Identification and optimization of N3,N6-diaryl-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamines as a novel class of ACK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.10.092

A new series of pyrazolo[3,4-d]pyrimidine-3,6-diamines was designed and synthesized as potent and selective inhibitors of the nonreceptor tyrosine kinase, ACK1. These compounds arose from efforts to rigidify an earlier series of N-aryl pyrimidine-5-carboxamides. The synthesis and structure-activity relationships of this new series of inhibitors are reported. The most promising compounds were also profiled for their pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2008.10.092

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6352–6356
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Bioorganic & Medicinal Chemistry Letters
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Identification and optimization of N3,N6-diaryl-1H-pyrazolo[3,4-d]
pyrimidine-3,6-diamines as a novel class of ACK1 inhibitors
a
a
a
a
e
David J. Kopecky a, , Xiaolin Hao , Yi Chen , Jiasheng Fu , XianYun Jiao , Juan C. Jaen ,
*
Mario G. Cardozo b, Jinsong Liu b, Zhulun Wang b, Nigel P. C. Walker b, Holger Wesche c,
Shyun Li c, Ellyn Farrelly d, Shou-Hua Xiao d, Frank Kayser a,
*
a Department of Chemistry, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
b Department of Molecular Structure, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
c Department of Oncology, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
d Department of Lead Discovery, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
e ChemoCentryx Inc., 850 Maude Avenue, Mountain View, CA 94043, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of pyrazolo[3,4-d]pyrimidine-3,6-diamines was designed and synthesized as potent and
selective inhibitors of the nonreceptor tyrosine kinase, ACK1. These compounds arose from efforts to
rigidify an earlier series of N-aryl pyrimidine-5-carboxamides. The synthesis and structure–activity rela-
tionships of this new series of inhibitors are reported. The most promising compounds were also profiled
for their pharmacokinetic properties.
Received 30 August 2008
Revised 16 October 2008
Accepted 20 October 2008
Available online 1 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
ACK1
Kinase inhibitor
Cancer
SAR
Pyrazolo[3,4,-d]pyrimidine-3,6-diamine
ACK1 (Activated Cdc42Hs-associated Kinase 1) is a nonreceptor
tyrosine kinase that was first identified by its interaction with
Cdc42 and was suggested to function as an effector of Cdc42,
Rac, and FAK in several integrin signaling pathways.1 ACK1 has also
been identified as a regulator of endocytosis via direct association
with clathrin.2 ACK1 is ubiquitously expressed, with highest
expression levels in the brain, and has been shown to be overex-
pressed in a number of human tumor cell lines potentially predis-
posing them to become metastatic.3 Recently it was suggested that
ACK1 activity is required for the survival of v-RAS-transformed
murine fibroblasts.3 Moreover, activation of ACK1 has been shown
to stimulate prostate tumorigenesis.4 These findings suggest that
inhibition of ACK1 could be a potential new point of intervention
in preventing the onset and spread of cancer. The development
of a potent and selective ACK1 inhibitor would allow the role of
ACK1 in tumor progression to be probed. Toward this end, herein
we report on a new class of ACK1 inhibitors.
During the course of our ACK1 inhibitor program, a high-
throughput screening (HTS) campaign of our internal sample col-
lection was conducted. A series of N-aryl pyrimidine-5-carboxam-
O
R2
R
O
O
N
O
O
N
R3
N
N
H
N
N
H
N
R1
N
H
N
N
H
I
1a: R=H
1b: R=(CH2)2OH
OMe
2
N
1
N
R2
N
NH
N
R3
3
4
N
N
N
N
H
N
H
6
R1
N
N
N
N
H
5
H
* Corresponding authors. Tel.: +1 650 244 2146; fax: +1 650 837 9369
(D.J.Kopecky); tel.: +1 650 244 2062 (F.Kayser).
E-mail addresses: dkopecky@amgen.com (D.J. Kopecky), fkayser@amgen.com (F.
Kayser).
II
2
Figure 1. N3,N6-diaryl-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamine lead structure II
where R2 is a functionalized aliphatic chain. Nitrogen atom numbering is shown.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.092

D. J. Kopecky et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6352–6356
6353
ides of the general structure I (Fig. 1) was identified. Earlier studies
of the structure–activity relationship (SAR) of I identified com-
pound 1a as one of the more potent inhibitors within this series
in both biochemical and cellular autophosphorylation assays.5–7
(ACK1 Ki = 0.02 lM, Cell IC50 = 0.1 lM). Unfortunately, this com-
pound series generally displayed poor selectivity against several
OMe
H2N
R1
R2
O
O
O
N
a, b, c
HO
NH
N
H
N
R2
N
H
O
N
Cl
other kinases (data not shown) and a poor pharmacokinetic profile
3
4
in male Sprague–Dawley rats (1a: Cl = 3.8 L/h/kg, AUC = 76 lg h/L;
0.4 mg/kg iv).
d
The X-ray co-crystal structure of the close analog 1b bound to
the ACK1 catalytic kinase domain was subsequently determined
at 2.3 Å resolution (Fig. 2). Inspection of the three-dimensional
structure of the complex revealed a binding mode in which the
aminopyrimidine moiety participates in key hinge interactions:
the pyrimidine nitrogen forms a hydrogen bond with the NH from
Ala208, and the carbonyl of Ala208 H-bonds to the NH of the 2-
amino group. The 4-position of the aryloxy moiety is oriented to-
wards a solvent-exposed area, while the dimethyl-phenyl group
is perfectly positioned to enter a deep hydrophobic pocket. Hydro-
phobic interactions within this selectivity pocket, combined with
an additional H-bond with the conserved threonine (Thr205) gate-
keeper amino-acid residue, are key features found in several potent
kinase inhibitors, such as those described for the related tyrosine
kinase p56lck (LCK).8
Molecular modeling suggested that the inhibitory activity of
series I might be improved upon by taking advantage of the addi-
tional Thr205 hydrogen bond. Following this rationale, several
modifications of I were designed which tied the amide carbonyl
and the ether oxygen into a cyclic structure, while rotating the
NH into a more optimal position to interact with Thr205. The
new designs were modeled into the ATP-binding site and the most
promising candidates were selected for synthesis. These efforts
identified N3,N6-diaryl-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamines
II as a novel series of inhibitors of ACK1, of which compound
2 is a representative example (Figs. 1 and 2).9
R1
R1
OMe
OMe
R2
R2
N
N
N
N
e
N
H
N
R2
R2
N
R3
N
H
N
N
N
Cl
H
2, 6-19
5
Scheme 1. Reagents and conditions: (a) PCl5, POCl3, reflux, quantitative; (b)
Amberlyst A-21 resin, 2,6-di(R2)-4-(R1)-aniline, EtOAc, 50 °C, 90%; (c) (Me)2
(MeO)CCH2CH2NHNH2, Et3N, THF, 75–85%; (d) PCl5, toluene, reflux, 65–80%; (e)
TFA, (R3)-aniline, 1,4-dioxane/n-BuOH, reflux, 50–70%.
ized aliphatic chain at N-1 of the N3,N6-diaryl-1H-pyrazolo[3,4-
d]pyrimidine-3,6-diamine core was beneficial for ACK1 inhibition.
Also, the replacement of the aromatic groups at either N-3 or N-6
of the 1H-pyrazolo[3,4-d]pyrimidine-3,6-diamine scaffold with an
alkyl group or a hydrogen led to significantly reduced levels of
ACK1 inhibition (data not shown).
Compound 2, which possesses a 3-methyl-3-methoxybutyl
substituent at N-1 (Table 1) showed significantly improved ACK1
inhibition over 1a in both biochemical and cellular autophosphory-
lation assays.6,7 Subsequent derivatives 6–19 demonstrated that
both methyl and chloro were well tolerated at R2 although hydro-
gen was not. Installation of larger groups at R1 such as methyl
(compounds 10 and 19) diminishes binding, presumably due to
steric crowding at the rear of the small hydrophobic pocket that
the N-3-substituent occupies. The effects of incorporating a smaller
fluorine atom at R1 are less pronounced (compounds 9 and 18).
The 4-position of the N-6-phenyl ring points out of the en-
zyme towards solvent, providing an area to introduce polar sol-
ubilizing groups for better cell penetration. As seen in Table 1, a
variety of N- and O-linked groups were tolerated in this posi-
tion without dramatically affecting enzyme inhibition. A 3-flu-
oro atom could also be introduced next to O-linked groups
without affecting potency (compounds 14–16). The 3-fluoro
was introduced in an attempt to impart metabolic stability to
the scaffold by stabilizing one of the two electron-rich aniline
moieties present in the molecule. Furthermore, in the case of
15, the other aniline moiety is simultaneously stabilized by
2,6-dichloro substitution.
Compounds of the general structure II were synthesized as out-
lined in Scheme 1 by a five-step procedure. Chlorination of 3 was
achieved with a mixture of PCl5 and POCl3. The resulting acid chlo-
ride was coupled with an appropriately substituted aniline in the
presence of an acid-scavenger and then treated with 3-methyl-3-
methoxybutylhydrazine10 to produce 4. Cyclization of 4 with PCl5
in refluxing toluene gave 5, and subsequent chloride displacement
with an appropriate aniline provided compounds 2 and 6–19. Early
exploratory work on the new series established that a functional-
Despite these efforts to prevent oxidative metabolic degrada-
tion, 15 and 16 showed only marginal improvements in metabolic
stability and still exhibited high plasma clearances as shown in Ta-
ble 2. High molecular weight as well as relative high clog P values
could be contributing factors to the poor PK profiles of 15 and re-
lated derivatives.11 We were pleased to discover several com-
pounds with reduced molecular weight where R3 is hydrogen
which retain high levels of biochemical and cellular potency (Table
1, compounds 7 and 8). At this stage, a thorough investigation of
the N-1 substituent was desired prior to readdressing the issue
of metabolic stability for these truncated analogs.
A high-resolution X-ray co-crystal structure of 2 complexed to
ACK1 was obtained (Fig. 3). As predicted in our earlier modeling,
two hydrogen bonds to the hinge region of the ACK1 ATP-binding
site along with an additional H-bond to the carbonyl oxygen of
Figure 2. Overlap of high-resolution X-ray co-crystal structure of original hit 1b
(green) bound to ACK1, and the binding mode model for compound 2 (cyan). RCSB
file name (3EQP).

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D. J. Kopecky et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6352–6356
Table 1
ACK1 inhibition for N3,N6-diaryl-N1-(3,3-dimethyl-3-methoxypropyl)pyrazolopyrimidine-3,6-diamines 2 and 6–19
R1
OMe
R2
N
N
N
H
R2
N
R3
N
N
H
R1
R2
R3
ACK1 Kia
(l
M)
ACK1 cell IC50 (lM)
b
Compound
2
6
7
8
9
10
11
12
13
14
15
16
17
18
19
H
H
H
H
F
Me
H
H
H
H
H
H
H
F
Me
H
Me
Cl
Me
Me
Cl
Me
Me
Me
Cl
4-Piperazin-1-yl
H
H
H
H
H
4-Piperazin-1-yl
4-(4-Methylpiperazin-1-yl)
4-O(CH2)2N(CH2)4
3-F, 4-O(CH2)2N(CH2)4
3-F, 4-O(CH2)2N(CH2)4
3-F, 4-O(CH2)3N(CH2)5
4-(4-Methylpiperazin-1-yl)
4-(4-Methylpiperazin-1-yl)
4-(4-Methylpiperazin-1-yl)
0.002
>4
0.003
0.007
0.026
>2.5
0.012
0.002
0.003
0.003
0.002
0.003
0.4
0.01
ND
0.02
0.04
5.5
ND
0.01
0.02
0.02
0.02
0.03
0.06
2.1
Me
H
Me
Me
0.018
0.012
0.02
0.05
Me
a
Values are means of at least two experiments.
Average of at least two IC50 values. ND, not determined.
b
Thr205 serve to anchor 2 into the binding pocket. In addition, the
dimethyl-phenyl group makes a deeper contact in the hydrophobic
selectivity pocket as compared to the amide series I. Moreover, two
regions of 2 are exposed to solvent. The polar piperazine moiety di-
rectly faces the solvent, creating a stabilizing interaction that aids
in inhibitor binding or recognition. Also, the terminus of the N-1
side chain is exposed to solvent by protruding through a lipophilic
channel present in the phosphate-binding region of the active site.
The latter observation served to guide subsequent SAR work: It
was hoped that binding could be improved by strengthening the
solubilizing interaction between the terminus of the N-1 side chain
and exposed solvent while maintaining a low overall molecular
weight by keeping the N-6 phenyl ring unsubstituted.
Table 2
Pharmacokinetic parameters following iv dose in male Sprague–Dawley rats.a,b
Compound
CL (L/h/kg)
Vss (L/kg)
MRT (h)
AUC (lgh/L)
A variety of N3-(aryl)-N6-(phenyl)-1H-pyrazolo[3,4-d]pyrimi-
dine-3,6-diamines possessing polar solubilizing groups linked to
N-1 by a one- to three-carbon spacer were synthesized and tested
(Table 3). While most of the cyclic amine derivatives 20–24 (two-
carbon spacer) were potent inhibitors of ACK1, inhibition was
weakened when an amide linker was employed (compounds 25
and 26). Non-cyclic amino groups attached to two- and three-car-
bon linkers were also investigated. While the N,N-dimethylamino-
ethyl derivative 27 exhibited slightly reduced cellular potency, the
N,N-dimethylaminopropyl derivatives 28 and 29 strongly inhibited
ACK1 (Ki of 5 nM and cellular IC50 of 20 nM for 28). Replacing the
dimethylamine with the more lipophilic diethylamine and pyrrol-
idine groups (compounds 30 and 31) led to reduced cellular activ-
ities compared to 29. Oxygenated carbon chains at R2 were also
investigated (Table 2, compounds 32–34). The diols 32 and 33
exhibited excellent biochemical and cellular potencies that were
comparable to those of 28 and 29. However, ACK1 inhibition was
weakened in converting the primary alcohol of 33 into a dimethyl-
amine (compound 34).
2
11
15
16
5.7
4.2
2.9
3.5
20.3
15.4
7.7
3.5
3.6
2.7
2
104
117
174
148
7.5
a
n = 3 animals per study.
Dosed at 0.5 mg/kg iv.
b
Compounds 20–24, 27, and 36–38 were each synthesized by a
five-step process from 3 and an appropriately mono-substituted
hydrazine10 as described above (Scheme 2). Compound 36 was fur-
ther transformed into either 25 or 26 by ester hydrolysis followed
by BOPCl-mediated amide formation (Scheme 3). The synthesis of
derivatives 28-34 is detailed in Scheme 4. Dihydroxylation of
either 37 or 38 with a mixture of OsO4 and NMO generated 32
and 33, respectively. Reductive amination of the aldehyde derived
from diol 32 or 33 produced 28–31, respectively. Compound 33
was transformed into 39 by selective tosylation of the primary
Figure 3. Actual high-resolution X-ray co-crystal structure of compound 2 bound to
the ACK1 kinase domain. RCSB file name (3EQR).

D. J. Kopecky et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6352–6356
6355
Table 3
H2N
R1
R2
ACK1 inhibition for N3-(aryl)-N6-(phenyl) pyrazolopyrimidine-3,6-diamines with
selected N-1 substituents R1.
O
O
O
N
a, b, c
HO
NH
N
H
N
R1
N
R2
R1
N
H
O
N
Cl
N
35
d, e
3
N
H
R1
N
N
N
H
Compound
R1
R2
ACK1 Kia
M)
ACK1 cell
IC50 (lM)
R1
N
R2
b
(
l
N
N
H
R1
N
7
Me
0.003
0.007
0.02
OMe
OMe
N
N
H
20-24, 27
8
20
21
22
23
Cl
0.04
0.03
0.03
0.07
0.05
36 R1=Cl, R2=CH2CO2Et
37 R1=Cl, R2=CH2CH2CH=CH2
38 R1=Me, R2=CH2CH2CH=CH2
N
Cl
0.01
0.05
0.05
0.03
N
Scheme 2. Reagents and conditions: (a) PCl5, POCl3, reflux, quantitative; (b)
Amberlyst A-21 resin, 2,6-di(R1)-aniline, EtOAc, 50 °C, 90%; (c) R2NHNH2, Et3N, THF,
55–85%; (d) PCl5, toluene, reflux, 50–80%; (e) TFA, aniline, 1,4-dioxane/n-BuOH or
1,4-dioxane, reflux, 55–90%.
OH
Cl
N
N
O
Me
Cl
OEt
N
R
O
O
Cl
Cl
N
N
N
N
a
N
N
H
N
H
Cl
Cl
N
CH2OH
N
N
H
N
N
H
36
25 R=NMe2
26 R=morpholin-1-yl
NMe2
24
Cl
0.01
0.02
N
N
Scheme 3. Reagents: (a) i—aq KOH, MeOH; ii—BOPCl, RH, Et3N, CH2Cl2, 90%.
25
26
27
28
29
30
31
Cl
0.03
0.07
0.005
0.005
0.03
0.04
0.1
0.09
0.14
0.14
0.02
0.04
0.14
0.3
O
alcohol followed by epoxide formation with NaOMe. Finally, 34
was obtained from 39 in low yield by exposure to dimethylamine
in ethanol.
The biological data in Table 3 show that structural manipulation
did not significantly affect ACK1 inhibitory activity (Table 1). How-
ever, a moderate improvement in PK properties was observed for a
key analog. The male Sprague–Dawley rat pharmacokinetic profile
of 32, one of the most potent analogs in Table 3, showed a signifi-
cant improvement in oral bioavailability relative to earlier analogs
(F = 30%; 4 mg/kg po dose).
Modulation of selectivity for ACK1 over other related kinases
was possible for this series by way of small structural changes.
As an example, compound 13 displayed good selectivity against
the related kinases KDR, Tie-2, and Jak3, but very potently inhibits
BTK and LCK, which possess high structural similarity to ACK1 (Ta-
ble 4). However, modification of the structure of 13 to include a
single fluorine atom on the N-6 phenyl ring (compound 14) elimi-
nated binding to KDR, Tie-2, and Jak3, while greatly diminishing
binding to both LCK and BTK relative to ACK1.
O
N
Cl
O
Cl
N
N
Cl
N
N
Me
Me
Me
N
In conclusion, we have demonstrated that N3,N6-diaryl-1H-
pyrazolo[3,4-d]pyrimidine-3,6-diamines represent a novel class
of ACK1 inhibitors. The presence of a polar substituent at C-4
of the N-6 aryl ring was shown to be unnecessary for maintain-
ing high levels of inhibitory activity. Furthermore, selectivity for
ACK1 over some related kinases could be achieved via minimal
structural modifications. Despite non-optimal pharmacokinetic
profiles, these highly potent analogs represent a promising new
lead series of ACK1 inhibitors which, with further study, may
prove useful for identifying compounds suitable for in vivo
experiments.
OH
32
33
Cl
0.01
0.02
OH
OH
OH
Me
Me
0.03
0.08
0.06
0.19
OH
N
34
a
Values are means of at least two experiments.
Average of at least two IC50 values.
b

6356
D. J. Kopecky et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6352–6356
OH
O
OH
R1
N
R1
N
a
c, d
N
N
N
N
N
H
N
H
N
H
R1
N
R1
N
N
N
N
H
N
N
H
N
N
H
37 R1=Cl
32 R1=Cl
39
38 R1=Me
33 R1=Me
b
e
R2
NMe2
OH
R1
N
N
N
N
H
N
R1
N
N
H
N
N
N
H
N
N
H
28 R1=Cl, R2=NMe2
29 R1=Me, R2=NMe2
34
30 R1=Me, R2=NE t
2
31 R1=Me, R2=N(CH2)4
Scheme 4. Reagents and conditions: (a) OsO4, NMO, acetone/H2O, 87%; (b) i—NaIO4, acetone/H2O, ii—R2HÁHCl, Et3N, NaBH(OAc)3, 1,2-dichloroethane, 51–52% (compounds 28
and 29) or R2H, AcOH, NaBH(OAc)3, 1,2-dichloroethane, 21–25% (compounds 30 and 31); (c) p-TsCl, pyridine, CH2Cl2, 64%; (d) NaOMe, CHCl3/MeOH, 0 °C, 88%; (e) MeNH2,
EtOH, 40 °C, 27%.
5. Unpublished results.
6. The Ki assay for ACK1 has been described in detail in: Xiao, S.-H.; Farrelly, F.;
Table 4
Kinase selectivity for compounds 1a, 13, and 14 (Ki, l
M).a
Anzola, J.; Crawford, D.; Jiao, J.X; Liu J.; Ayres, M.; Li, S.; Huang, L.; Sharma R.;
Kayser F.; Wesche, H.; Young, S.W. Anal. Biochem. 2007, 267, 179. Briefly,
histidine(6) tagged truncated ACK1 (aa 117–489) were over expressed and
purified to apparent homogeneity from SF9 cells using baculovirus expression
Kinase
1a
13
14
ACK1
LCK
BTK
KDR
Tie-2
Jak3
0.020
0.013
0.078
>25
>8.3
0.074
0.003
0.00004
0.00025
1.59
1.16
0.17
0.003
0.011
0.125
>12.5
>2.5
system.
A biotinylated peptide (AIRLKEEEI-YFFFFAKKK-amide) substrate is
phosphorylated by ACK1 in the presence of various concentrations of ATP. The
phosphorylated peptide is detected with electrochemiluminescence using
phospho-tyrosine-specific mAb-labeled with ruthenium (II) tribipyridine (BV-
TagTM), and streptavidin coated paramagnetic beads. Kis were generated by
fitting the dose response data to the Morrison equation.
>12.5
a
7. ACK1 cell-based assay IC50 determination: The assay relies upon the fact that
ACK1 is heavily autophosphorylated in proliferating, adherent cells, and upon
detachment ACK1 becomes de-phosphorylated very rapidly. When attached to
plastic, ACK1 auto-phosphorylation is detectable within minutes, and reaches a
maximum within 2 h. For this experiment, 293 cells stably expressing Flag-
tagged ACK1 are detached from their culture dish with PBS/EDTA. After a PBS
wash, cells are pre-incubated in suspension with DMSO or compound. Next, the
samples are either lysed directly (control) or allowed to re-attach in medium
containing 10% FCS on tissue culture dishes for 2 h prior to lysis. The inhibition
of ACK1 auto-phosphorylation is determined by anti p-Tyr WB after Flag IP.
8. (a) Snow, R. J.; Cardozo, M. G.; Morwick, T. M.; Busacca, C. A.; Dong, Y.; Eckner,
R. J.; Jacober, S.; Jakes, S.; Kapadia, S.; Lukas, S.; Panzenbeck, M.; Peet, G. W.;
Peterson, J. D.; Prokopowicz, A. S.; Sellati, R.; Tolbert, R. M.; Tschantz, M. A.;
Moss, N. J. Med. Chem. 2002, 45, 3394; (b) Goldberg, D. R.; Butz, T.; Cardozo, M.
G.; Eckner, R. J.; Hammach, A.; Huang, J.; Jakes, S.; Kapadia, S.; Kashem, M.;
Lukas, S.; Morwick, T. M.; Penzenbeck, M.; Patel, U.; Pav, S.; Peet, G. W.;
Peterson, J. D.; Prokopowicz, A. S.; Snow, R. J.; Sellati, R.; Takahashi, H.; Tan, J.;
Tschantz, M. A.; Wang, X.-J.; Wang, Y.; Wolak, J.; Xiong, P.; Moss, N. J. Med.
Chem. 2003, 46, 1337.
Values are means of at least two experiments.
Acknowledgments
We are grateful to our colleagues Merrill Ayres for ACK1 protein
expression, and George Tonn and Monica Sweany for generating PK
data.
References and notes
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9. All new compounds described herein were characterized by mass spectroscopy
and 1H NMR, and were determined to be of >95% purity by reverse-phase HPLC.
10. Monosubstituted hydrazines were synthesized by reacting hydrazine hydrate
with the appropriate alkyl halide in refluxing ethanol overnight followed by
distillation.
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11. For
a discussion of the relationship between physical properties with
successful drug development, see e.g.: Lipinski, C. A. J. Pharmacol. Toxicol.
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