N4-Phenyl modifications of N2-(2-hydroxyl)ethyl-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-d iamines enhance glucocerebrosidase inhibition by small molecules with potential as chemical chaperones for Gaucher disease

Article abstract of DOI:10.1016/j.bmcl.2007.08.050

A series of 1,3,5-triazine-2,4,6-triamines were prepared and analyzed as inhibitors of glucocerebrosidase. Synthesis, structure activity relationships and the selectivity of chosen analogues against related sugar hydrolases enzymes are described.

Full text of DOI:10.1016/j.bmcl.2007.08.050

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5783–5789
N4-Phenyl modifications of N2-(2-hydroxyl)ethyl-6-(pyrrolidin-
1-yl)-1,3,5-triazine-2,4-diamines enhance
glucocerebrosidase inhibition by small molecules
with potential as chemical chaperones for Gaucher disease
Wenwei Huang,a Wei Zheng,a Daniel J. Urban,b James Inglese,a Ellen Sidransky,b,*
Christopher P. Austina and Craig J. Thomasa,*
aNIH Chemical Genomics Center, National Human Genome Research Institute, NIH, 9800 Medical Center Drive,
MSC 3370, Bethesda, MD 20892-3370, USA
bMedical Genetics Branch, National Human Genome Research Institute, NIH, Bld 35 Rm1A213,
35 Convent Drive, Bethesda, MD 20892-3708, USA
Received 3 August 2007; revised 23 August 2007; accepted 23 August 2007
Available online 28 August 2007
Abstract—A series of 1,3,5-triazine-2,4,6-triamines were prepared and analyzed as inhibitors of glucocerebrosidase. Synthesis, struc-
ture activity relationships and the selectivity of chosen analogues against related sugar hydrolases enzymes are described.
Published by Elsevier Ltd.
Glycosphingolipids are cell membrane components that
maintain the stability of the lipid bilayer, function as cel-
lular recognition elements and play an important role in
cellular adherence.1 These important cellular constructs
are comprised of varying polysaccharide chains linked
to ceramide via a glycosidic bond. Glycosphingolipids
are ultimately targeted for degradation through the ac-
tion of selected lysosomal hydrolases.2 Several different
diseases arise when a defect in one or more of these
hydrolases causes non-degraded glycosphingolipids to
accumulate within the lysosome.3 These maladies are
commonly referred to as ‘lysosomal storage disorders’
and include Niemann-Pick, Tay–Sachs and Gaucher
diseases.
different mutations giving rise to Gaucher disease have
been identified, and it is likely that multiple misfolded
protein conformations exist.4 Current treatment for
Gaucher disease involves enzyme replacement therapy.5
While this therapy alleviates many of the systemic man-
ifestations of the disease, including hepatosplenomegaly,
anemia and thrombocytopenia, the inability of recombi-
nant enzyme to cross the blood–brain barrier prevents
amelioration of the CNS associated symptoms in neur-
onopathic forms of Gaucher disease. The subset of
mutations that result in protein misfolding and impro-
per trafficking of GC to the lysosome presents an oppor-
tunity for the use of ‘chemical chaperone’ therapy, and
importantly, could provide a therapeutic approach
capable of penetrating the blood–brain barrier.
Gaucher disease results from mutations in the glucocer-
ebrosidase (GC) gene. Many of these are point muta-
Chemical chaperones are small molecules that bind to
misfolded proteins, restoring correct structural conforma-
tion and enabling appropriate protein trafficking.6 Once
the chaperoned protein reaches its appropriate subcellu-
lar location, the small molecule chaperone must either
be displaced by native substrate to allow for the continued
presence of a correctly folded, active protein, or bind to an
allosteric site that does not disrupt substrate binding. The
utility of chaperone therapy in Gaucher, Sandhoff, Fabry
and Tay–Sachs diseases has been studied.7,8 Kelly and
coworkers,9 Overkleeft and coworkers10 and Fan and
tions that result in
a
misfolded protein with
diminished catalytic activity or aberrant trafficking from
the endoplasmic reticulum to the lysosome.2,3 Over 200
Keywords: Triazine; Gaucher disease; Glucocerebrosidase; Chemical
chaperone.
*
Corresponding authors. Tel.: +1 301 217 4079; fax: +1 301 217 5736
(C.J.T.); tel.: +1 301 451 0901, +1 301 496 0373; fax: +1 301 402 6438
(E.S.); e-mail addresses: sidranse@mail.nih.gov; craigt@mail.nih.gov
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.08.050

5784
W. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5783–5789
coworkers11 have advanced several iminosugars designed
to mimic the native glycosphingolipid substrate as
potential GC chaperones. Several of these small mole-
cules are currently being evaluated in clinical trials.8 In
addition, at least one reported non-sugar based small
molecule (1-phenyl-2-decanoylamino-3-morpholino-1-
propanol), which was designed as a ceramide mimic, is
also being studied clinically.8 We recently reported the
use of qHTS to identify three novel, structurally distinct
small molecule inhibitors of GC including 2-(4-(5-chloro-
2-methoxyphenylamino)-6-(pyrrolidin-1-yl)-1,3,5-triazin-
2-ylamino)ethanol (1), N-(4-methyl-2-morpholinoquino-
lin-6-yl)cyclohexanecarboxamide (2) and N-(5-ethyl-1,3,
4-thiadiazol-2-yl)-4-(phenylsulfonamido)benzamide (3)
(Fig. 1).12 These novel GC inhibitors possess potencies
comparable to the best iminosugars, including the clini-
cally relevant N-butyl-deoxynojirimycin (Miglustat) (4)
[for 1: IC50 = 0.70 lM; for 2: IC50 = 0.031 lM; for 3:
IC50 = 0.070 lM]. A significant property of these struc-
tures is their apparent selectivity versus related sugar
hydrolases including a-glucosidase, a-galactosidase and
b-hexosaminidase.
chaperone activity.12 This intriguing finding prompted
us to further explore and optimize this series of com-
pounds. To this end, we synthesized and evaluated
numerous analogues of 1 by examining three discrete
sections of the compounds; the aminoethanol moiety,
the pyrrolidinyl moiety and the substituted aniline
moiety.
The synthetic methods for the construction of substi-
tuted triazines are well documented.15 We utilized a
three step, one pot procedure (Scheme 1) whereby cyan-
uric chloride is treated first with substituted aniline (see
Table 3) in acetonitrile with (method B) or without
(method A) Hunig’s base.16 Following this transforma-
tion, primary or secondary amines (see Table 2) were
administered in either 3.3 equiv (method A) or 1.1 equiv
(method B) in DMA with Hunig’s base for 10 min. Fi-
nally, a large excess of primary and secondary alkyl
and cycloalkylamines (see Table 1) was added and stir-
red for 4–8 h and the resulting mixture was purified
via mass-directed preparative HPLC. Yields for the
one pot, three step procedure were typically in excess
of 60%. Each construct was examined by LCMS and
ELSD for purity and characterized via 1H NMR,
high-resolution mass spectrometry and 13C NMR for se-
lected analogues.17 Individual compounds were evalu-
ated biochemically18 from standard 10 lM DMSO
solutions via a GC enzyme assay utilizing a fluorogenic
resorufin b-D-glucopyranoside substrate as previously
reported.12
It has been demonstrated that the ability of iminosugars
to act as chemical chaperones is dependent upon the
nature of the specific mutant protein.13 Further, Kelly
and coworkers have shown that temperature alterations
have differential effects on the cellular trafficking of se-
lected GC variants.14 These studies suggest that differing
mutant forms of GC likely result in distinctive protein
misfolding. Taken together with the vast number of clin-
ically relevant GC mutations, these findings emphasize
the value of developing divergent small molecule chem-
otypes to target alternate folded proteins.
Our first investigations addressed the SAR of the etha-
nolamine moiety (Table 1). The qHTS screen revealed
1 as a potent inhibitor of GC from a collection of nearly
400 related triazine structures that did not maintain an
ethanolamine moiety. As anticipated, our subsequent at-
tempts to modify this substructure were collectively det-
rimental. It was also noted that deviations from any
substructure that did not contain a hydroxyl group
Previously, we evaluated the ability of these novel GC
inhibitors to act as chemical chaperones and found that
the least potent structural series (the 1,3,5-triazine-2,4,6-
triamine core represented by 1) possessed the greatest
OMe
H
H
N
OH
N
S
Cl
NH
N
H
O
O
N
N
O
N
N
N
N
N
HO
OH
HO
O
S
N
N
H
N
O
OH
1
2
3
4
Figure 1. Compound structures of previously reported inhibitors of glucocerebrosidase including 2-(4-(5-chloro-2-methoxyphenylamino)-6-
(pyrrolidin-1-yl)-1,3,5-triazin-2-ylamino)ethanol (1), N-(4-methyl-2-morpholinoquinolin-6-yl)cyclohexanecarboxamide (2) and N-(5-ethyl-1,3,4-
thiadiazol-2-yl)-4-(phenylsulfonamido)benzamide (3) and N-butyl-deoxynojirimycin (Miglustat) (4).
H
N
H
N
Cl
N
Cl
R
N
X
Cl
N
i
ii
R'
R'
N
N
N
N
N
N
iii
Cl
Cl
1, 5-47
Scheme 1. Reagents and conditions: Method A. (i) Substituted ArNH2 (1 equiv), ACN, rt 4 h; (ii) substituted amine (3.3 equiv), Hunig’s base,
DMA, rt 10 min; (iii) substituted amine (5–10 equiv), rt 4–8 h. Method B. (i) Substituted ArNH2 (1 equiv), Hunig’s base, ACN, rt 4 h; (ii) substituted
amine (1.05 equiv), Hunig’s base, DMA, rt 10 min; (iii) substituted amine (5–10 equiv), rt 4–8 h.

W. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5783–5789
Table 1. SAR surrounding the ethanolamine moiety of the core triazine scaffold
R'
5785
HN
N
N
R
N
N
Analogue #
R
R0
GC inhibition IC50 (lM) and SDa
1
5
–NHCH2CH2OH
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe
0.70 0.08
Inactive
Inactive
7.77 0.33
Inactive
Inactive
Inactive
Inactive
20.70
–NHCH2CH2OCH3
–N(CH2CH2OH)2
–NHCH2CH2CH2CH3
–NHCH2Ph
6
7
8
9
–NHCH2(2-furanyl)
–NH2
10
11
12
13
14
15
16
–NHCH2CH2NHCOCH3
–NHCH2CH(OH)CH3
–NHCH2CH(OH)CH2Ph
–NHCH2CONH2
2-OMe
2-OMe
6.80 0.83
Inactive
Inactive
Inactive
2-OMe
2-OMe
2-OMe
–NHCH2CH2CH2CH2OH
–NHCH2COOMe
a Error represents the standard deviation determined from three separate experiments.
Table 2. SAR surrounding the pyrrolidine moiety of the core triazine scaffold
R'
HN
N
N
HO
N
N
X
H
Analogue #
X
R0
GC inhibition IC50 (lM) and SDa
1
17
18
19
20
21
22
23
24
Pyrrolidin-1-yl
Morpholin-1-yl
N,N-Diethylamino
Piperidin-1-yl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe, 5-Cl
2-OMe
0.70 0.08
12.13
Inactive
31.10
Azepan-1-yl
N-(n-Butyl)amino
N-Benzylamino
Inactive
6.1 0.51
18.80
2-OMe
2-OMe
2-OMe
3,3-Difluoropyrrolidin-1-yl
Azetidin-1-yl
7.75 0.82
2.31 0.28
a Error represents the standard deviation determined from three separate experiments.
conferred difficulty with water solubility. This observa-
tion is supported by the calculated cLogP values of se-
lected analogues as illustrated by the comparison of 1
(calculated cLogP of 3.35) with 7 (calculated cLogP
of 5.23). Additional compounds were made and exam-
ined including the 2-aminoethyl analogue and showed
no apparent activity (data are not shown due to insuffi-
cient purity analysis—typically less than 80% pure by
HPLC UV/vis peak integration).
the same relative size, yet lost the rigid ring structure, re-
sulted in an analogue that was completely inactive.
Rings of a larger size (6 and more) were significantly less
potent and the only smaller ring system explored (the 4-
membered azetidinyl ring) was modestly potent, yet sig-
nificantly less than the core pyrrolidinyl moiety found in
1 and 31.
Finally, optimizations of the anilinyl moiety were exam-
ined (Table 3). Within this substructure we first focused
on the existing 2-methoxy, 5-chloro substitution pattern
by exploring selected replacements and deletions in
terms of mono- versus di-substitution and relative posi-
tion (ortho, meta and para). While the majority of these
analogues (for example 28–30, 31–34) remained mod-
estly potent, none surpassed the original ligand. We also
We next attempted to optimize the inhibitory potential
of this series through alterations at the pyrrolidinyl moi-
ety (Table 2). Here again, deviations from this core com-
ponent were found to be adverse in terms of GC
inhibition. Interestingly, altering a pyrrolidinyl to a
N,N-diethylamino moiety (analogue 18) that maintained

5786
W. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5783–5789
Table 3. SAR surrounding the aniline moiety of the core triazine
scaffold
of altering a 2-OMe group (analogue 31) to a 2-OEt
group (analogue 35). This change resulted in a modest
potency enhancement relative to the mono-substituted
2-OMe analogue. We further evaluated this trend by
expanding this moiety into a 2-OPh (analogue 36) and
2-Ot-Bu (analogue 37). Gratifyingly, 37 provided an
IC50 value of 0.33 lM, the lowest reported potency for
this structural class of GC inhibitors. Unfortunately,
this SAR was not found to be enhanced by incorpora-
tion of the previously noted disubstituted analogues
containing a 5-Cl substitution in conjugation with the
2-alkoxy series (analogues 50–52). Continued explora-
tions also revealed that a 2-OH, 5-Ph substitution (ana-
logue 49) conferred an IC50 value of 0.37 lM. Thus, the
two most potent analogues possessed structurally unre-
lated substitution patterns and only modestly related
calculated cLogP values (calculated cLogP of 3.72 for
37 and calculated cLogP of 4.22 for 49). Efforts to ascer-
tain the binding site through co-crystallization and
docking studies will be pursued.
R'
HN
N
N
HO
N
H
N
N
Analogue # R0
GC inhibition IC50 (lM) and SDa
1
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
2-OMe, 5-Cl
2-Me
3-Me
0.70 0.80
3.94 0.13
6.19 0.46
10.04
4-Me
2-Cl
28.97
3-Cl
4-Cl
6.69 0.27
6.34 0.06
1.08 0.13
8.25 0.21
8.80 0.64
2.00 0.14
0.77 0.04
21.67
2-OMe
3-OMe
4-OMe
2-OH
2-OEt
2-OPh
2-Ot-Bu
2-OBn
2-OCF3
2-SMe
Having found novel analogues with improved potency,
it was important to confirm that they maintained the
selectivity profile of the parent compound (1) versus
the related hydrolases a-glucosidase, a-galactosidase
and b-hexosaminidase.18 Importantly, 37 showed no
activity against any of these related enzymes at concen-
trations up to 77 lM (Fig. 2).
0.33 0.06
31.4
3.34 0.06
2.39 0.09
2-OMe, 5-OMe 1.53 0.08
2-OMe, 4-OMe 4.90 0.13
2-OMe, 5-CF3
2-OMe, 5-Ph
2-NO2
2.65 0.14
2.22 0.04
Inactive
Through manipulations of the N4-phenyl moiety, we
optimized the inhibitory activity of a small molecule
shown to possess strong chemical chaperone activity
and arrived at novel analogue 37 with an IC50 value of
330 nM. The expanded compound set provides numer-
ous analogues with GC enzyme inhibitory potencies
ranging from 0.3 lM to 2.0 lM. This family of inhibi-
tors, as well as those from ongoing studies based upon
2 and 3, will enable us to probe the relationship between
enzymatic inhibitory potency and chaperone activity.
Expanded compound sets of divergent structures that
maintain a range of potencies may represent crucial
probe compounds that will permit further characteriza-
tion of various GC mutants. Moreover, since different
mutant forms of GC are likely to require different chem-
ical chaperones, these expanded compound sets will
facilitate exploration of the potential for mutation-spe-
cific chemical chaperones.
2-CONH2
2-NHCOCH3
2-OH, 5-Cl
2-OH, 5-Ph
2-OEt, 5-Cl
2-Oi-Pr, 5-Cl
2-Ot-Bu, 5-Cl
H
Inactive
Inactive
0.73 0.09
0.37 0.05
0.96 0.02
3.44 0.08
1.04 0.05
16.47
a Error represents the standard deviation determined from three sep-
arate experiments.
explored various commonly applied substitutions
including methyl (analogues 25–27) di-methoxy (ana-
logues 41, 42) and trifluoromethoxy (analogue 39) with
limited success. We next investigated the consequence
120
100
80
60
40
20
0
O
Glucocerebrosidase
NH
α-galactosidase
N
N
HEX
HO
N
N
H
N
α-glucosidase
37
-20
-11 -10 -9
-8
-7
-6
-5
-4
-3
Log (Compound) M
Figure 2. Selective inhibition of glucocerebrosidase (e), a-glucosidase (Ç), a-galactosidase (D) and b-hexosaminidase (m) by 37. Data represent the
collective results of three independent experiments performed in triplicate.

W. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5783–5789
5787
17. Spectroscopic data for 1, 5–53 [Note: Several peaks within
the proton and carbon NMR spectrum existed as rotomers
that coalesce upon heating to 60 °C. This is a well-
established phenomenon associated with triazine scaffolds
(see Arvanitis et al. J. Med. Chem. 1999, 42, 805; Hajduk
et al. J. Med. Chem. 1999, 42, 3852 and Leftheris et al. J.
Med. Chem. 2004, 47, 6283 for examples)]: 1 1H NMR
(400 MHz, DMSO-d6) d 8.64 (d, 1H, J = 6.6 Hz), 7.37–
7.28 (m, 1H), 7.05–6.94 (m, 3H), 4.74–4.66 (m, 1H), 3.90
(s, 3H), 3.60–3.33 (m, 8H), 1.92 (br s, 4H); HRMS (ESI):
m/z calcd for C16H21ClN6O2 [M+1]+ 365.1493, found
365.1492. 5 1H NMR (400 MHz, DMSO-d6) d 8.63 (d, 1H,
J = 2.3 Hz), 7.39–7.28 (m, 1H), 7.05–6.98 (m, 3H), 3.91 (s,
3H), 3.51–3.30 (m, 11H), 1.91 (br s, 4H); HRMS (ESI): m/
Acknowledgments
The authors thank Jeremy Smith, Adam Yasgar and Paul
Shinn for assistance in compound management. The
authors thank Bryan Mott and Mary E. LaMarca for
helpful discussion and critical reading of this manuscript.
Thanks also to Bev Chamberlin at MSU Mass Spectrom-
etry Facility for HRMS analyses. This research was sup-
ported by the Molecular Libraries Initiative of the
National Institutes of Health Roadmap for Medical
Research and the Intramural Research Program of the
National Human Genome Research Institute, National
Institutes of Health.
z
calcd for C17H23ClN6O2 [M+1]+ 379.1649, found
379.1644. 6 1H NMR (400 MHz, DMSO-d6) d 8.60 (d,
1H, J = 2.3 Hz), 7.36 (s, 1H), 7.05 (d, 1H, J = 8.8 Hz), 6.99
(dd, 1H, J = 8.6, 2.5 Hz), 4.80–4.77 (m, 2H), 3.91 (s, 3H),
3.66 (br s, 8H), 3.56–3.46 (m, 4H), 1.93 (br s, 4H); HRMS
(ESI): m/z calcd for C18H25ClN6O3 [M+1]+ 409.1755,
found 409.1766. 7 1H NMR (400 MHz, DMSO-d6) d 8.68–
8.62 (m, 1H), 7.35–7.24 (m, 1H), 7.16–6.97 (m, 3H), 3.91
(s, 3H), 3.51–3.27 (s, 6H), 1.91 (br s, 4H), 1.57–1.50 (m,
2H), 1.42–1.32 (m, 2H), 0.93 (t, 3H, J = 7.4 Hz); HRMS
(ESI): m/z calcd for C18H25ClN6O [M + 1]+ 377.1857,
found 377.1860. 8 1H NMR (400 MHz, DMSO-d6) d 8.62–
8.56 (m, 1H), 7.74–7.62 (m, 1H), 7.39–7.20 (m, 6H), 7.04–
6.97 (m, 2H), 4.51–4.49 (m, 2H), 3.90 (br s, 3H), 3.49–3.27
(m, 4H), 1.92 (s, 4H); HRMS (ESI): m/z calcd for
C21H23ClN6O [M+1]+ 411.1700, found 411.1709. 9 1H
NMR (400 MHz, DMSO-d6) d 8.63 (d, 1H, d = 2.5 Hz),
7.38–7.28 (m, 1H), 7.05–6.97 (m, 3H), 4.06–3.98 (m, 1H),
3.90 (s, 3H), 3.82–3.28 (m, 8H), 1.96–1.78 (m, 7H), 1.68–
1.58 (m, 1H); HRMS (ESI): m/z calcd for C19H25ClN6O2
[M+1]+ 405.1806, found 405.1800. 10 1H NMR (400 MHz,
DMSO-d6) d 8.62 (d, 1H, J = 2.5 Hz), 7.30 (s, 1H), 7.05–
6.97 (m, 2H), 6.59 (br s, 2H), 3.90 (s, 3H), 3.52–3.38 (m,
4H), 1.92 (br s, 4H); HRMS (ESI): m/z calcd for
C14H17ClN6O [M+1]+ 321.1231, found 321.1237. 11 1H
NMR (400 MHz, DMSO-d6) d 8.47–8.44 (m, 1H), 7.96–
7.90 (m, 1H), 7.35–7.26 (m, 1H), 7.04–6.93 (m, 4H), 3.90
(s, 3H), 3.51–3.22 (m, 8H), 1.91 (br s, 4H), 1.83 (s, 3H);
HRMS (ESI): m/z calcd for C18H25N7O2 [M+1]+
372.2148, found 372.2147. 12 1H NMR (400 MHz,
DMSO-d6) d 8.46 (d, 1H, J = 7.2 Hz), 7.33–7.26 (m,
1H), 7.05–6.80 (m, 4H), 4.84–4.68 (m, 1H), 3.90 (br s, 3H),
3.89–3.68 (m, 1H), 3.60–3.17 (m, 6H), 1.91 (br s, 4H), 1.09
(d, 3H, J = 6.3 Hz); HRMS (ESI): m/z calcd for
C17H24N6O2 [M+1]+ 345.2039, found 345.2041. 13 1H
NMR (400 MHz, DMSO-d6) d 8.48–8.39 (m, 1H), 7.39–
7.24 (m, 6H), 7.08–6.80 (m, 4H), 5.63–5.44 (m, 1H), 4.86–
4.76 (m, 1H), 3.90 (br s, 3H), 3.60–3.22 (m, 6H), 1.96–1.86
(m, 4H); HRMS (ESI): m/z calcd for C22H26N6O2 [M+1]+
407.2195, found 407.2188. 14 1H NMR (400 MHz,
DMSO-d6) d 8.48–8.41 (m, 1H), 7.36–7.26 (m, 2H),
7.04–6.90 (m, 5H), 3.90 (s, 3H), 3.82 (d, 2H, J = 6.1 Hz),
3.58–3.34 (m, 4H), 1.91 (br s, 4H); HRMS (ESI): m/z calcd
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Alpan, O.; Simeonov, A.; Goldin, E.; Auld, D.; LaMarca,
M. E.; Inglese, J.; Austin, C. P.; Sidransky, E. Proc. Natl.
Acad. Sci. U.S.A. 2007, 104, 13192.
13. Sawker, A. R.; Adamski-Werner, S. L.; Cheng, W.-C.;
Wong, C.-H.; Beutler, E.; Zimmer, K.-P.; Kelly, J. W.
Chem. Biol. 2005, 12, 1235.
14. Sawker, A. R.; Schmitz, M.; Zimmer, K.-P.; Reczek, D.;
Edmunds, T.; Balch, W. E.; Kelly, J. W. ACS Chem. Biol.
2006, 1, 235.
15. Katiyar, S. B.; Srivastava, K.; Puri, S. K.; Chauhan, P. M.
S. Bioorg. Med. Chem. Lett. 2005, 15, 4957.
16. The majority of substituted anilines were commercially
available. However 5-chloro-2-ethoxyaniline, 5-chloro-2-
isopropoxyaniline, 2-tert-butoxyaniline and 2-tert-but-
oxy-5-chloroaniline required independent syntheses. The
method was modified from a procedure by Wandless and
coworkers (Woiwode, T. F.; Rose, C.; Wandless, T. J.
J. Org. Chem. 1998, 63, 9594). Substituted anilines were
utilized directly following a purifying extraction.
1
for C16H21N7O2 [M+1]+ 344.1835, found 344.1838. 15 H
NMR (400 MHz, DMSO-d6) d 8.50–8.47 (m, 1H), 7.31–
7.21 (m, 1H), 7.02–6.94 (m, 4H), 4.41 (br s, 1H), 3.90 (br s,
3H), 3.54–3.24 (m, 8H), 1.90 (s, 4H), 1.62–1.44 (m, 4H);
HRMS (ESI): m/z calcd for C18H26N6O2 [M+1]+
359.2195, found 359.2192. 16 1H NMR (400 MHz,
DMSO-d6) d 8.45–8.30 (m, 1H), 7.44–7.36 (m, 2H),
7.05–6.89 (m, 3H), 4.01–3.96 (m, 2H), 3.89 (s, 3H), 3.66
(s, 3H), 3.54–3.36 (m, 4H), 1.94–1.86 (m, 4H); HRMS
(ESI): m/z calcd for C17H22N6O3 [M+1]+ 359.1832, found
359.1838. 17 1H NMR (400 MHz, DMSO-d6)d 8.46 (s,
1H), 7.54–7.43 (m, 1H), 7.16–6.94 (m, 3H), 4.70 (br s, 1H),
NO2
NO2
NH2
R''-OH
F
OR''
OR''
Na2S2O4
H2O, MeOH
microwave heating
100 oC, 10 min.
KHMDS
THF, 0 oC to r.t.
2-4 h.
Y
Y
Y
Y = H or Cl
R'' = Et, iPr, tBu

5788
W. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5783–5789
3.89 (s, 3H), 3.71–3.35 (m, 12H); HRMS (ESI): m/z calcd
C16H22N6O2 [M+1]+ 331.1882, found 331.1883. 32 1H
NMR (400 MHz, DMSO-d6) d 8.97–8.85 (m, 1H), 7.77–
7.69 (m, 1H), 7.36–7.20 (m, 1H), 7.13 (t, 1H, J = 8.2 Hz),
6.73–6.66 (m, 1H), 6.50 (dd, 1H, J = 7.6, 1.8 Hz), 4.77–
4.66 (m, 1H), 3.76 (s, 3H), 3.71–3.32 (m, 8H), 1.91 (br s,
4H); HRMS (ESI): m/z calcd for C16H22N6O2 [M+1]+
331.1882, found 331.1879. 33 1H NMR (400 MHz,
DMSO-d6) d 8.81–8.70 (m, 1H), 7.51 (d, 2H, J = 8.2 Hz),
6.85 (d, 2H, J = 8.8 Hz), 6.62 (br s, 1H), 4.77–4.66 (m,
1H), 3.74 (s, 3H), 3.69–3.32 (m, 8H), 1.90 (br s, 4H);
HRMS (ESI): m/z calcd for C16H22N6O2 [M+1]+
331.1882, found 331.1888. 34 1H NMR (400 MHz,
DMSO-d6) d 10.68 (br s, 1H), 8.16–8.12 (m, 1H), 7.83–
7.75 (m, 1H), 6.93–6.76 (m, 4H), 4.74–4.68 (m, 1H), 3.56–
3.37 (m, 11H), 1.91 (br s, 4H); HRMS (ESI): m/z calcd for
C15H20N6O2[M+1]+317.1726, found 317.1729. 35 1H
NMR (400 MHz, DMSO-d6) d 8.50 (br s, 1H), 7.32–7.24
(m, 1H), 7.01–6.81 (m, 4H), 4.72 (br s, 1H), 4.20–4.13 (m,
2H), 3.56–3.34 (m, 8H), 1.91 (br s, 4H), 1.42 (t, 3H,
J = 7.8 Hz); HRMS (ESI): m/z calcd for C17H24N6O2
[M+1]+ 345.2039, found 345.2046. 36 1H NMR (400 MHz,
DMSO-d6) d 8.49 (d, 1H, J = 8.0 Hz), 7.54–7.36 (m, 3H),
7.19–7.14 (m, 2H), 7.10–6.98 (m, 3H), 6.94–6.86 (m, 1H),
6.82 (t, 1H, J = 5.7 Hz), 4.74–4.60 (m, 1H), 3.56–3.31 (m,
8H), 1.92–1.86 (m, 4H); HRMS (ESI): m/z calcd for
C21H24N6O2 [M+1]+ 393.2039, found 393.2033. 37 1H
NMR (400 MHz, DMSO-d6) d 8.57–8.49 (m, 1H), 7.34–
7.25 (m, 1H), 7.12–7.03 (m, 2H), 6.93–6.84 (m, 2H), 4.76–
4.64 (m, 1H), 3.57–3.34 (m, 8H), 1.91 (br s, 4H), 1.39 (s,
9H); 13C NMR (400 MHz, DMSO-d6, 60 °C) d 165.5,
163.2, 163.1, 143.3, 133.8, 122.8, 121.3, 120.6, 119.3, 79.6,
60.0, 45.4, 42.8, 28.4, 28.4, 24.4. HRMS (ESI): m/z calcd
for C16H21ClN6O3 [M+1]+ 381.1442, found 381.1439. 18
1H NMR (400 MHz, DMSO-d6) d 8.64–8.56 (m, 1H),
7.38–7.29 (m, 1H), 7.28–6.87 (m, 3H), 4.76–4.64 (m, 1H),
3.90 (s, 3H), 3.62–3.30 (m, 8H), 1.24–1.06 (m, 6H); HRMS
(ESI): m/z calcd for C16H23ClN6O2 [M+1]+ 367.1649,
found 367.1647. 19 1H NMR (400 MHz, DMSO-d6) d 8.50
(d, 1H, J = 2.3 Hz),7.45–7.34 (m, 1H), 7.08–6.94 (m, 3H),
4.72–4.64 (m, 1H), 3.90 (s, 3H), 3.78–3.68 (m, 4H), 3.57–
3.52 (m, 2H), 3.40–3.32 (m, 2H), 1.70–1.46 (m, 6H);
HRMS (ESI): m/z calcd for C17H23ClN6O2 [M+1]+
379.1649, found 379.1659. 20 1H NMR (400 MHz,
DMSO-d6) d 8.62–8.56 (m, 1H), 7.38–7.28 (m, 1H),
7.06–6.86 (m, 3H), 4.72–4.64 (m, 1H), 3.91 (s, 3H), 3.71–
3.28 (m, 8H), 1.81–1.68 (m, 4H), 1.58–1.46 (m, 4H);
HRMS (ESI): m/z calcd for C18H25ClN6O2 [M+1]+
393.1806, found 393.1805. 21 1H NMR (400 MHz,
DMSO-d6) d 8.45–8.43 (m, 1H), 7.40–6.62 (m, 6H), 4.72
(br s, 1H), 3.89 (s, 3H), 3.53–3.26 (m, 6H), 1.58–1.48 (m,
2H), 1.40–1.30 (m, 2H), 0.93 (t, 3H, J = 7.2 Hz); HRMS
(ESI): m/z calcd for C16H24N6O2 [M+1]+ 333.2039, found
1
333.2046. 22 H NMR (400 MHz, DMSO-d6) d 8.45–8.20
(m, 1H), 7.64–7.20 (m, 7H), 7.06–6.74 (m, 4H), 4.80–4.64
(m, 1H), 4.51 (d, 2H, J = 6.3 Hz), 3.87 (br s, 3H), 3.56–
3.30 (m, 4H); HRMS (ESI): m/z calcd for C19H22N6O2
[M+1]+ 367.1882, found 367.1889. 23 1H NMR (400 MHz,
DMSO-d6) d 8.40–8.32 (m, 1H), 7.50–7.40 (m, 1H), 7.06–
6.90 (m, 4H), 4.74–4.66 (m, 1H), 3.96–3.86 (m, 4H), 3.76–
3.68 (m, 2H), 3.56–3.34 (m, 5H), 2.55–2.45 (m, 2H);
HRMS (ESI): m/z calcd for C16H20F2N6O2 [M+1]+
367.1694, found 367.1695. 24 1H NMR (400 MHz,
DMSO-d6) d 8.42–8.38 (m, 1H), 7.41–7.33 (m, 1H),
7.04–6.93 (m, 4H), 4.79–4.64 (m, 1H), 4.09–3.98 (m,
4H), 3.89 (s, 3H), 3.56–3.50 (m, 2H), 3.40–3.32 (m, 2H),
2.34–2.40 (m, 2H); HRMS (ESI): m/z calcd for
C15H20N6O2 [M+1]+ 317.1726, found 317.1723. 25 1H
NMR (400 MHz, DMSO-d6) d 8.10–7.94 (m, 1H), 7.70–
7.50 (m, 1H), 7.20–7.13 (m, 2H), 7.04–6.96 (m, 1H), 6.64–
6.48 (m, 1H), 4.78–4.56 (m, 1H), 3.58–3.24 (m, 8H), 2.25
(s, 3H), 1.87 (br s, 4H); HRMS (ESI): m/z calcd for
C16H22N6O [M+1]+ 315.1933, found 315.1938. 26 1H
NMR (400 MHz, DMSO-d6) d 8.90–7.70 (m, 1H), 7.77–
7.54 (m, 2H), 7.12 (t, 1H, J = 7.6 Hz), 6.75–6.60 (m, 2H),
4.78–4.67 (m, 1H), 3.58–3.34 (m, 8H), 2.29 (s, 3H), 1.91
(br s, 4H); HRMS (ESI): m/z calcd for C16H22N6O [M+1]+
315.1933, found 315.1940. 27 1H NMR (400 MHz,
DMSO-d6) d 8.90–8.72 (m, 1H), 7.71 (d, 2H, J = 8.6 Hz),
7.06 (d, 2H, J = 8.0 Hz), 6.66 (br s, 1H), 4.82–4.60 (m,
1H), 3.58–3.28 (m, 8H), 2.26 (s, 3H), 1.90 (br s, 4H);
HRMS (ESI): m/z calcd for C16H22N6O [M+1]+ 315.1933,
found 315.1938. 28 1H NMR (400 MHz, DMSO-d6) d
8.26–8.18 (m, 1H), 7.84–7.70 (m, 1H), 7.47 (dd, 1H,
J = 8.0, 1.6 Hz), 7.32 (t, 1H, J = 7.2 Hz), 7.10–7.05 (m,
1H), 6.84–6.78 (m, 1H), 4.74–4.66 (m, 1H), 3.53–3.31 (m,
8H), 1.89 (br s, 4H); HRMS (ESI): m/z calcd for
C15H19ClN6O [M+1]+ 335.1387, found 335.1396. 29 1H
NMR (400 MHz, DMSO-d6) d 9.23–9.11 (m, 1H), 8.16–
8.10 (m, 1H), 7.71–7.69 (m, 1H), 7.26 (t, 1H, J = 7.8 Hz),
6.95 (d, 1H, J = 7.2 Hz), 6.83–6.77 (m, 1H), 4.75–4.65 (m,
1H), 3.58–3.36 (m, 8H), 1.92 (br s, 4H); HRMS (ESI): m/z
calcd for C15H19ClN6O [M+1]+ 335.1387, found 335.1391.
1
for C19H28N6O2 [M+1]+ 373.2352, found 373.2356. 38 H
NMR (400 MHz, DMSO-d6) d 8.47 (br s, 1H), 7.54–7.29
(m, 7H), 7.12–6.93 (m, 2H), 6.86–6.81 (m, 1H), 5.23–5.21
(m, 2H), 4.71–4.66 (m, 1H), 3.60–3.32 (m, 8H), 1.90 (br s,
4H); HRMS (ESI): m/z calcd for C22H26N6O2 [M+1]+
407.2195, found 407.2202. 39 1H NMR (400 MHz,
DMSO-d6) d 8.20 (d, 1H, J = 8.0 Hz), 8.10–7.96 (m, 1H),
7.38–7.33 (m, 2H), 7.18–7.13 (m, 1H), 6.77–6.75 (m, 1H),
4.72–4.63 (m, 1H), 3.48–3.33 (m, 8H), 1.89 (br s, 4H);
HRMS (ESI): m/z calcd for C16H19F3N6O2 [M+1]+
385.1600, found 385.1602. 40 1H NMR (400 MHz,
DMSO-d6) d 8.12–8.07 (m, 1H), 7.88–7.83 (m, 1H),
7.49–7.40 (m, 1H), 7.25 (t, 1H, J = 7.4 Hz), 7.10–7.08
(m, 1H), 6.79 (t, 1H, J = 5.7 Hz), 4.72–4.65 (m, 1H), 3.52–
3.30 (m, 8H), 2.42 (s, 3H), 1.90 (br s, 4H); HRMS (ESI):
m/z calcd for C16H22N6OS [M+1]+ 347.1654, found
347.1659. 41 1H NMR (400 MHz, DMSO-d6) d 8.25–
8.10 (m, 1H), 7.21–7.16 (m, 1H), 6.75–6.60 (m, 2H), 6.51
(br s, 1H), 4.78–4.64 (m, 1H), 3.86 (s, 3H), 3.75 (s, 3H),
3.57–3.26 (m, 8H), 1.94–1.84 (m, 4H); HRMS (ESI): m/z
calcd for C17H24N6O3 [M+1]+ 361.1988, found 361.1987.
1
42 H NMR (400 MHz, DMSO-d6) d 8.30–8.24 (m, 1H),
7.30–7.21 (m, 1H), 6.96–6.84 (m 2H), 6.50 (dd, 1H,
J = 8.8, 2.9 Hz), 4.78–4.64 (m, 1H), 3.86 (s, 3H), 3.77 (s,
3H), 3.56–3.24 (m, 8H), 1.90 (br s, 4H); HRMS (ESI): m/z
calcd for C17H24N6O3 [M+1]+ 361.1988, found 361.1991.
1
43 H NMR (400 MHz, DMSO-d6) d 9.01–8.95 (m, 1H),
7.47–7.39 (m, 1H), 7.33 (d, 1H, J = 8.4 Hz), 7.21 (d, 1H,
J = 8.4 Hz), 7.00–6.86 (m, 1H), 4.73–4.63 (m, 1H), 3.99 (s,
3H), 3.54–3.26 (m, 8H), 1.92 (br s, 4H); HRMS (ESI): m/z
calcd for C17H21F3N6O2 [M+1]+ 399.1756, found
399.1760. 44 1H NMR (400 MHz, DMSO-d6) d 8.99–
8.87 (m, 1H), 7.66 (d, 2H, J = 7.4 Hz), 7.48–7.26 (m, 5H),
7.12 (d, 1H, J = 8.4 Hz), 6.88–6.83 (m, 1H), 4.60–4.49 (m,
1H), 3.95 (s, 3H), 3.60–3.40 (m, 8H), 1.92 (br s, 4H);
HRMS (ESI): m/z calcd for C22H26N6O2 [M+1]+
407.2195, found 407.2192. 45 1H NMR (400 MHz,
1
30 H NMR (400 MHz, DMSO-d6) d 9.15–9.04 (m, 1H),
7.86 (d, 2H, J = 8.8 Hz), 7.29 (br s, 2H), 6.76 (br s, 1H),
4.76–4.67 (m, 1H), 3.56–3.32 (m, 8H), 1.91 (br s, 4H);
HRMS (ESI): m/z calcd for C15H19ClN6O [M+1]+
335.1387, found 335.1391. 31 1H NMR (400 MHz,
DMSO-d6) d 8.48–8.46 (m, 1H), 7.34–7.26 (m, 1H),
7.04–6.82 (m, 4H), 4.74–4.68 (m, 1H), 3.90 (s, 3H), 3.51–
3.32 (m, 8H), 1.92 (br s, 4H); HRMS (ESI): m/z calcd for

W. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5783–5789
5789
DMSO-d6) d 9.58 (d, 1H, J = 7.2 Hz), 8.51–8.26 (m, 1H),
8.07 (t, 1H, J = 8.6 Hz), 7.72–7.67 (m, 1H), 7.22–7.17 (m,
1H), 6.93–6.87 (m, 1H), 4.78–4.65 (m, 1H), 3.58–3.28 (m,
8H), 1.89 (br s, 4H); HRMS (ESI): m/z calcd for
C15H19N7O3 [M+1]+ 346.1628, found 346.1622. 46 1H
NMR (400 MHz, DMSO-d6) d 10.86–10.82 (m, 1H), 8.91–
8.83 (m, 1H), 8.18 (s, 1H), 7.74 (dd, 1H, J = 8.0, 1.6 Hz),
7.60 (s, 1H), 7.48–7.44 (m, 1H), 7.00–6.82 (m, 2H), 4.72–
4.70 (m, 1H), 3.57–3.33 (m, 8H), 1.91 (br s, 4H); HRMS
(ESI): m/z calcd for C16H21N7O2 [M+1]+ 344.1835, found
J = 8.6 Hz), 7.04–6.92 (m, 2H), 4.73–4.67 (m, 1H), 3.57–
3.31 (m, 8H), 1.93 (br s, 4H), 1.40 (s, 9H); HRMS (ESI):
m/z calcd for C19H27ClN6O2 [M+1]+ 407.1962, found
1
407.1966. 53 H NMR (400 MHz, DMSO-d6) d 8.97–8.86
(m, 1H), 7.85 (d, 2H, J = 8.6 Hz), 7.25 (t, 2H, J = 7.8 Hz),
6.92 (t, 1H, J = 7.4 Hz), 6.69 (br s, 1H), 4.76–4.66 (m, 1H),
3.57–3.31 (m, 8H), 1.91 (br s, 4H); HRMS (ESI): m/z calcd
for C15H20N6O [M+1]+ 301.1777, found 301.1774.
18. GC enzyme assay. Assays were conducted using recombi-
nant GC (CerezymeÒ, Genzyme Co., Cambridge, MA)
and resorufin-b-D-glucopyranoside (Km = 28 lM) in an
assay buffer composed of 50 mM citric acid/KH2PO4, pH
5.9, 10 mM sodium taurocholate and 0.01% Tween 20. GC
in assay buffer was added to a 1536-well black plate at
2 ll/well, followed by addition of 23 nl of compound in
DMSO with a pin-tool station (Kalypsys, San Diego).
After 5 min at RT (ꢀ21 °C), 1 ll/well of substrate was
added and incubated for 20 min at RT. Fluorescence
intensity was measured at an excitation of 570 ( 10) nm
and an emission of 610 ( 10) nm. The final concentrations
of enzyme and substrate were 1.9 nM and 30 lM, respec-
tively. Enzyme selectivity assays. Three additional hydro-
lases and their substrates, a-glucosidase (from rice) and
4-methylumbelliferyl a-D-glucopyranoside (4MU-a-Glc),
a-galactosidase (from coffee beans) and 4-methylumbel-
liferyl a-D-galactopyranoside (4MU-a-Gal), and b-N-acet-
ylglucosaminidase (from bovine kidney) (HEX) and
4-methylumbelliferyl N-acetyl-b-D-glucosaminide (4MU-
b-GSM) were obtained from Sigma–Aldrich. The buffer
for all three enzyme assays consisted of 50 mM citric acid/
KH2PO4, pH 4.5, 10 mM sodium taurocholate and 0.01%
Tween 20. The final enzyme concentrations for a-gluco-
sidase, a-galactosidase and b-N-acetylglucosaminidase
were 8, 1 and 8 nM, respectively. The substrate concen-
trations were similar to the Km values for these related
enzymes, at 0.4, 0.16 and 0.2 mM, respectively.
1
344.1839. 47 H NMR (400 MHz, DMSO-d6) d 9.78–9.70
(m, 1H), 8.13–8.01 (m, 1H), 7.85 (s, 1H), 7.43 (d, 1H,
J = 7.6 Hz), 7.17–7.03 (m, 2H), 6.82–6.77 (m, 1H), 4.71–
4.65 (m, 1H), 3.51–3.33 (m, 8H), 2.10 (s, 3H), 1.89 (br s,
4H); HRMS (ESI): m/z calcd for C17H23N7O2 [M+1]+
358.1991, found 358.1994. 48 1H NMR (400 MHz,
DMSO-d6) d 10.83 (br s, 1H), 8.19–8.09 (m, 1H), 7.90
(br s, 1H), 7.04–6.83 (m, 3H), 4.82–4.60 (m, 1H), 3.61–3.24
(m, 8H), 1.92 (br s, 4H); HRMS (ESI): m/z calcd for
1
C15H19ClN6O2 [M+1]+ 351.1336, found 351.1334. 49 H
NMR (400 MHz, DMSO-d6) d 10.74 (br, 1H), 8.34–8.15
(m, 2H), 7.63–7.60 (m, 2H), 7.45 (t, 2H,J = 7.6), 7.32 (t,
1H, J = 7.2 Hz), 7.24–7.20 (m, 1H), 6.96–6.94 (m, 2H),
4.71 (br s, 1H), 3.57–3.30 (m, 8H), 1.92 (br s, 4H); HRMS
(ESI): m/z calcd for C21H24N6O2 [M+1]+ 393.2039, found
393.2050. 50 1H NMR (400 MHz, DMSO-d6) d 8.67 (s,
1H), 7.34–7.26 (m, 1H), 7.06–6.93 (m, 3H), 4.74–4.64 (m,
1H), 4.20–4.12 (m, 2H), 3.58–3.31 (m, 8H), 1.93 (br s, 4H),
1.42 (t, 3H, J = 7.0 Hz); HRMS (ESI): m/z calcd for
1
C17H23ClN6O2 [M+1]+ 379.1649, found 379.1648. 51 H
NMR (400 MHz, DMSO-d6) d 8.69 (d, 1H, J = 2.3 Hz),
7.31–7.23 (m, 1H), 7.07–6.90 (m, 3H), 4.72–4.67 (m, 2H),
3.57–3.34 (m, 8H), 1.93 (br s, 4H), 1.34 (d, 6H,
J = 5.3 Hz); HRMS (ESI): m/z calcd for C18H25ClN6O2
[M+1]+ 393.1806, found 393.1815. 52 1H NMR (400 MHz,
DMSO-d6) d 8.69 (s, 1H), 7.34–7.25 (m, 1H), 7.12 (d, 1H,