Synthesis and evaluation of potent and selective human V1a receptor antagonists as potential ligands for PET or SPECT imaging

Article abstract of DOI:10.1016/j.bmc.2011.12.013

SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood-brain barrier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visualization and characterization of the role of th

Full text of DOI:10.1016/j.bmc.2011.12.013

Bioorganic & Medicinal Chemistry 20 (2012) 1337–1345
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of potent and selective human V1a receptor
antagonists as potential ligands for PET or SPECT imaging
Karine Fabio a, Christophe Guillon a,b, , Carl J. Lacey a, Shi-fang Lu b,c, Ned D. Heindel a, Craig F. Ferris b,d
,
⇑
Michael Placzek e, Graham Jones e, Michael J. Brownstein b, Neal G. Simon b,c
a Dept. of Chemistry, Lehigh University, Bethlehem, PA 18015, USA
b Azevan Pharmaceuticals, Inc., Bethlehem, PA 18015, USA
c Dept. of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA
d Dept. of Psychology, Northeastern University, Boston, MA 02115, USA
e Dept. of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood–brain bar-
rier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visu-
alization and characterization of the role of the V1a receptor in multiple stress-related disorders. Before
radiolabeling studies, cold reference analogs of SRX246 were prepared. This study describes the synthesis
and in vitro screening for human V1a receptor binding and permeability of fluoro, iodo, and methyl ref-
erence compounds for SRX246 and the preparation of a tin precursor. For each compound, the potential
utility of corresponding radiolabeled analogs for PET and SPECT imaging is discussed.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 12 October 2011
Revised 5 December 2011
Accepted 7 December 2011
Available online 20 December 2011
Keywords:
Arginine vasopressin
Vasopressin V1a antagonist
Radiolabeling
PET
SPECT
1. Introduction
the treatmentof depression, anxiety, Post-Traumatic Stress Disorder
(PTSD), and Intermittent Explosive Disorder.7–17
Arginine vasopressin (AVP) is a cyclic nonapeptide hormone
that modulates a broad range of physiological and behavioral ef-
fects by binding to specific GPCRs in the central nervous system
and certain peripheral tissues/sites.1,2 Three distinct AVP receptor
subtypes have been identified—V1a, V1b, and V2. V1a is the pre-
dominant AVP receptor found in the brain. Especially high levels
are found in the cerebral cortex, limbic system, hypothalamus,
and brainstem.1 The V1b receptor is located in the pituitary gland
and it is less widespread in the brain than the V1a receptor. The V2
receptor is found in kidneys, where it mediates the antidiuretic ef-
fects of vasopressin. It is not generally thought to be expressed in
the nervous systems of adult animals and humans. These findings
have led to considerable interest in V1a and V1b receptors as po-
tential targets for CNS therapeutics. The effects of AVP and its
mechanism of action have been extensively studied and discussed
elsewhere.3–6
Our interest is in the V1a receptor, where there is strong evi-
dence from preclinical animal models as well as observations in
humans indicating that this subtype may well play a central role
in these indications.1,3,4,6,18–32 We were the first group to report
the discovery of potent, highly selective, CNS-penetrating human
V1a (hV1a) receptor antagonists.18 The technology places us in a
strong position to build upon our earlier discoveries to develop
imaging agents for the hV1a system based in particular on
SRX246, one of our clinical candidates.
Both PET and SPECT protocols have proved very useful as tools
to investigate in vivo drug behavior including a recent example for
the vasopressinergic system focusing on PET and the human V1b
(hV1b) receptor.33,34 The current study presents our progress gen-
erating reference compounds and a versatile tin intermediate that
would allow for the preparation of both PET and SPECT tracers for
the hV1a receptor, together with preliminary work on 123I labeling.
Selective V1a and V1b receptor antagonists that cross the
blood–brainbarrier have attracted considerableinterest as potential
therapeutics because the compounds represent a novel approach for
2. Results and discussion
SRX246 is a mono beta-lactam derivative with an azetidone ring
as a central pharmacophore core around which four distinct
peripheral zones (Zone A, B, C and D) can be varied (Fig. 1). We
⇑
Corresponding author. Tel.: +1 610 758 4706; fax: +1 610 758 6536.
E-mail address: chg3@lehigh.edu (C. Guillon).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.013

1338
K. Fabio et al. / Bioorg. Med. Chem. 20 (2012) 1337–1345
accomplished through a three step process (Scheme 2) from com-
O
O
mercially available 3-fluoro or 3-methyl cinnamic acids.35 Because
of the lack of a reliable commercial source for 3-iodocinnamic acid,
1a was obtained via a Horner–Wadsworth–Emmons reaction on
the 3-iodobenzaldehyde instead.36 Reduction of the ethyl cinna-
mate esters to the alcohols 2a, 2b and 2c and further oxidation
with pyridinium dichromate lead to the meta-substituted-trans-
cinnamaldehyde 3a, 3b and 3c in high yields. This three step pro-
cess led to the target halogenated or alkyl derivatives in much
higher overall yields compared to published procedures,37 particu-
larly for the iodo derivative. The absolute stereochemistry of the
chiral methine carbons of the azetidone ring for compounds 11a,
11b and 11c was assigned based on previous findings and con-
firmed by the 1H and 13C NMR chemical shifts and coupling
constants.18
Zone A
Zone D
Zone B
Zone C
N
O
O
Me
N
N
H
N
N
O
Figure 1. Key structural features of SRX246.
intend to modify Zone B in SRX246 for radioisotope introduction
123I, 18F or 11C). This approach represents a less challenging
(
synthetic pathway as compared to introducing the same tracers
on the aryl group in either Zone A or C. We also know, based on
previously described hV1a SARs,18 that Zone B is more tolerant of
small structural modifications, especially at the meta-position of
the phenyl ring.
In practice, the synthetic route (Scheme 1) leading to
compounds 11a, 11b and 11c was adapted from that previously
published for the preparation of SRX246.18 The procedure is based
on a chiral Staudinger 2+2 cyclo-addition reaction that allows the
building of the azetidone ring. In this modified procedure, meta-
substituted-trans-cinnamaldehydes were used instead of trans-cin-
namaldehyde while the rest of the reaction sequence, reagents and
conditions were kept the same. The three target compounds were
obtained in consistent yield but required the preparation of the
corresponding meta-substituted-trans-cinnamaldehydes. This was
After an earlier attempt to directly convert 11a to 16, we
decided to evaluate the utility of our general synthetic pathway
for the preparation of such a tin compound. It became quickly
apparent that the preparation of the tin analog (compound 16) rep-
resented a challenge because the conditions used for the synthesis
of SRX246 were not directly transposable to the synthesis of the tin
compound. In particular, we had concerns about the potential det-
rimental cleavage of the tin substituent in the presence of an acid
such as the one used (formic acid) to cleave the tButyl ester distal
carboxyl acid protecting group off the D-aspartic acid and after the
cyclo-addition step. A distinct synthetic pathway was designed
(Scheme 3) that would circumvent this potential problem. In this
new approach, we decided to introduce the two amide functions
O
O
O
O
O
O
NH
a
NH
b
O
NH2
O
OH
NH
CH3
NH
O
O
O
O
O
6
5
O
O
N
O
X
X
CO2H
c'
O
O
X
O
c
N
O
d
N
O
O
N
N
+
COCl
NH
O
3a X = I
N
O
3b X = F
3c X = CH3
H
9a X = I
O
7a X = I
O
9b X = F
7b X = F
7c X = CH3
8
O
9c X = CH3
O
O
N
e
X
O
O
N
O
N
X
O
N
H
f
O
O
N
N
O
N
H
O
10a X = I
11a X = I
11b X = F
11c X = CH3
N
HO
10b X = F
10c X = CH3
Scheme 1. Syntheses of fluoro, iodo and methyl analogs of SRX246. Reagents and conditions: (a) (R)-methylbenzylamine, HOBt, EDCꢀHCl, CH2Cl2, rt, 18 h; (b) H2, Pd/C 5%,
MeOH, 18 h; (c) Na2SO4 anhydrous, CH2Cl2; (c0) oxalyl chloride, DMF cat., CH2Cl2; (d) NEt3, CH2Cl2, 0 °C to rt, 1 h; (e) HCO2H; (f) 4-(1-piperidinyl) piperidine, HOBt, EDC, HCl,
CH2Cl2, rt, 18 h.

K. Fabio et al. / Bioorg. Med. Chem. 20 (2012) 1337–1345
1339
OH
OEt
OH
O
a
b
c
O
O
1a X = I
2a X = I
3a X = I
3b X = F
X
X
1b X = F
2b X = F
2c X = CH3
X
X
X = F
X = CH
1c X = CH3
3c X = CH3
d
3
O
a'
O
4
I
SnMe3
Scheme 2. Preparation of meta-substituted-trans-cinnamaldehydes. Reagents and conditions: (a) EDCꢀHCl, DMAP, EtOH, CH2Cl2, rt, 18 h; (a0) triethyl phosphonoacetate,
nBuLi in hexanes, THF, ꢁ78 °C, 10 min; 3-iodobenzaldehyde, THF, ꢁ78 °C, 40 min, to rt 45 min; (b) DIBAL, CH2Cl2, ꢁ78 °C, 2 h; 10% aqueous NaOH, ꢁ78 °C to rt, 15 min; (c)
pyridinium dichromate, CH2Cl2; (d) hexamethylditin, Pd(PPh3)4, dioxane, 150 min.
NH2
O
O
NH
CH3
O
O
O
O
NH
c
a
b
O
O
NH
5
NH
CH3
N
HO
NH
CH3
N
O
12
N
O
N
13
14
O
O
O
d'
O
4
O
SnMe3
N
N
Me3Sn
CO2H
O
d
N
O
O
N
H
SnMe3
O
O
e
N
N
+
O
N
COCl
16
NH
N
15
O
N
N
8
Scheme 3. Synthesis of radiolabeling precursor 16. Reagents and conditions: (a) HCO2H; (b) 4-(1-piperidinyl) piperidine, HOBt, EDCꢀHCl, CH2Cl2, rt, 18 h; (c) H2, Pd/C 5%,
MeOH, 18 h; (d) Na2SO4 anhydrous, CH2Cl2; (d0) oxalyl chloride, DMF cat., CH2Cl2; (e) NEt3, CH2Cl2, 0 °C to rt, 1 h.
prior to the cyclo-addition step, which allowed us to avoid putting
our tin group in jeopardy because the use of formic acid was now
warranted in an earlier step of the process. In summary, it allowed
the conservation of the alkyl stannyl group through the multi-step
synthesis and also resulted in the 2+2 cyclo-addition being the fi-
nal step of the reaction sequence. To achieve this, we also had to
develop a method to prepare the new meta-trimethyltin-trans-cin-
namaldehyde (4) needed for this approach. It was synthesized by
direct modification of 3-iodocinnamaldehyde (3a) via a Stille
reaction with hexamethylditin in the presence of a catalytic
amount of tetrakis(triphenylphosphine)palladium.38,39 The desired
trimethylstannane aldehyde was obtained in high yield (80%) and
was easily purified by silica gel filtration. Following the prepara-
tion of the aldehyde and implementing this strategy, we were in-
deed able to obtain the target compound 16 in satisfactory
overall yield.
ation with [123I]INa was conducted to afford [123I]11a in 88% radio-
chemical yield. After RP-HPLC purification, the radiochemical
purity of [123I]11a was determined at 94% with a specific activity
of 20 mCi/nmol. The potential use of 16 for the generation of 18F
and 11C radiolabeled ligands for PET imaging was also assayed in
cold conditions only. Electrophilic fluorination of 16 with the well
known fluorinated agent SelectFluor™, which has been highlighted
recently as a promising fluorine source for 18F radiolabeling, affor-
ded 11b with a chemical yield of 63% (Scheme 4).41 Alternative ap-
proaches leading to high specific activity radioligands will also be
investigated in future studies.42–44 Conversion of 16 to 11c via a
Stille coupling reaction used for 11C radiolabeling45 also showed
promising results and confirmed the feasibility of 11C radiolabeling
(data not presented).
Having prepared compounds 11a, 11b and 11c the next step
was to evaluate their biological activity in vitro against the human
vasopressin V1a receptor and in the Parallel Artificial Membrane
Permeability Assays for the Blood–Brain Barrier (PAMPA-BBB) as-
say. As expected, the modifications in the cinnamyl part (Zone B)
of the clinical candidate SRX246 did not affect dramatically the
biological activity for the hV1a receptor (SRX246’s hV1a Ki was re-
ported previously at 0.3 nM).18 Compounds 11a, 11b and 11c dis-
played potent nanomolar activity and extremely high affinity
in vitro for the hV1a receptor (Table 1). Compound 11b was the
Conversion to the cold and hot reference compounds, 11a and
[
123I]11a, via demetallation reaction was probed to further validate
the potential use of 16 for 123I radiolabeled ligands generation for
SPECT imaging (Scheme 4). Preparation of 11a and [123I]11a from
16 was investigated following established methods.40 In the pres-
ence of chloramine T (CAT) as the oxidant and sodium iodide as
the iodine source, 16 was successfully converted to the cold iodo
compound 11a with a chemical yield superior to 98%. Radioiodin-

1340
K. Fabio et al. / Bioorg. Med. Chem. 20 (2012) 1337–1345
O
O
O
O
X
SnMe3
N
O
N
O
O
O
N
N
a,
or a',
or b
N
N
H
H
O
O
N
N
16
N
N
11a X = I (98%)
123I]11a X = I (88% radio chemical yield)
11b X = F(63%)
[
Scheme 4. Conversion of trimethyltin derivative 16 to 11a, [123I]11a and 11b. Reagents and conditions: (a) NaI, chloramine T, 1 M HCl, ethanol, rt, 5 min; (a0) [123I]NaI,
chloramine T, 1 M HCl, ethanol, rt, 5 min; (b) Selectfluor™ (BFꢁ4 ), AgOTf, acetone, rt, 30 min.
Table 1
Vasopressin receptor affinity and PAMPA-BBB results for potential SRX246 based PET and SPECT ligands
Compound
hV1a Ki (nM)
hV1b Ki (nM)
hV2 Ki (nM)
cLogP
Pe (10ꢁ6 cm sꢁ1
)
Predicted CNS penetration
11a
11b
11c
1.1
0.61
0.63
>1000
1154
1300
>1000
>10,000
>10,000
5.62
4.42
4.75
2.2
5.3
4.5
CNS
CNS+
CNS+
most active of the three potential radiolabeled ligands with a Ki of
0.61 nM. This is not surprising if one considers that fluorine is an
excellent isostere for hydrogen,46 creating one of the closest possi-
ble structural analogs of SRX246. In a screening assay for binding
to hV1b and hV2 receptors, all the tested compounds were found
with Ki greater than 1000 nM demonstrating excellent selectivity
for the V1a receptor over other vasopressin receptor subtypes (Ta-
ble 1). The lipophilicity was mildly affected by the addition of a
fluorine atom or a methyl group with cLogP increases of 0.16
and 0.49 units for 11b and 11c, respectively, compared to that of
SRX246 (cLogP = 4.26, Spartan06). One can therefore reasonably
predict that both compounds will exhibit bioavailability similar
to SRX246, the first orally bioavailable, selective hV1a vasopressin
antagonists with CNS penetration.47 A larger increase in lipophilic-
ity is observed for 11a (1.4 units for cLogP) which is generally the
case with the addition of an iodine atom.
In vitro PAMPA-BBB were also carried out as a predictive tool
for CNS penetration (Table 1). Both the fluorine and the methyl
derivative 11b and 11c exhibited high permeability values (Pe
>4.0 ꢂ 10ꢁ6 cm sꢁ1), which strongly suggest high predicted passive
Blood–Brain Barrier (BBB) permeation.48 Compound 11a exhibited
a lower value (2.0 ꢂ 10ꢁ6 < Pe < 4.0 ꢂ 10ꢁ6 cm sꢁ1), suggesting an
uncertain predicted BBB permeation. However, BOLD (Blood-Oxy-
gen-Level Dependence) imaging studies in rodents demonstrated
that this particular compound has a CNS modulatory effect, dem-
onstrating that it is capable of brain penetration.49
4. Experimental
4.1. Chemistry
4.1.1. (E)-Ethyl 3-(3-iodophenyl)acrylate (1a)
Triethyl phosphonoacetate (9.21 g, 41.12 mmol) in tetrahydro-
furan (65 mL) was treated with 2.5 M n-butyl lithium in hexanes
(12.82 mL, 32.07 mmol) at ꢁ78 °C. The resulting mixture was stir-
red for 10 min and added via a cannula to a pre-cooled solution at
ꢁ78 °C of 3-iodobenzaldehyde (4.77 g, 20.56 mmol) in tetrahydro-
furan (20 mL). The resulting mixture was stirred at ꢁ78 °C for
40 min and slowly warmed to ambient temperature for 45 min.
The reaction was quenched with saturated aqueous NH4Cl
(200 mL). The aqueous solution was extracted with diethyl ether.
The resulting organic layer was dried over magnesium sulfate
and evaporated. The residue was purified by silica gel chromatog-
raphy (90:10 hexanes/ethyl acetate) to give 5.71 g (92%) of com-
pound 1a as a yellow oil; 1H NMR (CDCl3) d 1.32 (t, J = 7.1 Hz,
3H); 4.24 (q, J = 7.1 Hz, 2H); 6.39 (d, J = 16.0 Hz, 1H); 7.09 (dd,
J = J0 = 7.8 Hz, 1H); 7.43–7.46 (m, 1H); 7.54 (d, J = 16.0 Hz, 1H);
7.64–7.69 (m, 1H); 7.83–7.86 (m, 1H).
4.1.2. General procedure for the formation of meta-substituted
cinnamaldehyde (3 steps)
4.1.2.1. Step 1. General procedure for the formation of meta-
substituted ethyl cinnamate ester.
A solution of 1 equiv of
meta-substituted cinnamic acid in dichloromethane (2 mL dichlo-
romethane/mmol acid) was treated by sequential addition of
0.1 equiv of 4-dimethylaminopyridine, 1.05 equiv of 1-[3-(dimeth-
ylamino)propyl]-3-ethylcarbodiimide hydrochloride and 10 equiv
of ethyl alcohol. The reaction was stirred at ambient temperature
until all of the reactants were consumed as measured by thin layer
chromatography (CH2Cl2 100%). When complete (approximately
18 h), the reaction mixture was concentrated under reduced pres-
sure. Water was added to the residue and the aqueous layer was
extracted with dichloromethane. The combined organic layers
were washed successively with 1 N hydrochloric acid, saturated
aqueous sodium bicarbonate, and saturated sodium chloride. The
organic layer was dried over magnesium sulfate, filtered, and
3. Conclusions
We have developed a reliable method to prepare a versatile pre-
cursor that should allow access to both PET (18F, 11C) and SPECT
(
123I) derivatives of SRX246, a highly selective and potent hV1a
antagonist. Furthermore, we have synthesized the F, I, and Me-
bearing reference compounds and have shown that they exhibit
strong hV1a receptor affinity, do not bind to V1b and V2 receptors,
and have a high likelihood of brain penetration based on PAMPA-
BBB results. These findings offer the potential to begin a broader
investigation into the role of the human vasopressin V1a receptor
in various CNS functions using relevant imaging tracers.

K. Fabio et al. / Bioorg. Med. Chem. 20 (2012) 1337–1345
1341
concentrated under reduced pressure. The residue was used di-
rectly for further reactions.
a white solid. 1H NMR (CDCl3) d 1.32 (t, J = 7.1 Hz, 3H); 2.35 (s,
3H); 4.24 (q, J = 7.1 Hz, 2H); 6.40 (d, J = 16.0 Hz, 1H); 7.15–7.19
(m, 1H); 7.22–7.28 (m, 1H); 7.28–7.37 (2H); 7.66 (d, J = 16.0 Hz,
1H).
4.1.2.2. Step 2. General procedure for the reduction of meta-
substituted ethyl cinnamate ester.
A solution of 1 equiv of
4.1.2.8.
(E)-3-(3-Fluorophenyl)prop-2-en-1-ol
(2b).
meta-substituted ethyl cinnamate ester in dichloromethane
(2 mL dichloromethane/mmol ester) was treated slowly by the
dropwise addition of 1 M diisobutylaluminium hydride (2.1 equiv)
solution in dichloromethane at ꢁ78 °C. The resulting mixture was
stirred 2 h at ꢁ78 °C and then quenched with 10% aqueous NaOH
(25 mL/10 mmol ester). The resulting mixture was slowly warm-
up to ambient temperature and the layers separated. The aqueous
layer was extracted with dichloromethane. The combined organic
layers were washed successively with water, 1 N aqueous hydro-
chloric acid, and saturated aqueous sodium chloride. The organic
layer was dried over magnesium sulfate, filtered, and concentrated
under reduced pressure. The residue was used directly for further
reactions or purified by chromatography from an appropriate sol-
vent system before use.
Compound 1b (2.2 g, 11.4 mmol) was treated according to the Gen-
eral Procedure Step 2 to give after flash column chromatography
purification (hexanes/dichloromethane 50:50 to dichloromethane
100%) 1.4 g (82%) of compound 2b as colorless oil. 1H NMR (CDCl3)
d 1.48 (t, J = 5.9 Hz, 1H); 4.30–4.34 (m, 2H); 6.35 (dt, J = 15.8 Hz,
J = 5.5 Hz, 1H); 6.57 (d, J = 15.9 Hz, 1H); 6.91 (ddd, J = J0 = 8.0 Hz,
J0 = 2.2 Hz, 1H); 7.04–7.09 (m, 1H); 7.11–7.14 (m, 1H); 7.23–7.29
(m, 1H).
4.1.2.9. (E)-3-(m-Tolyl)prop-2-en-1-ol (2c).
Compound 1c
(1.73 g, 9.09 mmol) was treated according to the General Proce-
dure Step 2 to give 1.24 g (92%) of compound 2c as colorless oil.
1H NMR (CDCl3) d 1.39 (t, J = 5.9 Hz, 1H); 2.33 (s, 3H); 4.28–4.32
(m, 2H); 6.34 (dt, J = 15.9 Hz, J = 5.7 Hz, 1H); 6.56 (d, J = 15.9 Hz,
1H); 7.03–7.08 (m, 1H); 7.15–7.25 (m, 3H).
4.1.2.3. Step 3. General procedure for the oxidation of
meta-substituted allyl alcohol.
A solution of 1 equiv of
4.1.2.10. (E)-3-(3-Fluorophenyl)acrylaldehyde (3b).
Com-
meta-substituted allyl alcohol in dichloromethane (2 mL dichloro-
methane/mmol alcohol) was treated with 1.5 equiv of pyridinium
dichromate under inert atmosphere. The reaction was stirred at
ambient temperature until all of the reactants were consumed as
measured by thin layer chromatography (CH2Cl2 100%). The result-
ing mixture was diluted with diethyl ether/hexanes (50:50) and fil-
tered through silica gel. The silica gel was rinsed with hexanes/
ethyl acetate (60:40) and the combined filtrate evaporated. The
residue obtained was dissolved in dichloromethane, washed with
0.1 N aqueous HCl, water and dried over magnesium sulfate. The
residue was used directly for further reactions or purified by chro-
matography from an appropriate solvent system before use.The
following compounds were prepared according to these
procedures.
pound 2b (1.4 g, 9.29 mmol) was treated according to the General
Procedure Step 3 to give 1.3 g (96%) of compound 3b as a colorless
oil. 1H NMR (CDCl3) d 6.68 (dd, J = 16.0 Hz, J = 7.5 Hz, 1H); 7.12
(ddd, J = J0 = 8.2 Hz, J00 = 0.95 Hz, 1H); 7.22–7.26 (m, 1H); 7.31–
7.34 (m, 1H); 7.37–7.42 (m, 1H); 7.42 (d, J = 16.0 Hz, 1H); 9.69
2
(d, J = 7.6 Hz, 1H). 13C NMR (CDCl3) d 114.50 (d, JC-F = 22.6 Hz,
2
4
1C), 117.87 (d, JC-F = 21.3 Hz, 1C), 124.19 (d, JC-F = 2.5 Hz, 1C),
3
4
129.39, 130.48 (d, JC-F = 8.8 Hz, 1C), 150.79 (d, JC-F = 2.5 Hz, 1C),
162.78 (d, JC-F = 247.7 Hz, 1C), 193.13.
1
4.1.2.11. (E)-3-(m-Tolyl)acrylaldehyde (3c).
Compound 2c
(1.24 g, 8.36 mmol) was treated according to the General Proce-
dure Step 3 to give after flash column chromatography purification
(hexanes 100% to hexanes/ethyl acetate 90:10) 0.68 g (55%) of
compound 3c as a colorless oil. 1H NMR (CDCl3) d 2.37 (s, 3H);
6.69 (dd, J = 15.9 Hz, J = 7.7 Hz, 1H); 7.22–7.40 (m, 3H); 7.43 (d,
J = 16.0 Hz, 1H); 9.68 (d, J = 7.7 Hz, 1H). 13C NMR (CDCl3) d 21.25,
125.69, 128.36, 128.93, 129.08, 132.10, 133.90, 138.78, 153.10,
193.81.
4.1.2.4. (E)-3-(3-Iodophenyl)prop-2-en-1-ol (2a).
Com-
pound 1a (5.71 g, 18.90 mmol) was treated according to the Gen-
eral Procedure Step 2 to give 4.88 g (99%) of compound 2a as a
yellow oil; 1H NMR (CDCl3) d 1.55 (t, J = 5.9 Hz, 1H); 4.29–4.33
(m, 2H); 6.32 (dt, J = 15.8 Hz, J = 5.5 Hz, 1H); 6.49 (d, J = 15.9 Hz,
1H); 7.02 (dd, J = J0 = 7.8 Hz, 1H); 7.29–7.32 (m, 1H); 7.53–7.56
(m, 1H); 7.71–7.72 (m, 1H).
4.1.3. (E)-3-(3-(Trimethylstannyl)phenyl)acrylaldehyde (4)
Compound 3 (0.5 g, 1.93 mmol) dissolved in dioxane (20 mL)
was treated with hexamethylditin (0.476 g, 1.45 mmol) and tetra-
kis(triphenylphosphine)palladium (0) (0.006 g, 0.0387 mmol). The
reaction was refluxed for 150 min and shielded from light. The
reaction mixture was then cooled down to ambient temperature
and filtered through celite, the celite was washed with dichloro-
methane and the solvent concentrated. The resulting oil was fil-
tered through silica gel (40 g) pre-equilibrated with a mixture of
light ether/dichloromethane (90:10), rinsed with this mixture
and dichloromethane and eluted with ethyl acetate/dichlorometh-
ane (30:70). The fractions collected were then evaporated to give
0.464 g (80%) of compound 4 as bright yellow oil. 1H NMR (CDCl3)
d 0.36 (s, with Sn satellites, d, J = 54.3 Hz, 9H); 6.73 (dd, J = 15.9 Hz,
J = 7.7 Hz, 1H); 7.44–7.56 (m, 3H); 7.64 (br s, 1H); 9.69 (d,
J = 7.7 Hz, 1H). 13C NMR (CDCl3) d ꢁ9.54, 128.09, 128.48, 128.54,
133.41, 135.98, 138.78, 143.67, 153.24, 193.74.
4.1.2.5. (E)-3-(3-Iodophenyl)acrylaldehyde (3a).
Compound
2a (4.88 g, 18.76 mmol) was treated according to the General
Procedure Step 3 to give 4.26 g (88%) of compound 3a as a yellow
solid. 1H NMR (CDCl3) d 6.67 (dd, J = 16.0 Hz, J = 7.6 Hz, 1H); 7.15
(dd, J = J0 = 7.8 Hz, 1H); 7.35 (d, J = 16.0 Hz, 1H); 7.49–7.53 (m,
1H); 7.72–7.77 (m, 1H); 7.87–7.91 (m, 1H); 9.68 (d, J = 7.5 Hz,
1H). 13C NMR (CDCl3) d 94.81, 127.44, 129.54, 130.69, 136.10,
137.22, 139.89, 150.59, 193.22.
4.1.2.6. (E)-Ethyl 3-(3-fluorophenyl)acrylate (1b).
3-Fluoro
cinnamic acid (2 g, 12.04 mmol) was treated according to the
General Procedure Step 1 to give 2.22 g (95%) of compound 1b as
colorless oil. 1H NMR (CDCl3) d 1.33 (t, J = 7.1 Hz, 3H); 4.27 (q,
J = 7.1 Hz, 2H); 6.42 (d, J = 16.0 Hz, 1H); 7.07 (ddd, J = J0 = 8.2 Hz,
J00 = 0.95 Hz, 1H); 7.14–7.24 (m, 1H); 7.28–7.31 (m, 1H); 7.32–
7.38 (1H); 7.63 (d, J = 16.0 Hz, 1H).
4.1.4. General procedure for amide formation from a carboxylic
acid
4.1.2.7.
(E)-Ethyl
3-(m-tolyl)acrylate
(1c).
3-Methyl
(R)-tert-Butyl 3-(((benzyloxy)carbonyl)amino)-4-oxo-4-(((R)-1-
phenylethyl)amino)butanoate (5).18 A solution of 0.6 g (1.75 mmol)
cinnamic acid (1.58 g, 9.77 mmol) was treated according to the
General Procedure Step 1 to give 1.85 g (93%) of compound 1c as
of N-benzyloxycarbonyl-D-aspartic acid b-t-butyl ester monohydrate

1342
K. Fabio et al. / Bioorg. Med. Chem. 20 (2012) 1337–1345
in 10 mL dichloromethane was treated by sequential addition of
0.238 mL (1.84 mmol) of (R)- -methylbenzylamine, 0.237 g
4.1.8. General procedure for formation of a 2-azetidinone from
an imine and an acetyl chloride
a
(1.84 mmol) of 1-hydroxy-7-benzotriazole, and 0.337 g (1.84 mmol)
of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride.
After 18 h stirring at ambient temperature, the reaction mixture was
washed sequentially with a saturated sodium bicarbonate solution
and with saturated sodium chloride solution. The organic layer was
evaporated to give 0.726 g (97%) of compound 5 as an off-white solid.
1H NMR (CDCl3) d 1.38 (s, 9H); 1.43 (d, J = 6.9 Hz, 3H); 2.54 (dd,
J = 17.2 Hz, J = 7.2 Hz, 1H); 2.87 (dd, J = 17.3 Hz, J = 4.0 Hz, 1H); 4.46–
4.50 (m, 1H); 4.99–5.15 (m, 3H); 5.92–5.96 (m, 1H); 6.78–6.82 (m,
1H); 7.21–7.33 (m, 10 H).
4.1.8.1. Step 1: General procedure for formation of an imine
from an amino acid derivative.
A solution of 1 equiv of an a-
amino acid ester or amide in dichloromethane was treated sequen-
tially with 1 equiv of an appropriate aldehyde, and a desiccating
agent, such as magnesium sulfate or sodium sulfate, in the amount
of about 14 equiv (2 g) of desiccating agent per gram of starting a-
amino acid ester or amide. The reaction was stirred at ambient
temperature until all of the reactants were consumed as measured
by thin layer chromatography (CH2Cl2 95%/MeOH 5%). When com-
plete, the reaction mixture was then filtered, the filter cake was
washed with dichloromethane, and the filtrate concentrated under
reduced pressure to provide the desired imine that was used di-
rectly in the subsequent step.
4.1.5. General procedure for hydrolysis of a tert-butyl ester
A solution of tert-butyl ester derivative in formic acid, typically
1 g in 10 mL, is stirred at ambient temperature until no more ester
is detected by thin layer chromatography (dichloromethane 95%/
methanol 5%), a typical reaction time being around 3 h. The formic
acid is then evaporated under reduced pressure to yield the corre-
sponding carboxylic acid.The following compound was prepared
according to this procedure.
4.1.8.2. Step 2: General procedure for the [2+2] cyclo-addition of
an imine and an acetyl chloride.
A dichloromethane solution
of the imine (10 mL dichloromethane/1 g imine) was cooled to 0 °C. To
this cooled solution was added 1.5 equiv triethylamine, followed by
the dropwise addition of a dichloromethane solution of 1.1 equiv of
an acetyl chloride 8 (10 mL of dichloromethane/1 g of 8). The reaction
mixture was allowed to warm to ambient temperature over 1 h and
was then quenched by the addition of a saturated aqueous solution
of ammonium chloride. The resulting mixture was partitioned be-
tween water and dichloromethane. The layers were separated and
the organic layer was washed successively with 1 N hydrochloric acid,
saturated aqueous sodium bicarbonate, and saturated aqueous sodium
chloride. The organic layer was dried over magnesium sulfate, filtered
and concentrated under reduced pressure. The residue was purified by
chromatography (hexanes/ethyl acetate or dichloromethane/metha-
nol) or by crystallization from dichloromethane/methanol.The follow-
ing compounds were obtained according to this sequence of
procedures.
4.1.5.1.
(R)-3-(((Benzyloxy)carbonyl)amino)-4-oxo-4-(((R)-1-
acid (12). Compound
phenylethyl)amino)butanoic
5
(0.726 g, 1.70 mmol) was hydrolyzed to give 0.614 g (97%) of compound
12 as a white solid; 1H NMR (MeOH-d4) d 1.40 (br s, 3H); 2.56–2.81 (m,
2H); 5.0–5.15 (m, 3H); 7.11–7.41 (m, 10H).
4.1.6. Benzyl ((R)-4-([1,40-bipiperidin]-10-yl)-1,4-dioxo-1-(((R)-
1-phenylethyl)amino)butan-2-yl)carbamate (13)
Compound 13 was prepared using the General procedure for
amide formation from a carboxylic acid except that N-benzyloxy-
carbonyl-
D
-aspartic acid b-t-butyl ester was replaced with com-
-methylbenzylamine
4.1.8.3. (R)-4-([1,40-Bipiperidin]-10-yl)-4-oxo-2-((3S,4R)-2-oxo-3-
((S)-2-oxo-4-phenyloxazolidin-3-yl)-4-((E)-3-(trimethylstan-
nyl)styryl)azetidin-1-yl)-N-((R)-1-phenylethyl)butanamide
pound 12 (0.614 g, 1.65 mmol) and (R)-
a
was replaced with 4-(1-piperidinyl)piperidine. Compound 13 was
obtained as a pink solid (0.718 g, 83%). 1H NMR (CDCl3) d 1.22–
1.34 (m, 1H); 1.34–1.47 (m, 7H); 1.47–1.62 (m, 4H); 1.72–1.90
(m, 2H); 2.33–2.60 (m, 6H); 2.80–3.01 (m, 1H); 3.06–3.20 (m,
1H); 3.66–3.83 (m, 1H); 4.46–4.67 (m, 2H); 4.95–5.17 (m, 3H);
6.22–6.38 (m, 1H); 7.11–7.40 (m, 10H).
(16).
Imine 15 prepared from 0.445 g (1.15 mmol) of 14 and
(E)-3-(3-(trimethylstannyl)phenyl)acrylaldehyde 4 was combined
with 2-(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chloride 8 to give
0.639 g (65%) of compound 16 as a yellow solid after flash column
chromatography (CH2Cl2 99% to 90% gradient/MeOH 1% to 10% gra-
dient, NH4OH <1%). 1H NMR (CDCl3) d 0.29 (s, with Sn satellites, d,
J = 54.2 Hz, 9H); 1.22–1.50 (m, 4H); 1.50–1.65 (m, 7H); 1.65–1.84
(m, 2H); 2.30–2.55 (m, 6H); 2.85–2.96 (m, 1H); 3.14–3.21 (m, 1H);
3.34–3.43 (m, 1H); 3.75–3.86 (m, 1H); 3.96–4.08 (m, 1H); 4.15 (t,
J = 8.5 Hz); 4.18–4.26 (m, 1H); 4.41–4.55 (m, 1H); 4.61–4.67 (m,
1H); 4.67–4.77 (m, 2H); 5.0–5.13 (m, 1H); 6.33–6.41 (m, 1H);
6.76–6.84 (m, 1H); 7.15–7.49 (m, 15H); 8.21–8.29 (m, 1H). 13C
NMR (CD3CN) d ꢁ9.63, 25.38/25.42, 26.96/27.01, 28.32/28.40,
28.69/28.90, 34.13/34.19, 42.09/42.19, 45.61/45.47, 50.25/50.26,
50.78/50.83, 55.60, 55.80, 61.27, 63.06, 63.09/63.14, 63.88/63.92,
71.90/71.92, 123.43/123.52, 126.76, 127.04, 127.63, 128.31/
128.36, 129.34, 130.09, 130.21, 135.76, 136.56/136.58, 136.96,
138.52/138.55, 138.97/139.04, 143.98, 145.75, 159.35/159.38,
164.62/164.62, 168.60/168.68, 169.15/169.21. HRMS (FAB) calcd
for C45H58N5O5Sn 868.3464, found 868.3466 (M+H)+.
4.1.7. General procedure for hydrogenolysis of a
benzyloxycarbonyl amine
4.1.7.1. (R)-4-([1,40-Bipiperidin]-10-yl)-2-amino-4-oxo-N-((R)-1-
phenylethyl)butanamide (14).
A suspension of 0.718 g
(1.37 mmol) of compound 13 and palladium (5 wt % on activated
carbon, 0.35 g) in 20 mL methanol was held under an atmosphere
of hydrogen for 18 h. The reaction was filtered to remove the pal-
ladium over carbon and the filtrate was evaporated to give 0.484 g
(87%) of compound 14 as a colorless oil. 1H NMR (MeOH-d4) d1.23–
1.39 (m, 1H); 1.39–1.52 (m, 7H); 1.52–1.76 (m, 4H); 1.80–1.92 (m,
2H); 2.42–2.61 (m, 7H); 2.61–2.69 (m, 1H); 2.69–2.80 (m, 1H);
2.87–3.03 (m, 1H); 2.64–3.74 (m, 1H); 3.88–4.00 (m, 1H); 4.49–
4.59 (m, 1H); 4.92–5.10 (m, 1H); 7.16–7.36 (m, 5H).The following
compound was obtained according to this procedure.
4.1.7.2.
ethyl)amino)butanoate (6).
(R)-tert-Butyl
3-amino-4-oxo-4-(((R)-1-phenyl-
Compound 5 (0.618 g, 1.45 m
4.1.8.4. (R)-tert-Butyl 3-((2R,3S)-2-((E)-3-iodostyryl)-4-oxo-3-
((S)-2-oxo-4-phenyloxazolidin-3-yl)azetidin-1-yl)-4-oxo-4-(((R)
mol) was hydrogenolized to give 0.407 g (96%) of 6 as an off-white
solid; 1H NMR (CDCl3) d 1.40 (s, 9H); 1.47 (d, J = 6.9 Hz, 3H); 1.98
(br s, 2H); 2.49 (dd, J = 7.9 Hz, J = 17.7 Hz, 1H); 2.83 (dd,
J = 3.6 Hz, J = 16.7 Hz, 1H); 3.69 (br s, 1H); 4.99–5.10 (m, 1H);
7.19–7.33 (m, 5H); 7.65–7.68 (m, 1H).
-1-phenylethyl)amino)butanoate (9a).
Imine 7a prepared
from 0.815 g (2.78 mmol) of 6 and of 3a was combined with 2-
(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chloride 8 to give 1.462 g
(71%) of compound 9a as a white solid after flash column chroma-
tography (hexanes 95% to 25% gradient/ethyl acetate 5% to 75%

K. Fabio et al. / Bioorg. Med. Chem. 20 (2012) 1337–1345
1343
gradient). 1H NMR (CDCl3) d 1.35 (s, 9H); 1.58 (d, J = 7.1 Hz, 3H); 3.09
(dd, J = 3.6 Hz, J = 17.8 Hz, 1H); 3.17 (dd, J = 10.7 Hz, J = 17.8 Hz, 1H);
3.92 (dd, J = 3.5 Hz, J = 10.7 Hz, 1H); 4.16 (dd, J = 7.6 Hz, J = 8.5 Hz,
1H); 4.25 (d, J = 5.0 Hz, 1H); 4.58 (dd, J = 5.0 Hz, J = 9.3 Hz, 1H);
4.67 (t, J = 8.7 Hz, 1H); 4.73 (t, J = 8.2 Hz, 1H); 5.04–5.13 (m, 1H);
6.29 (dd, J = 9.3 Hz, J = 15.8 Hz, 1H); 6.62 (d, J = 15.8 Hz, 1H); 7.02
(t, J = 7.8 Hz, 1H); 7.11–7.28 (m, 4H); 7.34–7.48 (m, 7H); 7.58–7.65
(m, 2H); 8.05–8.11 (m, 1H).
J = 16.0 Hz, 1H); 6.92–7.05 (m, 3H); 7.19–7.50 (m, 11H); 8.25–
8.31 (m, 1H). 13C NMR (CDCl3) d 21.66, 34.40, 49.83, 54.41, 61.02,
62.02, 63.44, 71.15, 113.29 (d, JC-F = 21.3 Hz, 1C), 115.76 (d, JC-
2
2
F = 21.3 Hz, 1C), 122.29, 122.83, 126.28, 127.19, 128.52, 129.76,
3
3
129.80, 130.33 (d, JC-F = 7.5 Hz, 1C), 135.82, 137.34 (d, JC-
1
F = 7.5 Hz, 1C), 138.10, 143.59, 158.04, 162.97 (d, JC-F = 246.5 Hz,
1C), 163.32, 167.28, 174.48.
4.1.9.3. (R)-3-((2R,3S)-2-((E)-3-Methylstyryl)-4-oxo-3-((S)-2-oxo
-4-phenyloxazolidin-3-yl)azetidin-1-yl)-4-oxo-4-(((R)-1-phenyl
4.1.8.5. (R)-tert-Butyl 3-((2R,3S)-2-((E)-3-fluorostyryl)-4-oxo-3-
((S)-2-oxo-4-phenyloxazolidin-3-yl)azetidin-1-yl)-4-oxo-4-
ethyl)amino)butanoic acid (10c).
Compound 9c (0.126 g,
(((R)-1-phenylethyl)amino)butanoate (9b).
pared from 0.330 g (1.13 mmol) of 6 and of 3b was combined with
2-(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chloride to give
Imine 7b pre-
0.20 mmol) was hydrolyzed to give 0.116 g (quantitative yield) of
compound 10c as an off-white solid; 1H NMR (CDCl3) d 1.59 (d,
J = 7.1 Hz, 3H); 2.34 (s, 3H); 3.22 (dd, J = 17.8 Hz, J = 8.1 Hz, 1H);
3.30 (dd, J = 17.8 Hz, J = 4.60 Hz, 1H); 3.92 (dd, J = 9.6 Hz,
J = 4.6 Hz, 1H); 4.15 (t, J = 8.2 Hz, 1H); 4.26 (d, J = 5.0 Hz, 1H);
4.52 (dd, J = 9.4 Hz, J = 5.0 Hz, 1H); 4.66 (t, J = 8.7 Hz, 1H); 4.73 (t,
J = 8.0 Hz, 1H); 5.04–5.14 (m, 1H); 6.29 (dd, J = 15.8 Hz, J = 9.6 Hz,
1H); 6.69 (d, J = 15.7 Hz, 1H); 7.01–7.47 (m, 14H); 8.29–8.34 (m,
1H). 13C NMR (CDCl3) d 21.36, 21.98, 34.45, 49.86, 54.29, 60.99,
62.00, 63.68, 71.07, 120.37, 126.19, 126.91, 127.19, 127.78,
128.44, 128.96, 129.40, 129.71, 135.10, 135.82, 138.37, 139.60,
143.69, 158.03, 163.43, 167.39, 174.64.
8
0.539 g (76%) of compound 9b as a white solid after flash column
chromatography (hexanes 100% to 70% gradient/ethyl acetate 0%
to 30% gradient). 1H NMR (CDCl3)
d 1.35 (s, 9H); 1.59
(d, J = 7.0 Hz, 3H); 3.10 (dd, J = 3.5 Hz, J = 17.5 Hz, 1H); 3.19
(dd, J = 9.5 Hz, J = 18.0 Hz, 1H); 3.92 (dd, J = 3.2 Hz, J = 10.8 Hz,
1H); 4.15 (t, J = 8.2 Hz, 1H); 4.25 (d, J = 5.0 Hz, 1H); 4.58 (dd,
J = 5.0 Hz, J = 9.5 Hz, 1H); 4.66 (t, J = 8.7 Hz, 1H); 4.73 (t,
J = 8.2 Hz, 1H); 5.06–5.14 (m, 1H); 6.31 (dd, J = 9.2 Hz, J = 15.7 Hz,
1H); 6.68 (d, J = 16.0 Hz, 1H); 6.92–7.02 (m, 3H); 7.15–7.30 (m,
4H); 7.35–7.47 (m, 7H); 8.07–8.14 (m, 1H).
4.1.10. Compounds 11a, 11b, and 11c were prepared according
to the ‘general procedure for amide formation from a carboxyl
4.1.8.6. (R)-tert-Butyl 3-((2R,3S)-2-((E)-3-methylstyryl)-4-oxo-3-
((S)-2-oxo-4-phenyloxazolidin-3-yl)azetidin-1-yl)-4-oxo-4-(((R)
ic acid’, except that N-benzyloxycarbonyl-D-aspartic acid a-t-bu
-1-phenylethyl)amino)butanoate (9c).
from 0.40 g (1.37 mmol) of 6 and of 3c was combined with 2-
(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chloride to give
Imine 7c prepared
tyl ester monohydrate was replaced with 10a, 10b and 10c and
(R)-a-methylbenzylamine replaced by 4-(1-piperidinyl) piperid
8
ine, all compounds exhibited an 1H and 13C NMR spectrum con
sistent with the assigned structure
0.469 g (55%) of compound 9c as a white solid after flash column
chromatography (hexanes 95% to 25% gradient/ethyl acetate 5%
4.1.10.1. (R)-4-([1,40-Bipiperidin]-10-yl)-2-((2R,3S)-2-((E)-3-iodo-
styryl)-4-oxo-3-((S)-2-oxo-4-phenyloxazolidin-3-yl)azetidin-1-
to 75% gradient) 1H NMR (CDCl3)
d 1.35 (s, 9H); 1.59 (d,
J = 7.1 Hz, 3H); 2.34 (s, 3H); 3.10 (dd, J = 3.5 Hz, J = 17.7 Hz, 1H);
3.19 (dd, J = 10.9 Hz, J = 17.8 Hz, 1H); 3.94 (dd, J = 3.5 Hz,
J = 10.8 Hz, 1H); 4.14 (t, J = 8.5 Hz, 1H); 4.24 (d, J = 5.0 Hz, 1H);
4.58 (dd, J = 5.0 Hz, J = 9.4 Hz, 1H); 4.65 (t, J = 8.8 Hz, 1H); 4.73 (t,
J = 8.4 Hz, 1H); 5.03–5.12 (m, 1H); 6.32 (dd, J = 9.5 Hz, J = 15.8 Hz,
1H); 6.72 (d, J = 15.8 Hz, 1H); 7.0–7.23 (m, 7H); 7.33–7.48 (m,
7H); 8.10–8.16 (m, 1H).
yl)-4-oxo-N-((R)-1-phenylethyl)butanamide
pound 11a was prepared using the ‘General procedure for amide
formation from a carboxylic acid’, except that N-benzyloxycar-
(11a).
Com-
bonyl-D-aspartic acid a-t-butyl ester monohydrate was replaced
with 10a (0.60 g, 0.88 mmol) and 3-(trifluoromethyl)benzylamine
was replaced with 4 (1-piperidinyl)piperidine. Compound 11a
(0.72 g, 98%) was obtained as an off-white solid after flash silica
gel column chromatography (CH2Cl2 99.5% to 88% gradient/MeOH
0.5% to 12% gradient, NH4OH <1%). 1H NMR (CDCl3) d 1.24–1.49 (m,
4H); 1.52–1.65 (m, 7H); 1.74–1.85 (m, 2H); 2.30–2.60 (m, 6H);
2.84–2.96 (m, 1H); 3.12–3.21 (m, 1H); 3.31–3.40 (m, 1H); 3.75–
3.83 (m, 1H); 3.98–4.04 (m, 1H); 4.16 (t, J = 7.8 Hz, 1H); 4.20–
4.26 (m, 1H); 4.40–4.52 (m, 1H); 4.63–4.77 (m, 3H); 5.05–5.14
(m, 1H); 6.26–6.34 (m, 1H); 6.61–6.69 (m, 1H); 6.99–7.06 (m,
1H); 7.09–7.14 (m, 1H); 7.17–28 (m, 4H); 7.32–7.49 (m, 1H);
7.57–7.65 (m, 2H); 8.16–8.25 (m, 1H). 13C NMR (CD3CN) d 22.88,
25.39/25.42, 26.95/ 27.00, 28.33/28.38, 28.69/28.89, 34.21/34.27,
42.11/42.19, 45.49/45.62, 50.10/50.11, 50.77/50.82, 55.52/55.71,
61.19, 63.05/63.08, 63.14/63.18, 63.62/63.64, 71.93/71.95, 95.23,
125.24/125.31, 126.85, 126.93, 127.82, 128.35/128.41, 129.46,
130.11, 130.22, 131.70, 136.47, 136.81/136.86, 138.20, 138.62/
138.64, 139.46, 145.67, 159.27, 164.64/164.66, 168.59/168.66,
169.10/169.58. HRMS (FAB) calcd for C42H49IN5O5 830.2773, found
830.2782 (M+H)+.
4.1.9. The following compounds were prepared according to the
procedure ‘general procedure for hydrolysis of a tert-butyl
ester’
4.1.9.1. (R)-3-((2R,3S)-2-((E)-3-Iodostyryl)-4-oxo-3-((S)-2-oxo-4-
phenyloxazolidin-3-yl)azetidin-1-yl)-4-oxo-4-(((R)-1-phenyleth
yl)amino)butanoic acid (10a).
Compound 9a (1.46 g,
1.98 mmol) was hydrolyzed to give 1.35 g (quantitative yield) of
10a as an off-white solid; 1H NMR (CDCl3) d 1.57 (d, J = 7.1 Hz,
3H); 3.25 (d, J = 7.6 Hz, 2H); 3.92 (t, J = 7.1 Hz, 1H); 4.16 (dd,
J = 8.4 Hz, J = 7.5 Hz, 1H); 4.27 (d, J = 5.0 Hz, 1H); 4.52 (dd,
J = 9.2 Hz, J = 5.0 Hz, 1H); 4.66 (t, J = 8.7 Hz, 1H); 4.72 (t,
J = 8.0 Hz, 1H); 5.03–5.11 (m, 1H); 6.25 (dd, J = 15.8 Hz, J = 9.3 Hz,
1H); 6.47 (d, J = 15.8 Hz, 1H); 7.02 (t, J = 7.8 Hz, 1H); 7.03–7.06
(m, 1H); 7.08–7.28 (m, 3H); 7.30–7.46 (m, 7H); 7.58–7.63 (m,
2H), 8.23–8.28 (m, 1H).
4.1.9.2. (R)-3-((2R,3S)-2-((E)-3-Fluorostyryl)-4-oxo-3-((S)-2-oxo-
4-phenyloxazolidin-3-yl)azetidin-1-yl)-4-oxo-4-(((R)-1-phenyl-
4.1.10.2. (R)-4-([1,40-Bipiperidin]-10-yl)-2-((2R,3S)-2-((E)-3-flu-
orostyryl)-4-oxo-3-((S)-2-oxo-4-phenyloxazolidin-3-yl)azetidin
ethyl)amino)butanoic acid (10b).
Compound 9b (0.535 g,
0.85 mmol) was hydrolyzed to give 0.487 g (quantitative yield) of
compound 10b as an off-white solid; 1H NMR (CDCl3) d 1.59 (d,
J = 7.0 Hz, 3H); 3.25 (d, J = 7.0 Hz, 2H); 3.92 (t, J = 7.2 Hz, 1H);
4.16 (t, J = 8.0 Hz, 1H); 4.27 (d, J = 5.0 Hz, 1H); 4.52 (dd, J = 5.0 Hz,
J = 9.5 Hz, 1H); 4.66 (t, J = 8.7 Hz, 1H); 4.72 (t, J = 8.0 Hz, 1H);
5.05–5.14 (m, 1H); 6.28 (dd, J = 15.5 Hz, J = 9.2 Hz, 1H); 6.66 (d,
-1-yl)-4-oxo-N-((R)-1-phenylethyl)butanamide (11b).
pound 11b was prepared using the ‘General procedure for amide for-
mation from a carboxylic acid’, except that N-benzyloxycarbonyl-
aspartic acid -t-butyl ester monohydrate was replaced with 10b
(0.030 g, 0.052 mmol) and 3-(trifluoromethyl)benzylamine was re-
placed with 4 (1-piperidinyl)piperidine. Compound 11b (0.037 g,
Com-
D-
a

1344
K. Fabio et al. / Bioorg. Med. Chem. 20 (2012) 1337–1345
quantitative yield) was obtained as an off-white solid after flash silica
gel column chromatography (CH2Cl2 99% to 90% gradient/MeOH 1% to
10% gradient, NH4OH <1%). 1H NMR (CDCl3) d1.26–1.47 (m, 4H); 1.49–
1.65 (m, 7H); 1.70–1.82 (m, 2H); 2.33–2.55 (m, 6H); 2.83–2.95 (m, 1H);
3.11–3.22 (m, 1H); 3.33–3.42 (m, 1H); 3.74–3.82 (m, 1H); 3.95–4.02
(m, 1H); 4.15 (t, J = 8.1 Hz); 4.19–4.25 (m, 1H); 4.40–4.50 (m, 1H);
4.62–4.75 (m, 3H); 5.05–5.14 (m, 1H); 6.29–6.38 (m, 1H); 6.69–6.75
(m, 1H); 6.90–7.01 (m, 3H); 7.15–7.29 (m, 4H); 7.31–7.47 (m, 7H);
8.20–8.28 (m, 1H). 13C NMR (CD3CN) d 22.79, 25.59, 27.21/27.18,
28.47/28.50, 28.81/29.03, 34.18/34.25, 42.20/42.28, 45.57/45.70,
50.09, 50.78/50.84, 55.64/55.84, 61.23, 62.96/62.98, 63.12/63.16,
1 ml/min; column, Eclipse XDB-C18 4.6 ꢂ 150 mm
5 lm; UV
detection, 257 nm.
4.2.2. Cold conversion to 11b via electrophilic
fluorodestannylation
Compound 16 (5 mg, 5.77 lmol) was treated with a solution of
SelectFluor tetrafluoroborate and silver triflate in anhydrous ace-
tone. The reaction was stirred 30 min at room temperature under
inert atmosphere (argon). The resulting mixture was concentrated
under reduced pressure and reconstituted in a mixture 1:1 meth-
anol/water prior to loading onto a conditioned Oasis HLB car-
2
63.63/63.67, 71.94/71.96, 114.00 (d, JC-F = 21.3 Hz, 1C), 115.99 (d,
tridge. The cartridge was washed three times with 500 lL
4
2JC-F = 21.3 Hz, 1C), 123.72 (d, JC-F = 2.5 Hz, 1C), 125.19/125.27,
water/methanol (95%/5%), eluted with acetonitrile (2 ꢂ 500
lL)
126.92, 127.83, 128.31/128.37, 129.38, 130.10, 130.22, 131.60 (d, 3JC-
and concentrated under reduced pressure. The mixture was ana-
lyzed by HPLC (method A) and compared to the previously pre-
pared fluorinated standard 11b. An estimated yield of 63% was
3
F = 7.5 Hz, 1C), 137.21/137.25, 138.56/138.60, 139.53 (d, JC-
F = 6.3 Hz, 1C), 145.66, 159.30/159.33, 163.94 (1JC-F = 243.9 Hz, 1C),
164.62/164.65, 168.59/168.66, 169.06/169.12. HRMS (FAB) calcd for
C
determined by RP-HPLC. MS (ESI), calcd for C42H49FN5O5 722.36,
+
42H49FN5O5 722.3712, found 722.3703 (M+H)+.
found 722.38 (M+H)
.
4.1.10.3. (R)-4-([1,40-Bipiperidin]-10-yl)-2-((2R,3S)-2-((E)-3-meth
ylstyryl)-4-oxo-3-((S)-2-oxo-4-phenyloxazolidin-3-yl)azetidin-1
4.2.3. Hot conversion to [123I]11a via iododestannylation
A solution of 16 (50 g, 0.057 mol) in ethanol (500
added to 500
L [123I]NaI (11.0 mCi) in aqueous NaOH (0.01 N),
mol) chloramine T trihydrate in 25 L H2O and
L aqueous HCl (1 M). The reaction was stirred for 5 min, then
L mobile phase A from HPLC was added and the resulting
l
l
lL) was
-yl)-4-oxo-N-((R)-1-phenylethyl)butanamide (11c).
Com-
l
pound 11c was prepared using the ‘General procedure for amide
formation from a carboxylic acid’, except that N-benzyloxycar-
bonyl-D-aspartic acid a-t-butyl ester monohydrate was replaced
26
20
500
l
l
l
g (0.11
l
l
with 10c (0.03 g, 0.053 mmol) and 3-(trifluoromethyl)benzylamine
was replaced with 4 (1-piperidinyl)piperidine. Compound 11c
(0.038 g, quantitative yield) was obtained as an off-white solid
after flash silica gel column chromatography (CH2Cl2 99% to 90%
gradient/MeOH 1% to 10% gradient, NH4OH <1%). 1H NMR (CDCl3)
d 1.29–1.53 (m, 4H); 1.56–1.76 (m, 7H); 1.83–1.94 (m, 2H); 2.33
(m, 3H); 2.41–2.69 (m, 6H); 2.85–2.98 (m, 1H); 3.11–3.22 (m,
1H); 3.32–3.42 (m, 1H); 3.78–3.89 (m, 1H); 3.97–4.06 (m, 1H);
4.13 (t, J = 8.3 Hz, 1H); 4.19–4.25 (m, 1H); 4.43–4.58 (m, 1H);
4.61–4.78 (m, 3H); 5.02–5.12 (m, 1H); 6.28–6.38 (m, 1H); 6.70–
6.79 (m, 1H); 6.99–7.23 (m, 1H); 7.29–7.47 (m, 7H); 8.21–8.30
(m, 1H). 13C NMR (CD3CN) d 21.48, 22.92, 25.62, 27.23/27.25,
28.49/28.53, 28.83, 29.06, 34.18/34.25, 42.21/42.29, 45.59/45.29,
50.14, 50.80/50.86, 55.58/55.79, 61.25, 62.98/63.00, 63.09/63.14,
63.92/63.96, 71.91/71.94, 123.12/123.18, 124.67, 126.89, 127.75,
128.32/128.38, 128.49, 129.39, 129.71, 130.11, 130.19, 130.22,
137.04, 138.56/138.60, 138.82/138.87, 139.41, 145.70, 159.33,
164.65/164.68, 168.61/168.68, 169.12/169.18. HRMS (FAB) calcd
for C43H52N5O5 717.3963, found 718.3954 (M+H)+.
solution purified by HPLC (method B). [123I]11a was obtained
with an estimated radiochemical yield of 88% and a specific activ-
ity of 9.7 mCi (94% radiochemical purity of 94%). HPLC method B:
instrument, Agilent 1120; mobile phase A, 10 mM ammonium
acetate in water; mobile phase B, 10 mM ammonium acetate in
acetonitrile; gradient method, 100/0% A/B to 20/80% A/B in
30 min, hold for 10 min; injection volume, 10 lL; flow rate:
1 ml/min; column, Eclipse Plus C18 4.6 ꢂ 250 mm 5
lm; UV
detection, 257 nm.
4.3. Biological evaluation
4.3.1. V1a receptor binding assay
The hV1a expressing cell line, cell culture conditions, and the
cell-based receptor binding assay procedures were performed
according to the methods described by Thibonnier et al.,50 with
modifications as reported earlier.18
4.2. Radiochemistry and conversions to cold analogs by
destannylation
4.4. Permeability assay
4.4.1. General procedure for the PAMPA-BBB assay
4.2.1. Cold conversion to 11a via iododestannylation
Parallel Artificial Membrane Permeability Assays (PAMPA) to
model the Blood–Brain Barrier (BBB) was run according to a pub-
lished procedure.48 Stock solutions of test compounds and stan-
dards (5 mg/mL) were prepared in DMSO. Stock solution in
A solution of 16 (1.27
with 1 equiv NaI (1.27 mol), 10 equiv of chloramine T trihydrate
(12.7 mol) and 100 L aqueous HCl (1 M). The reaction was stir-
red for 5 min and quenched by the addition of sodium thiosulfate
(100 L) and sodium bicarbonate (200 L). The mixture was loaded
onto a conditioned Oasis HLB cartridge. The cartridge was washed
three times with 250 L water/methanol (95%/5%), eluted with
acetonitrile (2 ꢂ 500 L) and concentrated under reduced pres-
lmol) in ethanol (200 lL) was treated
l
l
l
DMSO (5 mg/mL) was diluted 200-fold (10
versal buffer pH 7.4.51 Three hundred microliters of secondary
stock solution (25 g/mL) were transferred to wells in donor
lL in 1.99 mL) in uni-
l
l
l
l
plate. The donor plate used was a PTFE 96-well plate (catalog
No. MSSACCEPT0R, Millipore). A PVDF filter membrane plate (cat-
alog No. MAIPN4550, Millipore) was coated with 4 lL porcine
brain lipid (Avanti Polar) in dodecane (20 mg/mL). A PVDF filter
membrane plate served as the acceptor plate and was immedi-
ately filled with 300 lL universal buffer pH 7.4. The plate was left
undisturbed at room temperature for 18 h. Verapamil and the-
ophylline were used as standard for high and low permeability,
respectively. Effective permeability (Pe) was calculated according
to the equation reported by Faller.52
l
sure. The mixture was analyzed by HPLC (method A) and com-
pared to the previously prepared iodinated standard 11a. An
estimated yield of 98% was determined by RP-HPLC. MS (ESI), calcd
for C42H49IN5O5 830.27, found 830.29 (M+H)+. HPLC method A:
instrument, Agilent 1120; mobile phase A, methanol 10%/water
90%/TFA 0.05%; mobile phase B, methanol 90%/water 10%/TFA
0.05%; linear gradient method, 20/80% A/B to 0/100% A/B in
3 min, hold at 100% B for 4 min; injection volume, 5 lL; flow rate:

K. Fabio et al. / Bioorg. Med. Chem. 20 (2012) 1337–1345
1345
24. Wersinger, S. R.; Kelliher, K. R.; Zufall, F.; Lolait, S. J.; O’Carroll, A. M.; Young, W.
S. Horm. Behav. 2004, 46, 638.
Acknowledgments
25. Landgraf, R.; Wigger, A. Stress 2003, 111.
Supported in part by NIMH awards R44MH063663 and
R44MH087001. The technical assistance of Carrie Garippa is
recognized.
26. Keck, M. E.; Welt, T.; Müller, M. B.; Uhr, M.; Ohl, M.; Ohl, F.; Wigger, A.; Toschi,
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