Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood-brain barrier penetration

Article abstract of DOI:10.1016/j.bmc.2009.08.065

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood-brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.

Full text of DOI:10.1016/j.bmc.2009.08.065

Bioorganic & Medicinal Chemistry 17 (2009) 7593–7605
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effect of potential amine prodrugs of selective neuronal nitric oxide
synthase inhibitors on blood–brain barrier penetration
a
b
b
Richard B. Silverman a,b,c, , Graham R. Lawton , Hantamalala Ralay Ranaivo , Laura K. Chico ,
*
Jiwon Seo a, D. Martin Watterson b
a Department of Chemistry, Northwestern University, Evanston, IL 60208-3113, United States
b Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208-3113, United States
c Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208-3113, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric
oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the
charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide
and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a vari-
ety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and
as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary
amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS
applications.
Received 4 August 2009
Revised 27 August 2009
Accepted 29 August 2009
Available online 6 September 2009
Keywords:
Amine prodrug
Neuronal nitric oxide synthase inhibitor
Blood–brain barrier
Organic azide
Ó 2009 Elsevier Ltd. All rights reserved.
Prodrug
Azide prodrug
Amide prodrug
Carbamate prodrug
Neuronal nitric oxide synthase
1. Introduction
hydrogen bond donors and two positive charges; these character-
istics limit their ability to cross the BBB. We found we could lower
Central nervous system (CNS) disorders comprise the second
largest therapeutic area (behind cardiovascular disorders).1 These
diseases can be the most difficult to treat because drugs must pen-
etrate the blood–brain barrier (BBB) to be effective. The BBB pro-
tects the brain parenchyma from blood-borne agents, including
potential neurological therapeutics.2,3 In fact, it is estimated that
only about 2% of potential CNS compounds can penetrate the
BBB.4 The physicochemical requirements for a small molecule to
diffuse passively across the BBB have been well studied, and guide-
lines for these properties have been postulated.5 In general, the
BBB will not allow compounds to pass into the CNS that are highly
polar, have a large number of hydrogen bond donor groups, or that
carry several charges, unless they are specifically taken up by an
active transport system. This means that a great many potential
drugs with high efficacy in vitro have little or no activity in vivo.
In our search for selective inhibitors of neuronal nitric oxide
synthase (nNOS) we have designed compounds that possess high
in vitro activity, but at physiological pH they contain multiple
the number of hydrogen bond donors by one by substitution of an
oxygen atom for one of the secondary amino groups without a det-
rimental effect on potency;6 this resulted in only a small increase
in BBB penetration. To make a more substantial impact on BBB
penetration, we wished to eliminate one of the charges and in-
crease lipophilicity by various prodrug approaches. One approach
is by the substitution of an azido group for a primary amino group.
The potential benefits of this prodrug are that (1) the only by-prod-
uct of metabolic conversion to the drug would be molecular nitro-
gen; (2) it has a low molecular weight; (3) azides are easily
synthesized, and (4) azides are relatively inert in vivo.7 For the pur-
pose of CNS penetration, they are not charged, are nonpolar (rela-
tive to a primary amine), are not H-bond donors, do not add
rotatable bonds, and do not significantly affect the water solubility
of the molecule. However, they would only be suitable as prodrug
moieties if they become reduced to primary amines on a physio-
logically relevant time scale. There are numerous examples of com-
pounds that possess azido functional groups that are converted to
amines in vivo.8–13 For example, the azido group of the HIV drug
AZT is metabolized to an amine.12 With other nucleotide drugs,
amines have been masked as azides to prevent rapid deamination,
* Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491 7713.
E-mail address: Agman@chem.northwestern.edu (R.B. Silverman).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.065

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R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
and the azide is then enzymatically converted back to the amine.
Encouraged by these studies, we decided to investigate whether
an azide prodrug would be effective with our nNOS-selective
inhibitors.14 An azide version (1, Fig. 1) of one of our truncated
2-aminopyridine-based inhibitors (2)6 was chosen for the investi-
bromide to give ( )-15. Ozonolysis, followed by reduction of the
ozonide with NaBH4, gave alcohol ( )-16. The benzyl group was re-
moved (( )-17), and a Mitsunobu reaction using DPPA as a source
of azide was performed. A portion of the resulting azide (( )-18)
was reduced to amine 13. Both 18 and 19 were treated with acid
to remove the Boc groups and give 1 and 2, respectively.
The synthesis and activity of inhibitor 4 have been described
previously.15 Compound 3 was synthesized via the route shown
in Scheme 2. Activated carbonate 21 was synthesized as a racemic
mixture using 20 and mercuric pivalate.19 The nitrile group of 22
was reduced selectively with borane in THF. After Boc protection
of the resulting primary amine, the nitro group was reduced under
hydrogenation conditions to give 24. Coupling of Fmoc protected
nitroarginine to 24 proved troublesome, but could be achieved
by generating the acid fluoride using TFFH. Reduction of the amide
bond with borane gave 26. The Fmoc group was removed, and the
resulting primary amine was coupled to activated carbonate 21.
Removal of the Boc group with TFA gave 3 as a diastereomeric mix-
ture of salts.
gation. L
-Nitroarginine-based inhibitor 415 was synthesized with
a pivaloylethoxycarbamate prodrug moiety capping one of its pri-
mary amino groups (3) for comparative purposes and to determine
whether this type of prodrug is effective for CNS therapeutics.
A prodrug approach for our secondary amine selective nNOS
inhibitors, such as 5 (Fig. 2),16 also was attempted. Recent studies
on the ability of 5 to cross the BBB indicated that it does cross into
the brain, but only to a small degree.6 Compound 5 has a molecular
weight in the desirable range for bioavailability of a CNS drug
(372 Da), its number of hydrogen bond acceptors is favorable (5),
and its polar surface area (74) is in the appropriate range.5 The
pKa of the aminopyridine moiety is about 7, but that of the two sec-
ondary amines is about 9, indicating a total charge of +2 at physi-
ological pH. Also, at physiological pH, the two secondary amines
should be protonated, producing a total of 6 hydrogen bond do-
nors; most CNS drugs have fewer than four.5 Furthermore, the
log D, predicted to be approximately À0.1 for 5,6 is lower than
the desired range of 1–3 for a brain penetrant drug.17 Conse-
quently, we thought that a prodrug of 5, in which one of the sec-
ondary amino groups was neutralized as an amide or carbamate,
would be useful to enhance BBB penetration by lowering the total
charge to +1, by removing two hydrogen bond donors, and by rais-
ing the log D. A carbamate linkage is significantly less polar than an
amide bond;18 therefore, a carbamate should have more of an im-
pact on brain uptake, as the passive diffusion rate should be much
greater. It was anticipated that once the prodrug crossed the BBB, it
would be converted back to the dicationic form, which is needed
for potency and selectivity as a nNOS inhibitor.16 Three prodrug
analogues were synthesized (6–8, Fig. 2) for this purpose.
2.1.2. Metabolism of 1–4
Compounds 1 and 3 were incubated in fresh mouse plasma at
37 °C in the presence of a NADPH regenerating system, as azide
reduction is believed to be NADPH dependent. Compound 3 was
used mainly to show that the plasma was active. At certain time
intervals, the reactions were quenched, and the amount of prodrug
remaining was quantified by HPLC. Figure 3 shows the normalized
peak integrals for each compound. No significant metabolism of
azide 1 was observed over 80 min. This is to be expected as the
concentration of P450s in plasma is extremely low, and 1 should
be stable to hydrolysis. In contrast, the metabolism of 3 was rapid
because of the abundance of esterases in plasma. As a result of the
presence of a stereogenic center in the prodrug moiety, 3 is a mix-
ture of two diastereomers, which are separable by HPLC.
The two isomers (3A and 3B) are metabolized at different rates.
One diastereomer was metabolized with a half-life of approximately
10 min and was almost completely consumed by 80 min. The other
was hydrolyzed more slowly with a half-life of approximately
30 min and could still be quantified at the 80-min time point. The
peak in the HPLC corresponding to the parent compound (4), was
also analyzed (Fig. 4). A clear increase in peak area over time was
observed, indicating that the prodrug was indeed being converted
to the parent compound and that the mouse plasma was active.
Compounds 1 and 3 were incubated with fresh mouse brain
homogenate in phosphate buffered saline (pH 7.4) at 37 °C with
NADPH added in excess to investigate the extent of metabolism
in brain tissue. At various time points the reactions were stopped
by heating to 95 °C for 1 min. The mixtures were partially purified
by solid phase extraction (SPE), and the amount of compound was
quantified by HPLC (see Supplementary data). The amount of each
diastereomer of 3 decreased over the course of the experiment. As
in the case of the plasma metabolism, one of the diastereomers
was metabolized slightly faster than the other. However, the rate
of metabolism was not nearly as rapid as it was in plasma because
of the decreased abundance of esterases. The concentration of 1 did
not decrease over the course of the experiment, indicating that no
significant metabolism occurred to the azide.
2. Results
2.1. Azide prodrug (1)
2.1.1. Chemistry
Azide 1 was synthesized using chemistry described previously
up to ( )-15 (Scheme 1).6 Boc protected 2-amino-4,6-dimethylpyr-
idine 9 was deprotonated with n-BuLi and used to open epoxide
10. The carbamate group of the resulting alcohol (( )-11) was pro-
tected with a benzyl group (( )-12), and then a Mitsunobu reaction
was performed to install the oxygen with cis stereochemistry (( )-
13). The ester was hydrolyzed, and the resulting alcohol (( )-14)
was deprotonated with sodium hydride and alkylated with allyl
H
H
N
N
H2N
N
NH2
H2N
N
N3
O
O
( )-2
( )-1
HN
NHNO2
Since no azide reduction was observed in the brain homogenate
experiment, a simpler system with an abundance of reducing en-
zymes was investigated. Microsomes, a concentrated mixture of li-
ver enzymes rich in cytochrome P450s, were added to the
compounds at several concentrations, and the mixtures were incu-
bated in the presence of excess NADPH. At certain time points, the
reactions were stopped by the addition of acetonitrile. After centri-
fugation, the supernatant was analyzed by HPLC. Typically, micro-
somes tend to metabolize compounds very rapidly under these
HN
NHNO2
NH
NH
NH2
NH2
O
O
H
H
N
N
O
O
N
H
H2N
4
3
Figure 1. Primary amine prodrug analogues.

R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
7595
H
N
H
N
CH3
O
H
F
F
N
N
O
( )-5
O
N
N
H2N
H2N
( )-6
H
N
H
N
OCH3
O
O
O
F
F
N
N
O
( )-7
O
N
N
H2N
H2N
( )-8
Figure 2. Structure of nNOS inhibitor 5, and analogs 6–8, designed to test whether removing a charge and increasing lipophilicity leads to greater BBB penetration.
Boc
Boc
N
N
v
i
iii
BocN
Ph
N
BocN
R
N
BocHN
N
OR
OH
( )-13, R = Ac
( )-14, R = H
9
iv
( )-11, R = H
( )-12, R = Bn
ii
Boc
N
Boc
N
viii
vi
BocN
Ph
N
BocN
R
N
OH
O
O
( )-15
( )-16, R = Bn
( )-17, R = H
vii
Boc
Boc
N
N
vii
BocHN
N
BocHN
N
N3
NH2
O
O
Boc
N
( )-19
( )-18
ix
ix
O
10
H
H
N
N
H2N
N
H2N
N
N3
NH2
O
O
( )-1
( )-2
Scheme 1. Reagents and conditions: (i) n-BuLi, THF,À78 °C tort, 30 min, then 10, À78 °C to rt, 4 h; (ii) NaH, DMF, 0 °C, 30 min, then BnBr, rt, 16 h; (iii) PPh3, DIAD, AcOH, THF,
rt, 16 h; (iv) 1 N NaOH (aq), MeOH, rt, 16 h; (v) NaH, DMF, 0 °C, 30 min, then allylbromide; (vi) O3, MeOH, À78 °C, 1 h, then NaBH4, rt, 3 h; (vii) H2, Pd/C, MeOH, 2 d; (viii)
PPh3, DIAD, DPPA, THF, rt, 16 h; (ix) 4 N HCI, dioxanes, rt, 16 h.
conditions, and it was expected that the azide would be degraded
over time. It would then be necessary to prove whether the degra-
dation was due to reduction of the azide to the amine, or due to
some other type of metabolism. The experiment was performed
in parallel with a control, minaprine, a prescription drug that is
known to be rapidly metabolized under similar assay conditions,
to determine whether the microsomes contained active P450s. As
expected, the diastereomers of 3 were metabolized over time at
different rates (See Supplementary data). A peak corresponding
to the active compound (4) did grow in, but it was difficult to quan-
tify, as it was small and overlapped with other peaks. Because the
concentration of esterases in the mixture is not very high, and
microsomes denature fairly rapidly at 37 °C, 3 was not fully de-
graded, and the concentration of prodrug remaining leveled off
after 20 min. The minaprine was rapidly degraded, confirming
the P450 activity of the microsome mixture. However, no signifi-
cant loss of the azide was observed. It appears that this compound
is stable to P450 metabolism.
Because in vitro azide reduction by P450s is inhibited by oxy-
gen,11 the incubation was repeated with increasingly stringent
anaerobic conditions, ultimately using vials with rubber septa un-
der a balloon of nitrogen with the reaction run in deoxygenated
buffer. However, although minaprine was metabolized readily, no
statistically significant loss in azide was detected. Mass spectral
analysis of the reaction mixtures showed no peak corresponding
to the amine. The acyloxyalkylcarbamate prodrug (3) was cleaved
relatively quickly in plasma, but more slowly in brain homogenate
and with liver microsomes (data not shown).
2.2. Amido and carbamate prodrugs 6–8
2.2.1. Chemistry
Three analogues of 5 were synthesized (6–8) to determine the
effect of neutralization of one of the amino groups. The N-acetyl
analogue (6) was synthesized in a straightforward way starting
from ( )-283 as shown in Scheme 3.

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R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
Cl
i
O
O
O
O
O
O
O
O
O2N
O2N
( )-21
( )-20
HN
NHNO2
NH
NO2
NO2
NH2
NHBoc
ii
iv
iii
NHBoc
NHBoc
CN
H
N
FmocHN
22
24
23
O
25
HN
NHNO2
NH
HN
NHNO2
NH
NHBoc
NHBoc
v
vi
vii
3
O
O
(mixture of
diastereomers)
H
H
N
N
O
O
FmocHN
N
H
27
26
Scheme 2. Reagents and conditions: (i) Hg((BuCOO)2, pivalic acid, 80° C; (ii) BH3–THF, then Boc2O, p-dioxane, 10% NaHCO3; (iii) H2, Pd/C, MeOH/H2O/AcOH; (v) Fmoc–
Arg(NO2)–OH, TFFH, HOAt, CH2Cl2/aq NaHCO3; (v) BH3–THF, À10 °C; (vi) 20% piperidine, DMF, then 21, DIEA, CH3CN; (vii) TFA, CH2Cl2.
Plasma Metabolism of 1 and 3
The two carbamate prodrugs (7 and 8) were synthesized by the
routes in Scheme 4.
1.2
1
2.2.2. Metabolism of 5–8
0.8
0.6
0.4
0.2
0
Compound 5 was treated with rat liver microsomes in the pres-
ence and absence of NADPH, to determine whether any metabo-
lism observed was NADPH dependent. The results are shown in
Figure 5.
Clearly, there is some degree of metabolism under both sets of
conditions, suggesting that the metabolism is not NADPH depen-
dent. However, the amount of compound lost is relatively small;
approximately 20% is lost over 30 min. Given that the microsomes
are concentrated in enzymes, this equates to reasonable meta-
bolic stability. The background metabolism of 5 should not inter-
fere with possible prodrug conversion studies or BBB penetration
studies, especially since the compounds will not be administered
orally.
3A
3B
1
0
20
40
60
80
100
Incubation time (min)
Figure 3. Incubation of 1 and 3 in fresh mouse plasma. The area under the peak was
integrated and the value was normalized based on the integration at 0 min. Two
diastereoisomers of 3 are shown separately (3A and 3B).
Compounds 6–8 were compared to 5 for metabolic stability in
the same assay. All were comparably stable; greater than 80%
remained after a 40-min incubation at 37 °C. Compound 8 was less
stable, possibly the result of oxidation of the benzyl group. None-
theless, greater than 60% of 8 remained after a 40-min incubation
(See Supplementary data).
Formation of 4 during incubation of 3 in plasma
8
7
6
5
4
3
2
1
0
2.2.3. Brain penetration of 5–8
A mouse brain uptake study of 5 was carried out using LC–MS/
MS detection, which is approximately 1000 times more sensitive
than HPLC with UV detection and gives reliable and consistent
quantification. The internal standard selected was 35, as it is a
structural isomer of 5, yet elutes differently by LC. A known
amount of the internal standard was added prior to all extractions,
and the final quantification was performed as a ratio of the com-
pound of interest to the internal standard. Standard curves for
the concentration of 5 in plasma and in brain are shown in Supple-
mentary data.
0
20
40
60
80
100
Incubation time (min)
Figure 4. Formation of compound 4 from prodrug 3 during incubation in fresh
mouse plasma (not normalized).

R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
7597
Boc
N
Boc
N
O
i
H
iii
BocN
Bn
N
( )-6
N
F
BocN
R
N
N
F
O
O
( )-28
( )-29, R = Bn
( )-30, R = H
ii
Scheme 3. Reagents and conditions: (i) Ac2O, MeOH, rt, 1 h; (ii) H2, Pd(OH)2/C, MeOH, 60 °C, 1–2 d; (iii) 4 N HCl, dioxanes, rt 16 h.
Boc
N
Boc
N
O
OMe
i
ii
( )-7
H
BocHN
N
BocHN
N
N
F
N
F
O
O
( )-31
( )-32
Boc
N
Boc
N
O
OBn
iii
ii
H
( )-8
H2N
N
N
F
H2N
N
N
F
O
O
( )-33
( )-34
Scheme 4. Reagents and conditions: (i) CICOOMe, MeOH, rt, 4 h; (ii) 4 N HCl, dioxanes, rt 16 h; (iii) CICOOBn, MeOH, rt 4 h.
Metabolism of 5 by microsomes
Plasma concentration of 5
1.2
1
35
30
25
20
15
10
5
5 ox
5 red
minaprine
0.8
0.6
0.4
0.2
0
0
0
5
10
15
20
25
Time after administration (min)
0
10
20
30
40
50
Figure 6. Concentration of 5 in plasma after an ip dose of 3.7 mg/kg. Error bars
show standard error mean.
Incubation time (min)
Figure 5. Metabolic stability of 5 with microsomes at 37 °C. The experiment was
performed in the presence (5 red) and absence (5 ox) of additional NADPH.
Minaprine concentration at 20 min was >0 but was not quantifiable.
converted to lM for display to allow a straightforward comparison
between compounds. The conversion for calculating the concentra-
tion in the brain assumed that the brain density is 1.0 g/mL. The
density of brain may be greater than this, which would mean that
the real concentrations would be higher than shown.
H
N
A brain uptake study of 6 was carried out in mice (Figs. 8 and 7).
The blood profile of 6 does not match that of 5 in that the plasma
concentration appears to increase continually up for at least
20 min. This could mean that 6 is taking longer to diffuse across
the lining of the peritoneal cavity into the blood stream.
The brain concentration of 6 (Fig. 7) peaks at 5 min and then
drops, suggesting that the compound might be a substrate for ef-
flux pumps that are actively removing the compound from the
brain. Analysis of the brain samples showed no presence of 5, indi-
cating that the acetyl group was not cleaved during the 20-min
experiment.
F
O
N
H
N
H2N
( )-35
Six mice were given 5 by intraperitoneal (ip) injection at
3.7 mg/kg, a concentration at which no toxicity was observed.
Three mice were sacrificed 5 min after administration; the other
three were sacrificed 10 min after administration. Blood and brain
samples were taken from each mouse, and the compounds were
extracted. At a later date, three more mice were given 5 and sacri-
ficed 20 min after administration. The plasma data are shown in
Figure 6 and the brain data are in Figure 7. The amount of 5 was
Comparing the brain penetration curves of 5 and 6 (Fig. 7) gives
a surprising result. The concentration of 6 at 5 min is comparable
or slightly higher than that of 5, but drops to a level that is below
that of 5 by the 10-min time point, and so the AUC for each com-
calculated in
lg/mL of plasma, and lg/g of brain. This value was

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R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
Brain Concentrations of 5 and 6
Brain Concentrations of 5 and 7
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
0.14
0.12
0.1
5
6
5
7
0.08
0.06
0.04
0.02
0
0
5
10
15
20
25
0
5
10
15
20
25
Time after administration (min)
Time after administration (min)
Figure 9. Comparison of the brain concentrations of 5 and 7 after a dose of 3.7 and
Figure 7. Comparison of the brain concentrations of 5 and 6 after administration of
4.3 mg/kg, respectively. Error bars show the standard error mean.
3.7 mg/kg and 4.1 mg/kg respectively. Error bars show standard error mean.
that for 7. The levels at 5 min are extremely low, but are steadily
increasing throughout the course of the experiment. However,
even by the 20-min time point, the concentration of 8 is still far be-
low that of 5 (Fig. 10), and the AUC over the 20-min experiment is
much lower.
In the case of carbamates 7 and 8, the prodrug modification is
detrimental to brain uptake over the first 20 min. The brain levels
for both compounds increase, whereas those for 5 are beginning to
decrease, and so over the course of a longer uptake study the con-
centration of carbamate may eventually exceed that of 5. This is
not encouraging for the use of (acyloxy)alkyl carbamate prodrugs
such as 3, as these would be hydrolyzed before they could offer
any advantage for brain uptake.
A comparison of the in vivo concentrations of all of the com-
pounds tested, as well as 36,16 the amino analogue of 5, allows
some conclusions to be drawn. Figure 11 shows the plasma levels
of all of the compounds used in this study. For clarity, the error
bars have been omitted, and the 5-min point for 5 was omitted,
as it is much higher than the others.
Plasma Concentration of 6
10
9
8
7
6
5
4
3
2
1
0
0
5
10
15
20
25
Time after administration (min)
Figure 8. Concentration of 6 in plasma after a dose of 4.1 mg/kg. Error bars show
standard error mean.
pound over the 20-min experiment is roughly the same. As the two
compounds were administered at the same dose, this indicates
that the acetylation of 5 to give 6 results in no increase in brain up-
take. It was predicted that the elimination of one positive charge
would have a significant positive effect on brain uptake, but this
is not the case with 6.
A brain uptake study of the more lipophilic methyl carbamate 7
at 4.3 mg/kg was carried out using the same procedure as for 6
(Fig. 9). The plasma (see Supplementary data) and brain levels were
calculated by LC–MS/MS. Plasma levels of 7 peak at 5 min and de-
cline slowly over the next 15 min. The brain levels of 7 are very low
at 5–10 min but continue to rise at 20 min and might peak at a
higher level. These data seem to suggest that the rate at which 7
crosses the BBB is slow. Although the brain levels have not peaked,
it is unlikely that they will reach a level that is significantly higher
than that of 5 (Fig. 9). The AUC up to 20 min for 5 was much greater
than that for 7. This implies that for overall brain uptake the
methyl carbamate modification is even less effective than
acetylation.
H
N
H
F
N
N
H
N
H2N
36
Brain concentrations of 5 and 8
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
8
5
For the brain uptake study with 8, compound 7 was used as the
internal standard, as these two compounds are more similar than 8
and 35, and the chromatography was therefore more reliable. Com-
pound 8 was administered to mice at a dose of 5.1 mg/kg. Two
mice were sacrificed at each time point, and the plasma and brain
samples were analyzed by LC–MS (see Supplementary data). The
plasma concentration curve for 8 is similar to that for 7, peaking
0
5
10
15
20
25
Time after administration (min)
at around 1
lM and declining slowly over the course of the exper-
Figure 10. Comparison of the brain concentrations of 5 and 8 after a dose of 3.7 and
iment. The curve for the brain concentration of 8 also resembles
5.1 mg/kg, respectively. Error bars show the standard error mean.

R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
7599
Table 1
Comparison of plasma levels
Average concentrations of compounds 10 min after administration of an intraperi-
9
8
7
6
5
4
3
2
1
0
toneal dose of 10
lmol/g of compounds 5–8, and 36
Compound
Log D7.4
Plasma (
l
M)
Brain (nM)
Brain–blood
5
6
7
8
À1.46
0.01
0.81
2.65
À2.36
1.6 0.2
4.6 1.3
1.21 0.05
0.49 0.025
1.6 0.2
73
39
18
9
7
1
1
1
2
0.046
0.008
0.015
0.018
0.024
5
6
7
8
36
39
36
Errors are standard error mean.
3. Discussion
0
5
10
15
20
25
Time after administration (min)
Initially, thegoalof thisresearch wasto designprodrugsforselec-
tive nNOS inhibitors to allow for enhanced penetration into the
brain. When it became apparent that the prodrug moieties selected
were more stable than expected, the new goal was to determine if
enhancing the lipophilicity and lowering the charge of the molecules
would have a beneficial effect on BBB penetration. If so, other pro-
drug approaches could be considered. We were surprised to find that
making these changes had no effect on BBB penetration.
Ideally, a prodrug designed to increase brain uptake should be
stable in plasma and the liver, but become rapidly metabolized
in the brain. In reality, this is very difficult to achieve, as there
are few enzymes specific to the brain that could be used to convert
a prodrug to a drug. Azides are stable in plasma, but the rate of
reduction of 1 in brain appears to be slow or nonexistent. Although
there are reports of in vivo azide reduction activating azide pro-
drugs, in our hands, there was no evidence that this occurred with
1, suggesting that, at best, azides can be used as a prodrug only in
limited cases. If azide reduction is extremely sensitive to oxygen,
this further limits its use as a CNS prodrug moiety, as the brain is
well supplied with oxygenated blood.
Surprisingly, prodrug analogues of 5, namely 6–8, also exhibited
little metabolism by plasma or microsomes. Despite the observa-
tion that 1 and 6–8 were not converted back to their parent com-
pounds, it was important to determine what effect decreasing
charge and increasing lipophilicity had on BBB penetration, the ini-
tial purpose for selecting those prodrug moieties.
It was anticipated that neutralization of the secondary amine of
5 to give an acetylated prodrug (6) would increase passive diffu-
sion from the peritoneal cavity into the blood, leading to higher
plasma levels. This appears to be the case. However, it was also
anticipated that 6 would diffuse more readily across the BBB and
that the brain concentration would be higher than that of 5. In fact,
the brain concentration of 6 is lower than that of 5 and also of 36,
whose log D is much more negative. Even more surprisingly,
increasing the lipophilicity of 5 further as methyl- and benzyl car-
bamates to give 7 and 8, respectively (see Table 1), leads to a fur-
ther lowering of total brain concentration.
Figure 11. Comparison of the plasma levels of 5, 6, 7, 8, 36 after ip injection.
The two carbamates (7 and 8) behave similarly in that their lev-
els are fairly constant over 20 min but are lower than those of 5
and 36 (except at the 20-min time point). This may be caused by
a low rate of diffusion from the peritoneal cavity into the blood
or that these compounds bind to more proteins and cellular com-
ponents than 5 so that their free plasma concentrations are lower.
The compound that displays a different profile is 6, the acetylated
compound. In the case of this compound, the plasma levels are
higher than the other compounds and are actually increasing with
time. It could be that although this compound is diffusing slowly
into the blood, it is ultimately more bioavailable than the other
compounds.
A comparison of the brain levels of each compound is shown in
Figure 12. It is apparent that over the course of the 20-min exper-
iment there is not much difference among the overall brain con-
centrations of the compounds tested. As noted earlier,6 brain
uptake by 5 is greater than that for 36, presumably because of
the one less hydrogen bond donor in 5. Compounds 5 and 6 have
a similar AUC, indicating similar overall brain penetration. Surpris-
ingly, the brain concentrations of the two carbamates (7 and 8) are
less than the other compounds. Even though their plasma levels
were lower, it was expected that the brain levels would be higher,
as the compounds are more lipophilic (see Table 1 for calculated
log D values) and should diffuse across the BBB more readily. A
summary of the brain penetration results for compounds 5–8 is
shown in Table 1.
Comparison of Brain Levels
0.1
0.09
0.08
The simple hypothesis that neutralization of a secondary amine
and enhancement of lipophilicity will increase BBB penetration is
clearly not universal. There are more factors that must be consid-
ered when attempting to increase BBB penetration. From a molec-
ular properties standpoint, the addition of lipophilic functionality,
such as carbamates, adds molecular weight and hydrogen bond
acceptors. The carbamates 7 and 8 also have additional rotatable
bonds. Nonetheless, it was anticipated that the significant increase
in log D relative to 5 and 36 would overcome these problems. This
did not occur.
An alternative explanation for these results is that the increase
in the log D may be responsible for producing undesirable results.
For example, there could be increased non-specific binding to pro-
teins and cellular contents.20 This would reduce the free concentra-
tion in plasma so that less compound is available to diffuse across
0.07
5
6
7
8
0.06
0.05
0.04
0.03
0.02
0.01
0
36
0
5
10
15
20
25
Time after administration (min)
Figure 12. Comparison of brain concentrations of 5, 6, 7, 8, 36 after ip injection.

7600
R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
the BBB. Additionally, an increase in the number of nitrogen or
oxygen atoms and an increase in the molecular weight favor better
binding to efflux pumps.21 Given that similar results were obtained
with the parent amine as with the amide and carbamate prodrug
analogues, possibly the polarities of amide and carbamate groups
are sufficiently great to offset the decrease in charge by acylation
or carbamoylation.
0.2 mL/min. All water used was Milli-Q water obtained using a Bio-
cel A10 water purification system from Millipore Corporation (Bed-
ford, MA). Solid phase extraction was carried out using Waters
Oasis HLB or MCX 1 cc cartridges or MCX micro-elution plates
(Waters Chromatography, Milford, MA). Analysis of biological sam-
ples was carried out using an API 3000 liquid chromatography–
tandem mass spectrometry system (Applied Biosystems, Foster
City, CA) equipped with an Agilent 1100 series HPLC system (Agi-
lent Technologies, Wilmington, DE). Sample concentration was
performed in a Genevac EZ-2plus (Genevac Inc., Valley Cottage,
NY). NADPH regeneration solutions and rat liver microsomes were
purchased from Bectin-Dickinson.
4. Conclusions
In conclusion, two prodrug approaches were taken to enhance
BBB penetration of amine-containing nNOS inhibitors. The use of
an azido group (1) as a prodrug for the primary amino group of 2
was not effective because of the inability of a brain homogenate
or of liver microsomes to reduce the azide to the amine. The poor
brain uptake properties of nNOS inhibitor 5 were attributed to its
basic secondary amino groups that are charged at physiological
pH. It was thought that neutralization of one of the secondary
amines would lead to an increase in brain penetration. Acetylated
and carbamoylated prodrug analogues of 5 having increased lipo-
philicity and lower charge, surprisingly, caused a decrease in BBB
penetration. These results suggest that late stage optimization of
an amine-containing lead by conversion to an acetylated or carba-
moylated prodrug may not necessarily result in increased BBB
penetration.
5.2. tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-4-
methylpyridin-2-yl)methyl)-4-(2-hydroxyethoxy)-pyrrol-idine-
1-carboxylate (( )-16)
A solution of ( )-156 (220 mg, 0.41 mmol) in methanol (5 mL)
was cooled to À78 °C. Ozone was bubbled through the solution
for 1 h. NaBH4 (57 mg, 1.5 mmol) was added, and the mixture
was allowed to warm to room temperature. The solution was
poured into saturated NH4Cl and extracted with ethyl acetate
(3 Â 15 mL). The organic layers were combined, dried over anhy-
drous Na2SO4, and concentrated in vacuo. The crude product was
purified using flash column chromatography (silica gel, ethyl ace-
tate/hexanes, 1:1) to afford ( )-16 as an oil (133 mg, 0.246 mmol,
60%). 1H NMR (500 MHz, CDCl3) d 7.34 (m, 1H), 7.24 (m, 5H),
6.72 (s, 1H), 5.14 (s, 2H), 3.69–3.43 (m, 5H), 3.26–3.11 (m, 3H),
2.97 (m, 1H), 2.72 (m, 1H), 2.56 (m, 2H), 2.30 (s, 3H), 1.46 (s,
9H), 1.42 (s, 9H); 13C NMR (125 MHz, CDCl3) d 158.2, 155.0,
154.7, 154.2, 149.1, 139.9, 128.3, 127.3, 127.2, 126.8, 120.5,
118.0, 81.4, 79.5, 78.5, 70.6, 61.9, (51.1 + 50.7), 50.5, (49.6 +
49.1), (43.4 + 42.8), 34.6, 28.8, 28.4, 21.3; ESMS m/z = 542 (M+H)+.
5. Experimental section
5.1. General methods
Proton nuclear magnetic resonances (1H NMR) were recorded in
deuterated solvents on a Mercury 400 (400 MHz) or a Varian Inova
500 (500 MHz) spectrometer. Chemical shifts are reported in parts
per million (ppm, d) relative to tetramethylsilane (d 0.00). 1H NMR
splitting patterns are designated as singlet (s), doublet (d), triplet
(t), quartet (q). Splitting patterns that could not be interpreted or
easily visualized were recorded as multiplet (m) or broad (br). Cou-
pling constants are reported in hertz (Hz). Proton-decoupled car-
5.3. tert-Butyl 3-((6-(tert-butoxycarbonylamino)-4-methyl-
pyridin-2-yl)methyl)-4-(2-hydroxyethoxy)-pyrrolidine-1-
carboxylate (( )-17)
To a solution of ( )-16 (133 mg, 0.246 mmol) in ethanol (5 mL)
was added Pd(OH)2/C (ꢀ10 mg). The mixture was stirred under a
hydrogen atmosphere at 60 °C for two days. The mixture was fil-
tered through Celite, and the solvent was removed in vacuo. The
crude product was purified using flash column chromatography
(silica gel, ethyl acetate/hexanes, 3:2) to afford ( )-17 as an oil
(41 mg, 0.091 mmol, 37%). 1H NMR (500 MHz, CDCl3) d 8.07 (m,
1H), 7.72 (s, 1H), 6.66 (m, 1H), 5.59 (br, 1H), 3.84–3.51 (m, 6H),
3.33 (m, 1H), 3.19 (m, 2H), 2.64 (m, 1H), 2.31 (m, 4H), 1.52 (s,
9H), 1.46 (m, 9H); 13C NMR (125 MHz, CDCl3) d 158.2, 155.0,
154.8, 153.0, 152.2, 150.9, 119.0, 110.9, 81.0, 79.6, 78.2, 70.4,
61.0, (51.0 + 50.6), (49.7 + 49.3), (45.7 + 45.0), 34.2, 28.8, 28.5,
21.6; ESMS m/z = 452 (M+H)+.
bon
( a Mercury 400
13C NMR) spectra were recorded on
(100 MHz) or a Varian Inova 500 (125 MHz) spectrometer and
are reported in ppm using the solvent as an internal standard
(CDCl3, d 77.23). NMR spectra recorded in D2O were not normal-
ized. In many cases, the presence of rotamers made the NMR spec-
tra complex. In the case of two peaks that are clearly a pair of
rotamers, but are too far apart for an average to accurately repre-
sent the spectrum, the pair is written enclosed in parentheses.
Electrospray mass spectra (ESMS) were obtained using an LCQ-
Advantage with methanol as the solvent in positive ion mode, un-
less otherwise stated. For most compounds, 1H and 13C NMR and
ESMS data are presented.
All chemical reagents were purchased from Aldrich and were
used without further purification unless stated otherwise. NADPH,
calmodulin, and human ferrous hemoglobin were also obtained
from Sigma–Aldrich. Tetrahydrobiopterin (H4B) was purchased
from Alexis Biochemicals. HEPES, DTT, and some conventional or-
ganic solvents were purchased from Fisher Scientific.
5.4. tert-Butyl 3-(2-azidoethoxy)-4-((6-(tert-butoxy-carb-onyl-
amino)-4-methylpyridin-2-yl)methyl)-pyrrol-idine-1-carb-
oxylate (( )-18)
To a solution of ( )-17 (41 mg, 0.091 mmol) in anhydrous THF
Tetrahydrofuran (THF) was distilled from sodium and benzo-
phenone prior to use. Methylene chloride (CH2Cl2) was distilled
from calcium hydride prior to use, if dry solvent was required.
Dimethylformamide (DMF) was purchased as an anhydrous sol-
vent and used directly.
Purity of compounds and bioanalytical analyses (e.g., for micro-
some assay) were determined on a Dionex HPLC system (Dionex,
Sunnyvale, CA) using a Phenomenex (Torrance, CA) Luna C18 col-
(5 mL) were added PPh3 (26 mg, 0.1 mmol), DPPA (26
lL,
0.12 mmol) and DIAD (21 L, 0.11 mmol). The mixture was stirred
l
for 16 h. The solvent was removed in vacuo and the crude product
was purified using flash column chromatography (silica gel, ethyl
acetate/hexanes, 1:3) to afford ( )-18 as an oil (34 mg, 0.072 mmol,
79%). 1H NMR (500 MHz, CDCl3) d 7.60 (m, 1H), 7.17 (s, 1H), 6.69
(m, 1H), 3.99–3.63 (m, 3H), 3.53–3.28 (m, 5H), 3.15 (m, 1H), 2.95
(m, 1H), 2.76–2.60 (m, 2H), 2.31 (m, 3H), 1.52 (s, 9H), 1.45 (s,
9H); 13C NMR (125 MHz, CDCl3) d 158.4, 155.1, 152.7, 151.6,
umn (250 Â 2.0 mm; 5
lM) and guard column with a flow rate of

R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
7601
150.2, 119.6, 110.4, (81.1 + 80.3), (79.6 + 79.2), 70.3, (68.6 + 68.4),
51.3, (50.9 + 50.4), (43.5 + 42.8), (34.9 + 34.7), (28.8 + 28.5), 22.2,
21.6; ESMS m/z = 477 (M+H)+.
obtained (619 mg, 74%); mp 235.5–236.2 °C. A mixture of mercuric
trimethylacetate (1.64 g, 4.07 mmol), 20 (1.00 g, 4.07 mmol), and
trimethylacetic acid (13.5 g) was heated at 80 °C in an oil bath
overnight. The reaction mixture was cooled down, dissolved in
ether (50 mL), and filtered. The filtrate was washed several times
with 1% NaHCO3 (30 mL) to remove the trimethylacetic acid com-
pletely. The organic layer was concentrated in vacuo, and the res-
idue was purified by flash column chromatography (hexane/
EtOAc = 7:1, Rf = 0.30) to afford 21 (1.33 g, 91%) as yellowish crys-
tals; mp 53.9–54.3 °C. 1H NMR (500 MHz, CDCl3) d 8.30 (d,
J = 9.0 Hz, 2H), 7.41 (d, J = 9.5 Hz, 2H), 6.83 (q, J = 5.5 Hz, 1H),
1.63 (d, J = 5.5 Hz, 3H), 1.246 (s, 9H); 13C NMR (125 MHz, CDCl3)
d 176.6, 155.4, 150.7, 145.7, 125.5, 122.0, 92.7, 38.9, 27.0, 19.5.
5.5. tert-Butyl 3-(2-aminoethoxy)-4-((6-(tert-butoxy-carbonyl-
amino)-4-methylpyridin-2-yl)methyl)-pyrrolidine-1-
carboxylate (( )-19)
To a solution of ( )-18 (14 mg, 0.03 mmol) in ethanol (5 mL)
was added Pd/C (ꢀ10 mg). The mixture was stirred under a hydro-
gen atmosphere for 16 h. The mixture was filtered through Celite
and the solvent was removed in vacuo. The crude product was
purified using flash column chromatography (silica gel, ethyl ace-
tate/methanol, 9:1) to afford ( )-19 as a colorless oil (11 mg,
0.025 mmol, 83%). 1H NMR (500 MHz, CDCl3) d 7.55 (s, 1H), 7.20
(s, 1H), 6.58 (s, 1H), 3.73 (m, 1H), 3.58–3.20 (m, 6H), 3.08 (m,
1H), 2.82 (m, 2H), 2.61 (m, 1H), 2.46 (m, 1H), 2.22 (s, 3H), 1.45
(s, 9H), 1.39 (s, 9H); ESMS m/z = 451 (M+H)+.
5.8.3. [2-(2-Nitrophenyl)ethyl]carbamic acid tert-butyl ester
(23)
To 2-nitrophenylacetonitrile (22, 2.25 g, 13.9 mmol) was added
dropwise a BH3–THF solution (1.0 M, 100 mL) at 0 °C, and the mix-
ture was stirred at room temperature overnight under nitrogen.
The reaction mixture was cooled down to 0 °C, and an ice-cold
solution of 6 N HCl (60 mL) was added carefully, giving a white
precipitate. After evaporation of the THF, the aqueous phase was
washed with CH2Cl2, basified with 4 N NaOH to pH 10, and ex-
tracted twice with EtOAc. The organic phase was washed with
brine and dried over Na2SO4. After evaporation, the residue was
dried overnight to provide a brown oil (1.90 g, 11.4 mmol, 82%),
which was dissolved in a mixture of 1,4-dioxane (20 mL) and
10% aq NaHCO3 (5 mL). To this solution was added Boc2O (3.74 g,
17.2 mmol) in 1,4-dioxane (5 mL) dropwise at 0 °C. After being stir-
red for 24 h at room temperature, the reaction mixture was parti-
tioned between EtOAc (30 mL) and water (30 mL). The organic
layer was washed with brine, dried over Na2SO4, and concentrated.
The residue was purified by flash column chromatography (hex-
ane/EtOAc = 4:1, Rf = 0.35) to afford 23 (2.79 g, 92%) as a brown
oil. 1H NMR (500 MHz, CDCl3) d 7.86 (d, J = 7.5 Hz, 1H), 7.49 (t,
J = 7.0 Hz, 1H), 7.34 (m, 2H), 4.93 (s, 1H), 3.40 (q, J = 6.5 Hz, 2H),
3.04 (t, J = 7.0 Hz, 2H), 1.36 (s, 9H); 13C NMR (125 MHz, CDCl3) d
156.1, 149.7, 134.4, 133.3, 132.9, 127.8, 125.0, 79.4, 41.2, 29.0,
28.6; MS (ESI, CH3CN) [2 M+H+] = 533.0.
5.6. 6-((4-(2-Azidoethoxy)pyrrolidin-3-yl)methyl)-4-methyl-
pyridin-2-amine (( )-1)
A solution of ( )-18 (19 mg, 0.04 mmol) in 4 N HCl in dioxanes
(3 mL) was stirred for 16 h. The solvent was removed under a
stream of nitrogen and the residue was dissolved in water, washed
with ethyl acetate and concentrated to give ( )-1 as a white dihy-
drochloride salt (7.3 mg, 0.02 mmol, 50%). 1H NMR (500 MHz,
CDCl3) d 6.55 (s, 2H), 4.00 (m, 1H), 3.62 (m, 2H), 3.50 (m, 1H),
3.43 (m, 1H), 3.34 (m, 2H), 3.17 (m, 1H), 3.05 (m, 1H), 2.84 (m,
2H), 2.68 (m, 1H), 2.20 (s, 3H); ESMS m/z = 277 (M+H)+. HRMS
(ESI): m/z calcd for C13H21N6O (M+H)+ 277.1771; found 277.1774.
5.7. 6-((4-(2-Aminoethoxy)pyrrolidin-3-yl)methyl)-4-methyl-
pyridin-2-amine (( )-2)
A solution of ( )-19 (11 mg, 0.025 mmol) in 4 N HCl in dioxanes
(3 mL) was stirred for 16 h. The solvent was removed under a
stream of nitrogen and the residue was dissolved in water, washed
with ethyl acetate and concentrated to give ( )-2 as a white trihy-
drochloride salt (5.5 mg, 0.015 mmol, 60%). 1H NMR (500 MHz,
CDCl3) d 6.55 (s, 1H), 6.50 (s, 1H), 4.06 (m, 1H), 3.67 (m, 1H),
3.51 (m, 2H), 3.40 (m, 1H), 3.21–3.04 (m, 4H), 2.80 (m, 1H), 2.68
(m, 1H), 2.19 (s, 3H); ESMS m/z = 251 (M+H)+.
5.8.4. [2-(2-Aminophenyl)ethyl]carbamic acid tert-butyl ester
(24)
Compound 23 (769 mg, 2.89 mmol) was dissolved in a mixture
of MeOH/H2O/AcOH (7:2:1, 20 mL) and subjected to catalytic
hydrogenation on 10% Pd/C (80 mg) at 1 atm. After 2.5 h, the reac-
tion mixture was filtered through Celite, and the filtrate was con-
centrated to dryness to afford an oily residue 24 (648 mg, 95%).
Rf = 0.20 (hexane/EtOAc = 4:1); 1H NMR (500 MHz, CDCl3) d 7.05
(t, J = 7.5 Hz, 1H), 6.99 (d, J = 7.0 Hz, 1H), 6.70 (t, J = 7.0 Hz, 1H),
6.67 (d, J = 8.0 Hz, 1H), 5.26 (s, 1H), 4.33 (s, 2H), 3.27 (dd,
J = 8.0 Hz, J = 6.0 Hz, 2H), 2.70 (t, J = 7.0 Hz, 2H), 1.48 (s, 9H); 13C
NMR (125 MHz, CDCl3) d 156.8, 145.4, 130.4, 127.9, 123.3, 118.5,
115.9, 79.4, 40.3, 32.5, 28.7; MS (ESI, CH3CN) [M+H+] = 237.1.
5.8. 1-((S)-1-{2-[2-Aminoethyl]phenylamino}-5-nitroguanidin-
opentan-2-ylcarbamoyloxy)ethyl pivalate (3)
5.8.1. 1-Chloroethyl p-nitrophenyl carbonate (20)
To an ice-cold mixture of p-nitrophenol (100 mg, 0.72 mmol)
and pyridine (57 mg, 0.72 mmol) in CHCl3 (5 mL) was added 1-
chloroethyl chloroformate (113 mg, 0.79 mmol). The cooling bath
was removed after 30 min, and the reaction mixture was stirred
at room temperature for 18 h. After being washed successively
with water, 0.5% aq NaOH, and water, the chloroform layer was
dried over Na2SO4 and evaporated to a viscous yellowish oil. After
trituration with hexane, 20 was obtained as a white solid (151 mg,
78%); mp 71.0–71.7 °C. 1H NMR (500 MHz, CDCl3) d 8.30 (d,
J = 9.0 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 6.50 (d, J = 5.5 Hz, 1H),
1.92 (d, J = 5.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) d 155.2, 150.7,
145.9, 125.7, 121.9, 85.5, 25.3.
5.8.5. (S)-tert-Butyl 2-(2-{9H-fluoren-9-ylmethylcarbamoyl}-5-
nitroguanidinopentanamido)phenethylcarbamate (25)
To an ice-cold solution of Fmoc–Arg(NO2)–OH (168 mg,
0.38 mmol) and 24 (45 mg, 0.19 mmol) dissolved in a mixture of
CH2Cl2 (4 mL) and aq Na2CO3 (0.17 M, 4 mL), was added a solution
of TFFH (127 mg, 0.48 mmol) and HOAt (52 mg, 0.38 mmol) in
CH2Cl2 (3 mL). The reaction mixture was stirred at room tempera-
ture for three days under nitrogen. An excess amount of CH2Cl2
(20 mL) was added. The organic layer was separated, washed with
water (20 mL) and brine (20 mL) and dried over Na2SO4. The solu-
tion was concentrated in vacuo, and the residue was purified by
flash column chromatography (hexane/EtOAc = 1:3, Rf = 0.25) to af-
5.8.2. 1-(Trimethylacetoxy)ethyl p-nitrophenyl carbonate (21)
Mercuric trimethylacetate was prepared on a small scale as fol-
lows: A mixture of yellow mercuric oxide (449 mg, 2.07 mmol) and
trimethylacetic acid (424 mg, 4.15 mmol) was heated at 85 °C in
CCl4 overnight. After filtration and evaporation, a white solid was

7602
R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
ford 25 (107 mg, 85%) as a white foam; mp 100.2–102.0 °C. 1H
NMR (500 MHz, CDCl3) d 8.90 (s, 1H), 8.74 (s, 1H), 7.92 (s, 1H),
7.72 (t, J = 6.0 Hz, 2H), 7.57 (d, J = 7.0 Hz, 2H), 7.31–7.37 (m, 2H),
7.24 (m, 3H), 7.08–7.13 (m, 2H), 6.70 (s, 1H), 5.28 (s, 1H), 4.64 (s,
1H), 4.39 (d, J = 6.0 Hz, 2H), 4.17 (m, 1H), 3.42 (s, 1H), 3.33 (s,
1H), 3.10 (m, 2H), 2.77 (s, 2H), 2.00 (m, 1H), 1.87 (s, 1H), 1.77 (s,
2H), 1.42 (s, 9H); 13C NMR (125 MHz, CDCl3) d 171.5, 159.6,
157.4, 157.0, 143.9, 143.8, 141.5, 135.7, 130.4, 128.0, 127.8,
127.3, 125.3, 120.2, 80.6, 67.4, 60.7, 47.3, 41.5, 40.8, 32.9, 28.6,
21.3, 14.5; MS (ESI, CH3CN) [M+Na+] = 682.6.
washed with CH2Cl2 and lyophilized to give 3 (25 mg, 93%) as a
white solid; mp 76.3–78.5 °C. TLC (CH2Cl2/MeOH/Et3N, 10:1:0.1)
Rf = 0.30; 1H NMR (500 MHz, CD3OD) d 7.15 (t, J = 7.0 Hz, 1H),
7.04 (d, J = 7.5 Hz, 1H), 6.78 (m, 1H), 6.67 (m, 1H), 3.86 (s, 1H),
3.28 (s, 2H), 3.15–3.24 (m, 5H), 2.87 (m, 2H), 1.77 (br s, 2H), 1.66
(br s, 1H), 1.52 (m, 1H), 1.47 and 1.43 (2 Â d, J = 5.0 Hz, 3H), 1.18
and 1.17 (2 Â s, 9H), diastereomers; HRMS (ESI): m/z calcd for
C22H38N7O6 (M+H+) 496.2884; found 496.2882. Anal. Calcd for
C22H37N7O6Á2.5TFAÁ1.5H2O: C, 40.15, H, 5.30, N, 12.14. Found: C,
40.17, H, 5.30, N, 12.24.
5.8.6. (S)-tert-Butyl 2-(2-{9H-fluoren-9-ylmethylcarbamoyl}-5-
nitroguanidinopentylamino)phenethylcarbamate (26)
5.9. tert-Butyl 3-((6-(benzyl(tert-butoxy-carbonyl)-amino)-4-
methylpyridin-2-yl)methyl)-4-(2-(N-(3-fluorophenethyl)-
acetamido)ethoxy)-pyrrolidine-1-carboxylate (( )-29)
Compound 25 (0.83 g, 1.25 mmol) was treated dropwise with a
solution of 1.0 M BH3 (13 mL) in THF at À10 °C under argon. The
reaction temperature was allowed to rise to 0 °C. After being stir-
red at 0 °C for 10 h, the residual BH3 was quenched by cautious
addition of MeOH (30 mL) at 0 °C, and the mixture was stirred
overnight at room temperature. The solution was evaporated un-
der reduced pressure and treated three times with MeOH
(50 mL), evaporating to dryness after each addition to remove boric
acid as trimethyl borate. The residue was purified by flash column
chromatography (hexane/EtOAc = 1:2, Rf = 0.25) to afford 26
(0.38 g, 46%) as an oily residue. 1H NMR (500 MHz, CDCl3) d 8.77
(s, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 6.0 Hz, 2H), 7.38 (m,
2H), 7.27 (t, J = 7.5 Hz, 2H), 7.16 (t, J = 7.5 Hz, 1H), 7.00 (d,
J = 7.0 Hz, 1H), 6.67 (t, J = 7.0 Hz, 1H), 6.60 (d, J = 8.0 Hz, 1H), 6.39
(d, J = 8.5 Hz, 1H), 5.23 (s, 1H), 4.93 (s, 1H), 4.37 (d, J = 6.5 Hz,
2H), 4.07 (s, 1H), 3.39 (m, 1H), 3.33 (m, 1H), 3.16–3.23 (m, 4H),
2.73 (t, J = 7.0 Hz, 2H), 1.85 (br s, 1H), 1.76 (m, 1H), 1.69 (m, 2H),
1.45 (s, 9H); 13C NMR (125 MHz, CDCl3) d 171.6, 159.6, 157.5,
157.4, 146.4, 144.0, 141.5, 130.3, 128.4, 128.0, 127.3, 125.4,
122.6, 120.3, 117.1, 110.2, 80.3, 67.1, 60.7, 48.1, 47.4, 41.2, 40.1,
33.3, 28.7, 21.3, 14.5.
To
(5 mmol) was added acetic anhydride (13
ethylamine (20 L, 0.14 mmol). The mixture was stirred for 4 h.
a
solution of ( )-28 (79 mg, 0.12 mmol) in methanol
lL, 0.14 mmol) and tri-
l
The solvent was removed in vacuo and the residue was dissolved
in sat NaHCO3 and extracted with ethyl acetate (3 Â 20 mL). The
organic layers were combined, dried over anhydrous Na2SO4
and concentrated in vacuo. The crude product was purified using
flash column chromatography (silica gel, ethyl acetate/hexanes,
4:1) to afford ( )-29 as a colorless oil (84 mg, 0.12 mmol, quant).
1H NMR (500 MHz, CDCl3) d 7.41 (m, 1H), 7.23 (m, 6H), 6.98–6.86
(m, 3H), 6.60 (m, 1H), 5.15 (s, 2H), 3.57 (m, 4H), 3.39 (m, 4H),
3.15 (m, 2H), 3.03 (m, 1H), 2.83 (m, 3H), 2.69–2.50 (m, 2H),
2.28 (m, 3H), (2.11 + 1.94 + 1.91) (s, rotamers, 3H), 1.44 (m, 9H),
1.41 (s, 9H); 13C NMR (125 MHz, CDCl3) d 171.1, 164.1, 162.2,
157.8, 155.0, 154.6, 154.2, 148.9, 142.1, 140.1, 130.6, 130.2,
128.3, 127.2, 127.1, 127.0, 126.8, 124.7, 120.1, 117.4, 115.9,
113.4, 81.5, 79.9, 79.6, 79.1, 61.7, 52.0, 50.7, 50.2, 49.4, 48.1,
46.5, 42.8, 35.2, 33.9, 28.8, 28.4, 22.1, 21.5, 21.3; ESMS m/
z = 705 (M+H)+.
5.8.7. 1-((S)-1-{2-[2-(tert-Butoxycarbonylamino)ethyl]phenyl-
amino}-5-nitroguanidinopentan-2-ylcarbamolyoxy)ethyl
pivalate (27)
5.10. tert-Butyl 3-((6-(tert-butoxycarbonyl-amino)-4-methyl-
pyridin-2-yl)methyl)-4-(2-(N-(3-fluorophenethyl)acet-amido)-
ethoxy)-pyrrolidine-1-carboxylate (( )-30)
Piperidine (1 mL) was added to a solution of 26 (240 mg,
0.37 mmol) in DMF (4 mL), and the mixture was stirred at room
temperature for 0.5 h (monitoring by TLC). The volatile compo-
nents were removed under reduced pressure. The residue was dis-
solved in dry CH3CN (7 mL). To this solution was added
successively DIEA (48 mg, 0.37 mmol) and 21 (115 mg, 0.37 mmol)
dissolved in dry CH3CN (1 mL). The reaction mixture was stirred at
room temperature overnight under nitrogen. The solution was
evaporated under reduced pressure, and the residue was purified
by flash column chromatography (hexane/EtOAc = 1:2, Rf = 0.25)
to afford 27 (183 mg, 83%) as a white foam; mp 73.2–74.0 °C. 1H
NMR (500 MHz, CDCl3) d 8.66 (s, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.97
(d, J = 7.0 Hz, 1H), 6.75–6.82 (m, 1H), 6.62 (t, J = 7.0 Hz, 1H), 6.57
(d, J = 8.0 Hz, 1H), 6.39–6.48 (m, 1H), 5.28 (t, J = 6.5 Hz, 1H), 4.98
and 4.90 (2 Â s, 1H), 4.06 (m, 1H), 3.26–3.40 (m, 4H), 3.15 (t,
J = 6.0 Hz, 2H), 2.70 (s, 2H), 1.84 (s, 1H), 1.72 (s, 1H), 1.65 (s, 2H),
1.47 and 1.46 (2 Â s, 9H), 1.39 (m, 3H), 1.15 and 1.13 (2 Â s, 9H),
diastereomers.
To a solution of ( )-29 (84 mg, 0.12 mmol) in ethanol (5 mL)
was added Pd(OH)2/C (ꢀ10 mg). The mixture was stirred under a
hydrogen atmosphere at 60 °C for two days. The mixture was fil-
tered through Celite, and the solvent was removed in vacuo. The
crude product was purified using flash column chromatography
(silica gel, ethyl acetate/hexanes, 5:1) to afford ( )-30 as a colorless
oil (55 mg, 0.09 mmol, 75%). 1H NMR (500 MHz, CDCl3) d 7.59 (m,
1H), 7.43 (m, 1H), 7.25 (m, 1H), 7.00–6.88 (m, 3H), 6.58 (m, 1H),
3.76–3.24 (m, 10H), 3.08 (m, 1H), 2.85 (m, 3H), 2.67–2.48 (m,
2H), 2.27 (m, 3H), (2.16 + 1.96 + 1.93) (s, rotamers, 3H), 1.52 (s,
9H), 1.43 (m, 9H); ESMS m/z = 615 (M+H)+.
5.11. N-(2-(4-((6-Amino-4-methylpyridin-2-yl)-methyl)pyrroli-
din-3-yloxy)ethyl)-N-(3-fluoro-phenethyl)acetamide (( )-6)
A solution of ( )-30 (55 mg, 0.09 mmol) in 4 N HCl in dioxanes
(3 mL) was stirred for 16 h. The solvent was removed under a
stream of nitrogen and the residue was dissolved in water, washed
with ethyl acetate and concentrated to give ( )-6 as a white dihy-
drochloride salt (29 mg, 0.06 mmol, 67%). mp 59–61 °C. 1H NMR
(500 MHz, D2O) d 7.21 (m, 1H), 6.89 (m, 3H), (6.53 + 6.47) (s, rota-
mers, 1H), (6.41 + 6.34) (s, rotamers, 1H), 3.97 (m, 1H), 3.69 (m,
1H), 3,62–3,42 (m, 5H), 3.39–3.15 (m, 4H), 3.01 (m, 1H), 2.79–
2.60 (m, 4H), (2.17 + 2.13) (s, rotamers, 3H), (2.02 + 1.71) (s, rota-
mers, 3H); ESMS m/z = 415 (M+H)+. HRMS (M+H)+ calcd for
C23H32FN4O2 415.2504; found 415.2522.
5.8.8. 1-((S)-1-{2-[2-Aminoethyl]phenylamino}-5-
nitroguanidinopentan-2-ylcarbamoyloxy)ethyl pivalate (3)
Compound 27 (27 mg, 0.05 mmol) was treated with trifluoro-
acetic acid/CH2Cl2 (1 mL/2 mL) at 0 °C under argon. The reaction
temperature was maintained at 0 °C, and stirring continued for
20 min. Excess TFA and solvent were removed by evaporation in
vacuo. The residue was repeatedly dissolved in CH2Cl2 (10 mL)
and the solvents evaporated to remove traces of TFA. The residue
was dissolved in a small amount of water, and the solution was

R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
7603
5.12. tert-Butyl 3-((6-(tert-butoxycarbonyl-amino)-4-methyl-
pyridin-2-yl)methyl)-4-(2-(3-fluoro-phenethylamino)-ethoxy)-
pyrrolidine-1-carboxylate (( )-31)
with ethyl acetate and concentrated to give ( )-7 as a greasy color-
less solid (8.4 mg, 0.02 mmol, 23%). 1H NMR (500 MHz, D2O) d 7.16
(q, J = 6 Hz, 1H), 6.83 (m, 3H), 6.48 (s, 1H), 6.33 (s, 1H), 3.91 (m,
1H), 3.66 (m, 1H), 3.58 (s, 1H), 3.52–3.40 (m, 4H), 3.33–3.13 (m,
7H), 2.99 (m, 1H), 2.70–2.63 (m, 4H), 2.12 (s, 3H); ESMS m/z =
431 (M+H)+. HRMS (ESI): m/z calcd for C23H32FN4O3 (M+H)+
431.2453; found 431.2460.
To a solution of ( )-28 (199 mg, 0.3 mmol) in ethanol (5 mL) was
added Pd(OH)2/C (ꢀ10 mg). The mixture was stirred under a hydro-
gen atmosphere at 60 °C for two days. The mixture was filtered
through Celite, and the solvent was removed in vacuo. A mixture
of ( )-31 and ( )-33 were formed. The crude products were purified
using flash column chromatography (silica gel, ethyl acetate/meth-
anol, 1:9) to afford ( )-31 (61 mg, 0.11 mmol, 37%) and ( )-33
(42 mg, 0.09 mmol, 30%) as white solids. Compound ( )-31 1H
NMR (500 MHz, CDCl3) d 7.60 (d, J = 10 Hz, 1H), 7.28–7.18 (m,
2H), 6.90 (m, 3H), 6.58 (s, 1H), 3.67 (m, 2H), 3.46 (m, 2H), 3.34
(m, 1H), 3.24 (dd, J = 4, 12 Hz, 1H), 3.08 (m, 1H), 2.95–2.73 (m,
6H), 2.57 (m, 2H), 2.47 (m, 1H), 2.30 (s, rotamers, 3H), 1.52 (s,
9H), 1.45 (s, 9H); 13C NMR (125 MHz, CDCl3) d 164.1, 162.1, 158.4,
154.9, 152.9, 151.9, 150.0, 142.7, 130.1, 124.6, 119.0, 115.7, 113.2,
110.3, 80.8, 79.5, 78.6, 68.8, 50.9, 50.5, (49.5 + 49.2), (44.1 + 43.3),
36.3, 29.0, 28.8, 28.3, 21.5; ESMS m/z = 573 (M+H)+.
5.16. Benzyl 2-(4-((6-amino-4-methylpyridin-2-yl)-methyl)-
pyrrolidin-3-yloxy)ethyl(3-fluoro-phenethyl)carbamate (( )-8)
A solution of ( )-34 (45 mg, 0.075 mmol) in 4 N HCl in dioxanes
(3 mL) was stirred for 16 h. The solvent was removed under a
stream of nitrogen, and the residue was dissolved in methanol,
which was then removed in vacuo. Anhydrous diethyl ether was
added, causing a white precipitate to form. The ether was dec-
anted, and the precipitate was dried to give ( )-8 as a greasy color-
less solid (18 mg, 0.031 mmol, 42%). HPLC and LC–MS revealed that
( )-8 was contaminated with ( )-5 (ꢀ10%). It is unclear whether
this was caused by loss of the CBZ group during deprotection, or
whether it is from an intermediate being carried through. 1H
NMR (500 MHz, CD3OD) d 7.35 (m, 6H), 6.97 (m, 3H), 6.67 (s,
1H), 6.50 (m, 1H), 5.08 (m, 2H), 4.21 (m, 1H), 3.99 (m, 1H), 3.75–
3.23 (m, 9H), 3.23 (m, 1H), 2.88 (m, 2H), 2.78 (m, 2H), 2.32 (s,
3H); ESMS m/z = 507 (M+H)+. HRMS (ESI): m/z calcd for
C29H35FN4O3 (M+H)+ 507.2766; found 507.2768.
5.13. tert-Butyl 3-((6-(tert-butoxy-carbonyl-amino)-4-methyl-
pyridin-2-yl)methyl)-4-(2-((3-fluorophenethyl)-(methoxy-car-
bonyl)amino)ethoxy)pyrrolidine-1-carboxylate (( )-32)
To a solution of ( )-31 (61 mg, 0.11 mmol) in CH2Cl2 (5 mL)
were added methyl chloroformate (10
lL, 0.12 mmol) and TEA
(16 L, 0.11 mmol). The mixture was stirred for 4 h. The solvent
l
5.17. HPLC analysis
was removed in vacuo, and the residue was dissolved in sat NaH-
CO3 and extracted with ethyl acetate (3 Â 20 mL). The organic lay-
ers were combined, dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude product was purified using flash
column chromatography (silica gel, ethyl acetate/hexanes, 1:1) to
afford ( )-32 as a colorless oil (55 mg, 0.087 mmol, 91%). 1H NMR
(500 MHz, CDCl3) d 7.60 (m, 1H), 7.28 (m, 2H), 6.91 (m, 3H), 6.57
(s, 1H), 3.70 (m, 4H), 3.59–3.25 (m, 8H), 3.10 (m, 1H), 2.84 (m,
3H), 2.60 (m, 3H), 2.28 (m, 3H), 1.52 (s, 9H), 1.44 (m, 9H); 13C
NMR (125 MHz, CDCl3) d 164.1, 162.2, 158.4, (157.0 + 156.8),
(155.0 + 154.8), (152.7 + 151.7), 150.1, 141.8, 130.1, 124.7, 119.3,
115.9, 113.5, 110.5, 81.0, 79.9, 79.5, 68.3, 60.6, 52.8, 50.8, 50.4,
49.5, 49.1, 48.4, 47.9, 43.7, 35.2, 28.9, 28.4, 21.5; ESMS m/z = 631
(M+H)+.
See Supplementary data for chromatograms.
5.18. Determination of metabolism of 1 and 3 in plasma
Three mice were sacrificed under anesthesia by exsanguination
and decapitation. The blood was collected in a pre-heparinized
tube. The tubes were centrifuged to precipitate the blood cells,
and the plasma supernatant was transferred to separate vials. To
an aliquot of plasma (84
tion A (5 L), NADPH regeneration solution B (1
3 (10 L of 200 M stock). Samples were incubated at 37 °C and re-
moved at various time points (t = 0, 10, 20, 40 min). To quench the
sample, acetonitrile (400 L) and 35 (10 L of 200 M stock) were
lL) was added NADPH regeneration solu-
l
l
L), and either 1 or
l
l
l
l
l
added. The samples were centrifuged, and the supernatant was
transferred to borosilicate tubes and concentrated to dryness using
a Genevac. The residues were reconstituted in 2% formic acid/
5.14. tert-Butyl 3-((6-amino-4-methylpyridin-2-yl)-methyl)-4-
(2-((benzyloxycarbonyl)-(3-fluoro-phenethyl)amino)ethoxy)-
pyrrolidine-1-carboxylate (( )-34)
water (50 lL) and the mixtures were analyzed by HPLC monitoring
at 310 nm for 1 and 35, and at 260 nm for 3. For each sample, the
area under the peak corresponding to the compound of interest
(3A, 3B, or 1) and to the internal standard (35) was integrated.
The ratio of compound of interest to internal standard was mea-
sured and normalized by dividing by the ratio at t = 0 min.
To a solution of ( )-33 (42 mg, 0.09 mmol) in methanol (3 mL)
was added benzyl chloroformate (20 lL, 0.1 mmol). The mixture
was stirred for 4 h. The solvent was removed in vacuo, and the res-
idue was dissolved in sat NaHCO3 and extracted with ethyl acetate
(3 Â 20 mL). The organic layers were combined, dried over anhy-
drous Na2SO4 and concentrated in vacuo. The crude product was
purified using flash column chromatography (silica gel, ethyl ace-
tate/methanol, 9:1) to afford ( )-34 (45 mg, 0.075 mmol, 83%) as
a white solid. 1H NMR (500 MHz, CDCl3) d 7.34 (m, 5H), 7.21 (m,
1H), 6.88 (m, 3H), 6.25 (m, 1H), 6.14 (m, 1H), 5.13 (m, 2H), 4.33
(m, 2H), 3.78–3.25 (m, 10H), 3.07 (m, 1H), 2.90–2.75 (m, 3H),
2.57 (m, 2H), 2.15 (m, 3H), 1.43 (m, 9H); ESMS m/z = 507 (M+H)+.
5.19. Determination of metabolism of 1 and 3 in brain
homogenate
Three fresh mice brains were homogenized, each in PBS (phos-
phate buffered saline, pH 7.4, 1 mL per brain) with NADPH regen-
eration buffer A (50
The homogenates were combined to form one stock. To an aliquot
of homogenate (190 L) was added either 1 or 3 (10 L of 200
lL) and NADPH regeneration buffer B (10 lL).
l
l
lM
5.15. Methyl 2-(4-((6-amino-4-methylpyridin-2-yl)-methyl)-
pyrrolidin-3-yloxy)ethyl(3-fluoro-phenethyl)carbamate (( )-7)
stock). The samples were incubated at 37 °C for varying lengths of
time (0, 10, 20 and 40 min.) At each time point, a sample was re-
moved from the water bath and heated to 95 °C for 1 min. A mix-
A solution of ( )-32 (55 mg, 0.087 mmol) in 4 N HCl in dioxanes
(3 mL) was stirred for 16 h. The solvent was removed under a
stream of nitrogen and the residue was dissolved in water, washed
ture of 2% formic acid in water (500
followed by 35 (20 L of 100 M stock). The 1 samples were loaded
onto 1 cc Oasis MCX SPE columns. The columns were washed with
lL) was added to each sample,
l
l

7604
R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
2% formic acid in water (1 mL), and with 80% acetonitrile, 2% for-
mic acid in water (1 mL), and the compounds were eluted with
5% NH4OH in methanol (2 mL). The eluents were concentrated to
(500
tion plate. The well was washed with 2% formic acid (500
and methanol (500 L), and the compound was eluted with 5%
NH4OH in methanol (2 Â 400 L). The solvent was removed under
a stream of nitrogen and the residue was reconstituted in LC–MS/
MS mobile phase (200 L, see below for constitution). An aliquot
(20 L) was analyzed by LC–MS. Standards were run in triplicate.
l
L) and loaded onto a preconditioned Oasis MCX micro-elu-
lL),
l
dryness and reconstituted in 2% formic acid (50
lL). The 3 samples
l
were loaded onto 1 cc Oasis HLB SPE columns. The columns were
washed with 2% formic acid in water (1 mL), and the compounds
were eluted with 80% acetonitrile, 2% formic acid in water
(2 mL). The eluents were concentrated to dryness and reconsti-
l
l
tuted in 2% formic acid (50
lL). The mixtures were analyzed by
5.23. Preparation of brain standard curve
HPLC monitoring at 310 nm for 1 and 35, and at 260 nm for 3.
For each sample, the area under the peak corresponding to the
compound of interest (3A, 3B, or 1) and to the internal standard
(35) was integrated. The ratio of compound of interest to internal
standard was measured and normalized by dividing by the ratio
at t = 0 min.
To a homogenizer was added 2% formic acid in water/acetoni-
trile (2:1, 700
of interest was added to obtain a final concentration in
based on a total volume of 1 mL. A typical standard curve would
contain the concentrations 0.001, 0.005, 0.01, 0.05 and 0.1 g/mL,
but additional measurements were made as needed. For example,
to obtain a concentration of 0.005 g/mL of 6, 12.08 L of 1
lL) and 35 (10
l
L of 100
l
M stock). The compound
lg/mL
l
l
l
lM
5.20. Standard microsome assay
stock was added. To the solution was added a mouse brain, a rat
pup brain, or a piece of pig brain weighing between 300 and
500 mg. The brain was homogenized and transferred to a centri-
fuge tube. The homogenizer was washed with 2% formic acid in
water/acetonitrile (2:1, 300 lL), which was also added to the cen-
trifuge tube. The samples were centrifuged (10 krpm, 12 min), and
To PBS (257
(15 L) and NADPH regeneration buffer B (3
pound of interest, for example, minaprine (10
The tubes were vortexed and equilibrated at 37 °C. Rat liver micro-
somes (15 L) were added, and the samples were incubated at
37 °C for varying lengths of time (0, 10, 20, 40 min.). To quench
the reaction, acetonitrile (500 L) was added, and the tubes were
l
L) was added NADPH regeneration buffer A
L), and the com-
L of 300 M stock).
l
l
l
l
l
the supernatant was transferred to a separate tube. An aliquot
(400
well was washed with 2% formic acid (500
(500 L), and the compound was eluted with 5% NH4OH in meth-
anol (2 Â 400 L). The solvent was removed under a stream of
nitrogen, and the residue was reconstituted in the LC–MS/MS mo-
bile phase (200 L). An aliquot (20 L) was analyzed by LC–MS.
Standards were run in triplicate.
lL) was loaded onto an Oasis MCX micro-elution plate. The
l
lL) and methanol
chilled to 0 °C to precipitate out the proteins. The samples were
centrifuged (14 krpm, 10 min), and the supernatants were trans-
ferred to separate tubes. The samples were concentrated to dry-
ness and reconstituted in 2% formic acid (50 lL). The mixtures
were analyzed by HPLC. Peak integrals were normalized by divid-
l
l
l
l
ing by the area for the peak at t = 0 min.
5.24. Administration of compounds to mice
5.21. Anaerobic microsome assay for azide metabolism
Compounds were diluted in PBS to form a 1 mM solution. Mice
Nitrogen gas was bubbled through PBS (2.35 mL) for 15 min to
deoxygenate it. To the buffer was added NADPH regeneration buf-
were weighed and then given the compound (100
intraperitoneal injection. Three minutes before the desired time of
sacrifice, the mouse was injected with pentobarbital (100 L of a
lL per 10 g) via
fer A (125
vials with PTFE faced rubber septa were added the deoxygenated
PBS mixture (240 L) and the azide (30 L of 100 M stock). The
lL) and NADPH regeneration buffer B (25 lL). To 2 mL
l
50 mg/mL solution in PBS). Once the animal was no longer respon-
sive, its thoracic cavity was opened, and blood was removed from
the right ventricle of the heart using a syringe that had been trea-
ted with heparin and transferred to Microtainer PST pre-heparin-
ized tubes. The tubes were centrifuged (5 min at 6 krpm), and
the plasma supernatants were transferred to labeled tubes. The
brain was perfused with PBS by inserting a needle through the left
ventricle and allowing PBS under pressure to flow into it. The right
atrium was cut to relieve the pressure. The mouse was decapitated,
and the brain was removed. All samples were flash-frozen in liquid
nitrogen and stored at À20 °C until analyzed.
l
l
l
vials were sealed, and the volume above the solution in the vial
was purged with nitrogen. A balloon was used to maintain a nitro-
gen atmosphere inside the vial. The vials were equilibrated in a
water bath at 37 °C. Microsomes (30
rubber septum, and the mixtures were incubated at 37 °C for var-
ious lengths of time (0, 10, 20 and 40 min). Acetonitrile (600 L)
lL) were added through the
l
was added to quench the reaction. The mixtures were transferred
to centrifuge tubes and cooled to 0 °C to precipitate the proteins.
The samples were centrifuged at 14 krpm for 10 min. The superna-
tants were concentrated to dryness and the residues were reconsti-
tuted in 2% formic acid (50 lL) and analyzed by HPLC. Peak
integrals were normalized by dividing by the area for the peak at
5.25. Preparation of plasma samples for quantification
t = 0 min.
To an aliquot of the sample (100
M stock). The mixture was diluted with 2% acetic acid
(500 L) and loaded onto a preconditioned Oasis MCX micro-elu-
tion plate. The well was washed with 2% formic acid (500 L)
and methanol (500 L), and the compound was eluted with 5%
NH4OH in methanol (2 Â 400 L). The solvent was removed under
a stream of nitrogen, and the residue was reconstituted in the LC–
MS/MS mobile phase (200 L). An aliquot (20 L) was analyzed by
LC–MS. Quantification of the compounds was carried out by calcu-
lating the ratio of the mass spectral peak intensity of the com-
pound to the mass spectral peak intensity of the internal
standard and comparing it to the standard curve.
lL) was added 35 (10 lL of
10
l
5.22. Preparation of plasma standard curve
l
l
Samples were prepared to obtain final concentrations in
A typical standard curve contained the concentrations 0.01, 0.05,
0.1, 0.5 and 1.0 g/mL, but additional points were measured as
needed. For example, to prepare a sample of 0.01 g/mL of 5 in
plasma, 5 (13.45 L of 1 M stock) was added to blank plasma
(487 L). The molecular weight of 5 is 372; therefore, 1 g =
0.00269 mol, and 1 g/mL = 2.69 M. An aliquot of 13.45 L in
500 L of 1 M stock = 0.0269 M = 0.01 g/mL.
To an aliquot of the sample (100 L) was added 35 (10
M stock). The mixture was diluted with 2% acetic acid
lg/mL.
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
lL of
The data were originally obtained in
lg/mL, but were converted
10
l
to
lM for ease of comparison. The error bars on the charts repre-

R. B. Silverman et al. / Bioorg. Med. Chem. 17 (2009) 7593–7605
7605
sent the standard error mean. This was calculated by dividing the
standard deviation of the data for each time point by the square
root of the number of animals used for that point.
for 8. The retention time of 7 was 3.58 min and that of 8 was
15.7 min.
Acknowledgments
5.26. Preparation of brain samples for quantification
The authors are grateful to the National Institutes of Health
(GM49725) for financial support of this research. The authors wish
to thank Dr. Michael Avram and Lynn Luong of the Northwestern
Clinical Pharmacology Core Facility and the Pharmaceutical Chem-
istry Translational Resource of the Center for Drug Discovery and
Chemical Biology for carrying out the SPE and LC–MS experiments.
Brains were thawed and weighed. The brain was added to a
homogenizer containing 2% formic acid/acetonitrile (2:1, 700
and 35 (10 L of 100 M). The brain was homogenized and trans-
ferred to a centrifuge tube. The homogenizer was washed with 2%
formic acid in water/acetonitrile (2:1, 300 L), which was also
lL)
l
l
l
added to the centrifuge tube. The samples were centrifuged
(10 krpm, 12 min), and the supernatant was transferred to a sepa-
Supplementary data
rate tube. An aliquot (400
cro-elution plate. The well was washed with 2% formic acid
(500 L) and methanol (500 L), and the compound was eluted
with 5% NH4OH in methanol (2 Â 400 L). The solvent was re-
moved under a stream of nitrogen, and the residue was reconsti-
tuted in the LC–MS/MS mobile phase (200 L). An aliquot (20 L)
lL) was loaded onto an Oasis MCX mi-
Chromatographic conditions and chromatograms for various
compounds; standard curves for quantification of 5 in plasma
and brain; brain concentration of 5, 6, 7, 8; plasma concentration
of 7 and 8 are available. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2009.08.065.
l
l
l
l
l
was analyzed by LC–MS. Quantification of the compounds was car-
ried out by calculating the ratio of the peak intensity of the com-
pound to the peak intensity of the internal standard and
comparing it to the standard curve. The final value in
divided by the mass of the brain to obtain a concentration in
of compound/g of brain. For display on the charts, this was con-
verted to a concentration in M by assuming that the density of
lg/mL was
References and notes
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the brain tissue is approximately 1 g/mL.
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5.27. LC–MS/MS conditions
Samples containing 5, 6, and 35 were eluted isocratically from a
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containing 0.1% TFA at a flow rate of 250 lL/min. The tandem mass
spectrometer was operated with its electrospray source in the posi-
tive ionization mode. The mass to charge ratios of the precursor-to-
product ion reactions monitored were 373.3?123.1 for 5 and 35 and
415.3?208.2 for 6. The retention time of 5 was 2.38 min, that of 35
was 4.74 min, and that of 6 was 6.12 min.
Samples containing 36, 35, and 7 were eluted isocratically from
a Phenomenex MAX column (50 Â 2.0 mm, Phenomenex, Torrance,
CA) with a mobile phase consisting of water and methanol (75:25)
containing 0.1% TFA at a flow rate of 125 lL/min. The tandem mass
spectrometer was operated with its electrospray source in the po-
sitive ionization mode. The mass to charge ratios of the precursor-
to-product ion reactions monitored were 372.3?123.2 for 36,
415.3?208.2 for 7. The retention time of 36 was 2.46 min, that
of 35 was 4.74 min, and that of 7 was 9.27 min.
Samples containing 8 and 7 were eluted isocratically from a
Phenomenex MAX column (50 Â 2.0 mm, Phenomenex, Torrance,
CA) with a mobile phase consisting of water and methanol
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(70:30) containing 0.1% TFA at a flow rate of 125 lL/min. The tan-
dem mass spectrometer was operated with its electrospray source
in the positive ionization mode. The mass to charge ratios of the
precursor-to-product ion reactions monitored were 507.3?91.1