtained in enantio-
merically pure form, while 14 was prepared as the
racemate.
Since high levels of nonspecific staining were observed
in our laboratory for δ-selective fluorescent opioid
receptor ligands,22 it was postulated that the lipophi-
licity of the fluorescent probes may lead to high levels
of nonspecific binding as a result of receptor internaliza-
tion and solubility in membranes. In order to minimize
this possibility, the polarity of the ligands was modu-
lated through the insertion of a glycyl or oligoglycyl
linker between the fluorophore and the pharmacophore.
In addition, we thought that longer linkers might also
minimize unfavorable interactions of the fluorophore
with the receptor.
† Department of Medicinal Chemistry.
Because fluorescein is unstable in light and difficult
to purify, conjugation to fluorescein isothiocyanate
(FITC-I) was performed in the last step of the prepara-
‡ Minneapolis Medical Research Foundation.
§ Department of Pharmacology.
X Abstract published in Advance ACS Abstracts, March 1, 1996.
0022-2623/96/1839-1729$12.00/0 © 1996 American Chemical Society

1730 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Chang et al.
Sch em e 1
Biologica l Resu lts
tion of the fluorescent probes (Scheme 1). The glycyl
Sm ooth Mu scle Assa ys. Target compounds were
tested on the electrically stimulated guinea pig longi-
tudinal ileal muscle25 (GPI) and mouse vas deferens26
(MVD) preparations as described previously.27 In wash-
resistant activity studies, the agonist-treated smooth
muscle preparation was incubated with naltrexone (500
nM) for 10 min before the tissue was washed with buffer
to remove the naltrexone (NTX).
and oligoglycyl linkers were first attached by DCC/
23,24
HOBT coupling of N-CBZ-(Gly)n
(n ) 1, 2, and 4) to
10 and 14. The resulting conjugates 11-13 and 15-
17 and the parent amines 10 and 14 were deprotected
by catalytic hydrogenolysis and coupled to FITC-I in the
presence of triethylamine to afford the meta- or para-
substituted fluorescent probes 2-9.
In GPI the meta series of fluorescent probes (2-5)
generally were highly potent opioid agonists and com-
parable to the parent amine 10 and the unsubstituted
ligand 1 (Table 1). The ligand with the diglycyl linker
(4) exhibited the lowest potency in this series. On the
other hand, the para-substituted series 6-9 were sub-
stantially less potent by factors ranging from 14 to 1100.
Both the meta and para series were generally less
potent agonists in the MVD relative to the GPI.
Fluorescent probes 2-5 were tested for wash-resis-
tant agonist activity by washing the treated preparation
with buffer or naltrexone (Table 2). The agonist activity
of unsubstituted compounds 1 and fluorescent probes 2
and 4 in the GPI preparation was only partially reversed
by extensive washing with buffer, but the activity of 3
and 5 could be fully reversed by washing with naltrex-
one, followed by washing with buffer to remove the NTX.
In the MVD preparation, the agonist activities of 2-5
could be removed by treatment with buffer alone.
Bin d in g Stu d ies. The opioid receptor binding af-
finities and selectivities of 3 and 5 were determined by
competition with radioligands in guinea pig brain
membranes by employing a modification of the method
of Werling et al.28 Binding to κ receptors was evaluated
using 1 nM [3H]-(5R,7R,8â)-(-)-N-methyl-N-1-pyrroli-
din yl-1-oxa spir o[4.5]dec-8-ylben zen ea cet a m ide29
([3H]U69,593), to µ receptors with 2 nM [3H][D-
Ala2,MePhe4,Gly-ol6]enkephalin30 ([3H]DAMGO), to δ1
receptors with 5 nM [3H][D-Pen2,D-Pen5]enkephalin31
([3H]DPDPE), and to δ2 receptors with 2 nM [3H]Tyr-

Arylacetamide-Derived Fluorescent Probes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1731
Ta ble 1. Agonist Potencies in Smooth Muscle Preparations
IC50 (nM) ( SEMa
Ta ble 3. Opioid Receptor Binding Selectivities in Guinea Pig
Brain
Ki (nM)a
Ki selectivity ratio
compd
GPI
MVD
compd
κ
µ
δ1
δ2
µ/κ
δ1/κ
δ2/κ
1
2
3
4
5
6
7
8
9
0.27 ( 0.09b
0.37 ( 0.18
0.17 ( 0.03
1.2 ( 0.3
0.15 ( 0.06
5.2 ( 0.88
62 ( 19
48 ( 14
166 ( 45
0.17 ( 0.08
0.65 ( 0.20
2.5 ( 1.7b
1.2 ( 0.19
1.2 ( 0.34
1.5 ( 0.12
6.5 ( 1.3
3
5
0.31
0.91
286
631
18.0
96.2
43.8
264
922
693
58
105
141
290
a
Values are geometric means of three experiments.
12.6 ( 2.1
102 ( 69
sity was also reduced by 61%, as determined by the
following formula: [(total fluorescence - background
fluorescence) - (fluorescence in the presence of norBNI
- background fluorescence)]/(total fluorescence - back-
ground fluorescence). The addition of â-FNA (500 µM)
did not significantly affect the percent of cells positively
labeled or mean fluorescence intensity by 2 (50 µM) and
5 (50 µM) (Table 4).
170 ( 15
421 ( 110
0.12 ( 0.01
4.5 ( 1.4
10
14
a
Values are arithmetic means of at least three experiments.
Reference 21.
b
Ta ble 2. Percent Recovery of Twitch Height in the GPI and
MVD after Wash Treatmenta
Discu ssion
GPI
MVD
compd
bufferb
NTXc
bufferb
The attachment of fluoroscein, through a glycyl or
oligoglycyl linker, to a κ-selective pharmacophore has
afforded a series of fluorescent ligands. Members of the
meta-substituted series (2-5) generally retained κ ago-
nist potency in smooth muscle preparations, whereas
the corresponding members of the para series (6-9)
were considerably less potent.
Since a high degree of nonspecific binding to cellular
components would render the ligands ineffective as
fluorescent probes, we investigated the facility with
which the agonist activity of 2-5 could be removed from
the GPI. This was evaluated by measuring the residual
agonist effect after repeated washing of the preparation
with buffer alone or with naltrexone. The finding that
the agonist effect of 3 and 5 on the GPI could be removed
upon washing, while the remaining members of the
meta series exhibited wash-resistant agonism, led us
to investigate these compounds further in an effort to
develop them as fluorescent probes. This was based on
the assumption that wash-resistant agonism may in
part be due to the partitioning of the ligand into the
cell membrane and to internalization.
After the κ selectivity of 3 and 5 was verified through
binding studies using guinea pig membranes, the most
hydrophilic (5) and the most hydrobic (2) members of
the meta series were evaluated by flow cytometry on
microglial cells which have been reported34 to contain
κ receptors. The fluorescent labeling of microglial cells
by 5 was found to be κ-selective, as indicated by
significant decreases in the fraction of cells stained and
in the staining intensity upon exposure to the κ antago-
nist, norBNI. In contrast to 5, the staining of microglial
cells by compound 2 was not changed by norBNI
treatment. This is consistent with a previous report13
on the staining of mouse immune cells by the racemic
form of 2.
The results of the flow cytometry studies clearly
demonstrate the advantage of the tetraglycyl linker in
5. This hydrophilic linker most likely reduces non-
specific staining by limiting internalization and mem-
brane partitioning of the fluorescent probe. In contrast,
the considerably more hydrophobic ligand 2 may more
easily penetrate membranes and the cell so that the
total binding far exceeds specific binding.
1
2
3
4
5
20
21
39
34
44
32
61
97
37
131
100
79
100
100
a
b
n ) 1. Percent recovery of twitch height after 30-40 washes
with buffer alone. c Percent recovery after treatment with 500 nM
of NTX and removal of NTX by 40 washes with buffer.
D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET).32 As shown in
Table 3, both 3 and 5 bind the κ opioid receptor with
high affinity and selectivity.
F lu or escen t La belin g Stu d ies
Direct fluorescent labeling of mouse microglial cells
by the fluorescent probes 2 and 5 was analyzed by flow
cytometry using a Coulter XL100 instrument (Coulter,
Miami, FL) with an excitation wavelength of 488 nm
and an emission wavelength of 525 nm. Microglial cells
have been shown to constitutively express the κ opioid
receptor33 and were prepared as previously reported.34
Because the microglial cells are believed to be function-
ally equivalent to monocytes or tissue macrophages of
the somatic immune system, the conditions of the
fluorescence studies are as previously reported in stud-
ies with mouse immune cells.13 Specificity of the
fluorescent labeling was determined by including the
κ-selective antagonist, norbinaltorphimine (norBNI),35
or the µ-selective antagonist, â-funaltrexamine (â-
FNA),36 in the fluorescent labeling studies. Mean
fluorescence intensity was determined from the percent
positively labeled cells. The background fluorescence
intensity (1.53) was determined from cells not treated
with any fluorescent probe. NorBNI was used at a
concentration of 1 mM because the 500 µM to 2 mM
range was previously shown to be optimal in similar
studies with mouse immune cells.13 In each sample,
about 10 000 cells were analyzed. Fluorescent probes
2 and 5 were studied to determine whether the tetra-
glycyl linker in 5 had increased the polarity sufficiently
to allow observation of κ-selective fluorescent labeling.
First, the concentration-dependence studies indicated
that both 2 and 5 are nearly equipotent in their ability
to label the microglial cells (Figure 1). However, when
norBNI (1 mM) was included, the percent of cells that
were positively labeled by 5 (50 µM) was significantly
decreased by 41% (Table 4). Mean fluorescence inten-
In conclusion, this study presents the first report of
direct selective staining of κ receptors by a fluorescent
nonpeptide opioid ligand (5). The reduction of non-

1732 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Chang et al.
1
(c ) 0.28, MeOH); H NMR (DMSO-d6) δ 1.90-1.95 (m, 4H,
CH2CH2), 2.76 (s, 3H, NCH3), 3-4.1 (complex, 10H, 5 CH2),
5.01 (s, 2H, ArCH2O), 6.07 (m, 1H, CH), 6.94 (m, 1H, amide
H), 7.25-7.56 (complex, 12H, aromatic H), 10.1 (s, 1H, amide
H); MS (FAB) m/z 597.2. Anal. (C31H34N4O4Cl2‚HCl) C, H,
N, Cl.
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1S)-1-[N-(N-CBZ-
glycylglycin a m id o)-3-a m in op h en yl]-2-(1-p yr r olid in yl)-
eth yl}a ceta m id e (12). Compound 12 was prepared from
N-CBZ-GlyGly (0.3056 g, 1.15 mmol), HOBT (0.1565 g, 1.16
mmol), DCC (0.2416 g, 1.17 mmol), and 1021 (0.3108 g, 0.76
mmol) in dry CH2Cl2 (28 mL). The procedure and reaction
conditions are similar to those employed for the preparation
of 11. After 18 h, the mixture was filtered through Celite, and
the filtrate was washed with saturated NaHCO3 before it was
dried (Na2SO4), filtered, and evaporated. Column chromatog-
raphy with CHCl3:2% NH3:5% MeOH followed by evaporation
of a 2-propanol solution of the HCl salt yielded 12‚HCl (0.3308
g, 63%): mp 224 °C; [R]25D +105.9° (c ) 0.31, MeOH); 1H NMR
(DMSO-d6) δ 1.94 (br s, 4H, CH2CH2), 2.77 (s, 3H, NCH3),
3.06-4.07 (complex, 12H, 6 CH2), 5.0 (s, 2H, ArCH2O), 6.07
(m, 1H, CH), 6.95 (d, J ) 7.8 Hz, 1H, amide H), 7.26-7.58
(complex, 12H, aromatic), 8.23 (m, 1H, amide H), 10.02 (s, 1H,
amide H); MS (FAB) m/z 654.2. Anal. (C33H37N5O5Cl2‚HCl)
C, H, N, Cl.
F igu r e 1. Concentration dependence of the percent of cells
that are positively labeled by 2 or 5. Microglial cells expressing
high autofluorescence (about 4-8% of total cell population)
were eliminated by gating. Data are mean values of ap-
proximately 10 000 cells/group and are representative of two
separate experiments.
Ta ble 4. Microglial Cell κ Opioid Receptors Demonstrated by
Immunofluorescencea
% cells
positively
labeled
mean
fluorescence
intensity
compd
treatment
5
medium
+ NorBNI
+ â-FNA
57.5
33
62
2.63
1.04
3.03
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1S)-1-[N-(N-CBZ-
glycylglycylglycylglycin a m id o)-3-a m in op h e n yl]-2-(1-
p yr r olid in yl)eth yl}a ceta m id e (13). A solution of DCC
(0.1273 g, 0.62 mmol) in dry EtOAc (12 mL) was added to an
ice-cold solution of N-CBZ-GlyGlyGlyGly (0.2204 g, 0.58 mmol)
and HOBT (0.0800 g, 0.59 mmol) in dry DMF (50 mL), and
the mixture was stirred in ice bath under N2 for 1 h. A solution
of 1021 in dry EtOAc (10 mL) was added, and the mixture was
stirred at 25 °C under N2 for 21 h before it was evaporated to
dryness. The residue was then suspended in hot EtOAc and
filtered, and the filtrate was washed with saturated NaHCO3
before it was dried (Na2SO4), filtered, and evaporated. Some
remaining dicyclohexylurea (DCU) was removed by flash-
column chromatography with CHCl3:2% NH3:10% MeOH, and
evaporation of an iPrOH solution of the HCl salt yielded
2
medium
+ NorBNI
+ â-FNA
69.6
68.9
71.4
3.53
3.52
3.88
a
Microglia cells (2 × 104) were treated with 50 µM of 5 or 2 in
the absence (medium) or presence of norBNI (1 mM) or â-FNA
(500 µM) and analyzed by flow cytometry. Values are expressed
both as the percent of cells positively labeled and mean fluores-
cence intensity. The mean background fluorescence intensity
(1.53), determined from cells not treated with any fluorescent
probe, was subtracted. Microglial cells having high autofluores-
cence (about 4-8% of total cell population) were eliminated by
gating.
13‚HCl (91.7 mg, 29%): mp 210 °C; [R]25 +88.42° (c ) 0.19,
specific staining by an oligoglycyl linker that joins the
pharmacophore to the fluorophore suggests this may be
used as a general strategy to diminish internalization
and nonspecific binding of nonpeptide ligands. Fluo-
rescent probe 5 may prove to be a useful tool in
pharmacologic and biochemical studies.
D
1
MeOH); H NMR (DMSO-d6) δ 1.95 (br s, 4H, CH2CH2), 2.77
(br s, 3H, NCH3), 3.03-4.04 (broad, 16H, 8 CH2), 5.0 (br s,
2H, ArCH2O), 6.07 (m, 1H, CH), 6.95 (m, 1H, amide H), 7.31-
7.52 (broad, 12H, aromatic), 8.24 (broad, 3H, amide H), 9.94
(br s, 1H, amide H); MS (FAB) m/z 768.2. Anal. (C37H43N7O7-
Cl2‚HCl‚H2O).
2-(3,4-Dich lor oph en yl)-N-m eth yl-N-{(1R,S)-1-[N-(N-CBZ-
glycin a m id o)-4-a m in op h en yl]-2-(1-p yr r olid in yl)et h yl}-
a ceta m id e (15). Compound 15 was prepared from N-CBZ-
Gly (0.3167 g, 1.514 mmol), HOBT (0.2087 g, 1.544 mmol),
DCC (0.3222 g, 1.562 mmol), and 1421 (0.3996 g, 0.9834 mmol)
in dry CH2Cl2 (26 mL). The procedure and reaction conditions
are similar to those employed for the preparation of 11. After
15 h, H2O was added to destroy excess active esters. The
suspension was then filtered through Celite, and the DCU was
washed with a little CH2Cl2. The filtrate was then washed
with saturated NaHCO3 before it was dried (Na2SO4), filtered
through Celite, and evaporated. Upon concentration, more
DCU precipitated and was removed by filtration through a
cotton-plugged pipet. The oil remaining was purified by flash-
column chromatography using CHCl3:2% NH3:5% MeOH
before it was converted to the HCl salt with Et2O‚HCl and
crystallized from MeOH:Et2O to yield 15‚HCl (0.4636 g,
72%): mp 145 °C dec; 1H NMR (DMSO-d6) δ 1.87-1.95 (m,
4H, CH2CH2), 2.74 (s, 3H, NCH3), 3.0-4.1 (complex, 10 H, 5
CH2), 5.01 (s, 2H, benzylic), 6.04 (m, 1H, CH), 7.15-7.58
(complex, 12 H, aromatic); MS (FAB) m/z 597.1. Anal.
(C31H34N4O4Cl2‚HCl) C, H, N, Cl.
Exp er im en ta l Section
All NMR spectra were recorded on a GE 300 MHz spec-
trometer or a Varian-500 MHz spectrometer at room temper-
ature. Optical rotations were measured using a Rudolph
Research Autopol III automatic polarimeter. Melting points
were determined using a Thomas Hoover capillary melting
point apparatus and are uncorrected. Elemental analyses
were conducted by M-H-W Laboratories in Phoenix, AZ. Mass
spectra were recorded by the Chemistry Mass Spec Labs at
the University of Minnesota’s Chemistry Department. Fluo-
rescein isothiocyanate isomer I was purchased from Aldrich
Chemical Co. N-CBZ-Gly and N-CBZ-GlyGly were purchased
from Sigma Chemical Co.
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1S)-1-[N-(N-CBZ-
glycin a m id o)-3-a m in op h en yl]-2-(1-p yr r olid in yl)et h yl}-
a ceta m id e (11). To an ice-cold solution of N-CBZ-Gly (0.2449
g, 1.17 mmol) in dry CH2Cl2 under N2 was added HOBT
(0.1599 g, 1.18 mmol), followed by a CH2Cl2 solution of DCC
(0.2467 g, 1.20 mmol). After 1 h at 0 °C, a CH2Cl2 solution of
1021 (0.3174 g, 0.781 mmol) was added, and the reaction
mixture was stirred at 25 °C under N2 for 4 days before it was
filtered through Celite. The residue after evaporation of the
filtrate was dissolved in EtOAc and washed successively with
saturated NaHCO3 and water, and the organic fraction was
dried (MgSO4), filtered, and evaporated. Flash-column chro-
matography with CHCl3:2% NH3:5% MeOH and crystallization
2-(3,4-Dich lor oph en yl)-N-m eth yl-N-{(1R,S)-1-[N-(N-CBZ-
glycylglycin a m id o)-4-a m in op h en yl]-2-(1-p yr r olid in yl)-
eth yl}a ceta m id e (16). To an ice-cold solution of 1421 (0.3043
g, 0.7489 mmol) in dry CH2Cl2 (20 mL) were sequentially
added N-CBZ-GlyGly (0.2993 g, 1.124 mmol), HOBT (0.1546
g, 1.144 mmol), DCC (0.2389 g, 1.158 mmol), and dry CH2Cl2
(4 mL) with stirring under N2. The mixture was allowed to
afforded 0.3178 g (68%) of 11‚HCl: mp 220 °C; [R]25 +113.6°
D

Arylacetamide-Derived Fluorescent Probes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1733
slowly warm to 25 °C overnight. After 64 h at 25 °C, H2O
was added to destroy excess active esters before the suspension
was filtered through Celite, and the DCU was washed with a
little CH2Cl2. The filtrate was diluted with CHCl3 to 200 mL
and washed with saturated NaHCO3 before it was dried
(Na2SO4), filtered through Celite, and evaporated. More DCU
precipitated and was removed by filtration through a cotton-
plugged pipet to yield an oil which was flash-column chro-
matographed using CHCl3:2% NH3:5% MeOH and then puri-
fied on 2 mm chromatotron plates using CHCl3:2% NH3:3%
MeOH. The product was converted to the HCl salt with
Et2O‚HCl and solidified by addition and evaporation of iPrOH
to yield 16‚HCl (0.1590 g, 31%): mp 137 °C dec; 1H NMR
(DMSO-d6) δ 1.94 (m, 4H, CH2CH2), 2.74 (s, 3H, NCH3), 3.1-
4.0 (complex, 12H, 6 CH2), 5.01 (s, 2H, benzylic), 6.05 (m, 1H,
CH), 7.17-7.56 (complex, 12H, aromatic); MS (FAB) m/z 654.3.
Anal. (C33H37N5O5Cl2‚HCl) C, H, N, Cl.
CH); MS (FAB) m/z 852.1 (MH+). Anal. (C44H39N5O7SCl2‚
(C2H5)3N) C, H, N, Cl.
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1S)-1-[N-(N-flu o-
r e s c e in y l-5-t h io u r e id o g ly c y lg ly c in a m id o )-3-a m in o -
p h en yl]-2-(1-p yr r olid in yl)eth yl}a ceta m id e (4). Interme-
diate 12‚HCl (0.3170 g, 0.485 mmol) was deprotected by
hydrogenation with 49 mg of 10% Pd/C in MeOH (14 mL)
under 10 psi for 69 h. To a solution of FITC-I (0.0841g, 0.216
mmol) in dry THF (1 mL) was added a solution of the free
base of deprotected 12 (0.1080 g, 0.2075 mmol) in absolute
ethanol (2 mL) and Et3N (1 mL) with stirring at 25 °C under
N2. After stirring for 24 h in the dark, the reaction mixture
was filtered through a cotton plug in a pipet, diluted with
absolute ethanol, and concentrated under reduced pressure.
Additional absolute ethanol and dry CH3CN were added, and
the mixture was concentrated again. This was repeated twice
before the solution was evaporated to dryness, and the residue
was suspended in absolute ethanol and separated by filtration.
The solid was washed thoroughly with absolute ethanol and
2-(3,4-Dich lor oph en yl)-N-m eth yl-N-{(1R,S)-1-[N-(N-CBZ-
glycylglycylglycylglycin a m id o)-4-a m in op h e n yl]-2-(1-
p yr r olid in yl)eth yl}a ceta m id e (17). To an ice-cold mixture
of 1421 (0.3013 g, 0.7415 mmol) and N-CBZ-GlyGlyGlyGly
(0.5612 g, 1.475 mmol) in anhydrous DMF (100 mL) were
added HOBT (0.2040 g, 1.510 mmol) and DCC (0.3139 g, 1.521
mmol) with stirring under N2. The mixture was allowed to
slowly warm to 25 °C overnight. After 21 h, the reaction was
incomplete, and N-CBZ-GlyGlyGlyGly (0.2808 g, 0.7383 mmol),
HOBT (0.1020 g, 0.7548 mmol), and DCC (0.1570 g, 0.7609
mmol) were added with cooling in ice-H2O. The mixture was
stirred at 0 °C for 1 h and at 25 °C for 15 h before H2O was
added to destroy excess active esters. After evaporation of
DMF, the residue was partitioned between 0.5 N HCl and
CHCl3 (200 mL) before the aqueous fraction was basified with
NaOH(s) and extracted with EtOAc (400 mL), which was dried
(Na2SO4), filtered through Celite, and evaporated. The residue
was purified by flash-column chromatography using CHCl3:
2% NH3:10% MeOH and converted to the HCl salt with
Et2O‚HCl to yield 17‚HCl (0.0699 g, 12%): mp 144 °C dec; 1H
NMR (DMSO-d6) δ 1.94 (br s, 4H, CH2CH2), 2.74 (s, 3H,
-NCH3), 3.0-4.0 (complex, 16H, 8 CH2), 5.00 (s, 2H, benzylic),
6.05 (m, 1H, CH), 7.15-7.61 (complex, 12H, aromatic); MS
(FAB) m/z 768.3. Anal. (C37H43N7O7Cl2‚HCl) C, H, N.
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1S)-1-[N-(flu or es-
cein yl-5-th iou r eid o)-3-a m in op h en yl]-2-(1-p yr r olid in yl)-
eth yl}a ceta m id e (2). With stirring at 25 °C under N2, a
solution of 1021 (0.1992 g, 0.4902 mmol) in absolute ethanol
(6 mL) and Et3N (2 mL) was added to a solution of fluorescein
isothiocyanate isomer I (FITC-I, 0.1526 g, 0.3919 mmol) in
freshly distilled THF (3 mL) and absolute ethanol (1 mL). After
stirring for 16 h in the dark, the solvent was evaporated, and
Et3N was removed azeotropically with dry CH3CN. The solid
was then washed thoroughly with absolute EtOH and MeOH
before it was purified by 0.25 mm reverse-phase-18 TLC plates
using 100% MeOH as the solvent to yield 2‚Et3N (80.1 mg,
dried to yield 4‚Et3N (0.1540 g, 73%): mp 205 °C dec; [R]25
D
+66.7° (c ) 0.15, MeOH with 1 drop Et3N); 1H NMR (DMSO-
d6) δ 1.62 (br s, 4H, CH2CH2), 2.68 (s, 3H, NCH3), 5.81 (br m,
1H, CH); MS (FAB) m/z 909.3 (MH
+). Anal. (C46H42N6O8SCl2‚
(C2H5)3N) C, H, N, Cl.
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1S)-1-[N-(N-flu o-
r escein yl-5-th iou r eid oglycylglycylglycylglycin a m id o)-3-
a m in op h en yl]-2-(1-p yr r olid in yl)eth yl}a ceta m id e (5). In-
termediate 13‚HCl (0.2037 g, 0.265 mmol) was deprotected by
hydrogenation with 30 mg of 10% Pd/C in MeOH (16 mL)
under 10 psi for 3 days. With stirring at 25 °C under Ar,
FITC-I (0.0735 g, 0.189 mmol) was added to a mixture of the
monohydrochloride salt of deprotected 13 (0.1209 g, 0.1802
mmol) and Et3N (4 mL) in freshly distilled THF (6 mL) and
absolute ethanol (4 mL). After stirring for 63 h in the dark,
more fluorescein isothiocyanate isomer I (0.0699 g, 0.180
mmol) was added, and the reaction was complete (by reverse-
phase TLC) after 28 h more of stirring. After evaporation of
solvent and azeotropic removal of Et3N with dry CH3CN, the
product was dissolved in MeOH:Et3N (99:1) and purified on
0.25 mm reverse-phase-18 TLC plates using 100% MeOH as
the solvent to yield 5‚Et3N (112.9 mg, 55.7%): mp 205 °C dec;
1
[R]25 ) +66.7° (c ) 0.12, MeOH with 1 drop Et3N); H NMR
D
(DMSO-d6) δ 1.5-1.7 (m, 4H, CH2CH2), 5.78-5.82 (m, 1H,
CH); MS (FAB) m/z 1123.3 (MH+). Anal. (C50H48N8O10SCl2‚
(C2H5)3N) C, H, N, Cl.
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1R,S)-1-[N-(flu o-
r escein yl-5-th iou r eido)-4-am in oph en yl]-2-(1-pyr r olidin yl)-
et h yl}a cet a m id e (6). A mixture of 1421 (0.1088 g, 0.2678
mmol), FITC-I (0.1085 g, 0.2786 mmol), and Et3N (2 mL) in
anhydrous EtOH:THF (5 mL, 3:2) was stirred at 25 °C under
N2 for 17 h before it was evaporated in the dark, and Et3N
was azeotropically removed with dry CH3CN to yield a solid
that was suspended in absolute EtOH (4 mL) and collected by
filtration. Washing successively with absolute EtOH (8 mL)
23%): mp 185 °C dec; [R]25 ) +118° (c ) 0.1, MeOH with 1
D
drop Et3N); 1H NMR (DMSO-d6) δ 1.6 (br s, 4H, CH2CH2),
5.79-5.82 (m, 1H, CH); MS (FAB) m/z 795.1 (MH+). Anal.
(C42H36N4O6SCl2‚(C2H5)3N) C, H, N, Cl.
N (0.1441 g, 60%): mp
and anhydrous ethyl ether yielded 6‚Et3
1
194 °C dec; H NMR (DMSO-d6) δ 1.63 (br s, 4H, CH2CH2),
2.72 (s, 3H, NCH3), 2.40-4.0 (complex, 8H, 4 CH2), 5.79 (m,
1H, CH); MS (FAB) m/z 795.3 (MH+). Anal. (C42H36N4O6SCl2‚
(C2H5)3N) C, H, N, Cl.
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1S)-1-[N-(N-flu o-
r escein yl-5-t h iou r eid oglycin a m id o)-3-a m in op h en yl]-2-
(1-p yr r olid in yl)eth yl}a ceta m id e (3). Intermediate 11‚HCl
(0.3050 g, 0.511 mmol) was deprotected by hydrogenation with
48 mg of 10% Pd/C in MeOH (10 mL) under 10 psi for 65 h.
To a solution of FITC-I (0.0808 g, 0.208 mmol) in dry THF (1
mL) and absolute ethanol (1 mL) was added a solution of the
free base of deprotected 11 (0.0928 g, 0.200 mmol) in absolute
ethanol (2 mL) and Et3N (1 mL) with stirring at 25 °C under
N2. After stirring for 23 h in the dark, the reaction mixture
was diluted with absolute ethanol and concentrated under
reduced pressure. Once concentrated, dry CH3CN was added
to remove Et3N azeotropically. The solution was then evapo-
rated to dryness, and the residue was suspended in absolute
ethanol and separated by filtration. The solid was washed
thoroughly with absolute ethanol and dried to yield 3‚Et3N
(0.1228 g, 64%): mp 200 °C dec; [R]25D +71.6° (c ) 0.11, MeOH
with 2 drops Et3N); 1H NMR (DMSO-d6) δ 1.655-1.977 (br m,
4H, CH2CH2), 2.720 (s, 3H, NCH3), 5.785-6.176 (br m, 1H,
2-(3,4-Dich lor op h en yl)-N-m et h yl-N-{(1R,S)-1-[N-(N-
flu or escein yl-5-th iou r eid oglycin a m id o)-4-a m in op h en yl]-
2-(1-pyr r olidin yl)eth yl}acetam ide (7). Intermediate 15‚HCl
(0.2024 g, 0.3193 mmol) and 30 mg of 10% Pd/C were mixed
in MeOH (12 mL) and hydrogenated at 25 °C under 10 psi for
70 h before the mixture was filtered through Celite, and the
Pd/C was washed thoroughly with hot MeOH (100 mL). After
evaporation of the filtrate, the residue was partitioned between
2 N NaOH (200 mL) and EtOAc (300 mL), and the organic
fraction was dried (Na2SO4), filtered through Celite, and
evaporated. To a solution of the deprotected intermediate
(0.1229 g, 0.2652 mmol) in absolute EtOH (3 mL) and Et3N (1
mL) were added FITC-I (0.1072 g, 0.2753 mmol) and freshly
distilled THF (1 mL) with stirring under N2. The mixture was
evaporated after 48 h in the dark, and Et3N was removed
azeotropically with dry CH3CN to yield a solid which was
suspended in 95% EtOH and collected by filtration. The solid

1734 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Chang et al.
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was washed successively with 95% EtOH and anhydrous ethyl
ether to yield 7‚Et3N (0.1538 g, 60.8%): mp 203 °C dec; 1H
NMR (DMSO-d6) δ 1.62 (br s, 4H, CH2CH2), 5.79 (m, 1H, CH);
MS (FAB) m/z 852.3 (MH+). Anal. (C44H39N5O7SCl2‚(C2H5)3N)
C, H, N, Cl.
2-(3,4-Dich lor op h en yl)-N-m et h yl-N-{(1R,S)-1-[N-(N-
flu or escein yl-5-th iou r eid oglycylglycin a m id o)-4-a m in o-
p h en yl]-2-(1-p yr r olid in yl)eth yl}a ceta m id e (8). Interme-
diate 16‚HCl (0.1195 g, 0.1729 mmol) and 24 mg of 10% Pd/C
were mixed in MeOH (6 mL) and hydrogenated at 25 °C under
10 psi for 138 h before the mixture was filtered through Celite,
and the Pd/C was washed thoroughly with hot MeOH. After
evaporation of the filtrate, the residue was partitioned between
1 N NaOH (150 mL) and EtOAc (300 mL), and the organic
fraction was dried (Na2SO4), filtered through Celite, and
evaporated. To a solution of the deprotected intermediate
(0.0615 g, 0.1182 mmol) in absolute EtOH (3 mL) and Et3N (2
mL) were added FITC-I (0.0479 g, 0.1229 mmol) and freshly
distilled THF (1 mL) with stirring at 25 °C under N2. After 7
days in the dark, the mixture was evaporated, and Et3N was
removed azeotropically with dry CH3CN (16 mL) to yield a
solid which was suspended in absolute EtOH (8 mL) and
collected by filtration. Washing with anhydrous ethyl ether
and drying yielded 8‚Et3N (0.0887 g, 74%): mp 208 °C dec;
1H NMR (DMSO-d6) δ 1.61 (m, 4H, CH2CH2), 5.80 (m, 1H, CH);
MS (FAB) m/z 909.3 (MH+). Anal. (C46H42N6O8SCl2‚(C2H5)3N)
C, H, N, Cl.
2-(3,4-Dich lor op h en yl)-N-m et h yl-N-{(1R,S)-1-[N-(N-
flu or escein yl-5-th iou r eidoglycylglycylglycylglycin am ido)-
4-a m in op h en yl]-2-(1-p yr r olid in yl)et h yl}a cet a m id e (9).
Intermediate 17‚HCl (0.0729 g, 0.0905 mmol) and 16 mg of
10% Pd/C were mixed in MeOH (20 mL) and hydrogenated at
25 °C under 10 psi. After 72 h, the mixture was filtered
through Celite. The Pd/C was washed thoroughly with hot
MeOH (100 mL), and the combined filtrate was evaporated.
To a solution of the 1 HCl salt of the deprotected intermediate
(0.0500 g, 0.0745 mmol) in absolute EtOH (5 mL) and Et3N (4
mL) was added FITC-I (0.0309 g, 0.0794 mmol) in dry THF (2
mL) with stirring at 25 °C under N2 in the dark. After 3 days,
more fluorescein isothiocyanate isomer I (0.0298 g, 0.0765
mmol) and 100% EtOH (4 mL) were added, and the stirring
continued for 2 days. The mixture was then evaporated, and
Et3N was azeotropically removed with dry CH3CN (8 mL). The
solid was purified on 0.25 mm reverse-phase-18 TLC plates
eluting with 100% MeOH. The residue solidified upon addition
of dry CH3CN, and the solid was collected by filtration to yield
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1
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of the compounds. We also acknowledge funding from
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