Structure-activity relationship of benzodiazepine derivatives as LXXLL peptide mimetics that inhibit the interaction of vitamin D receptor with coactivators

Article abstract of DOI:10.1016/j.bmc.2012.11.042

Suppression of vitamin D receptor (VDR)-mediated transcription is expected to be of therapeutic value in Paget's disease of bone. It is known that interaction between VDR and coactivators is necessary for VDR transactivation, and the interaction occurs wh

Full text of DOI:10.1016/j.bmc.2012.11.042

Accepted Manuscript
Structure-activity relationship of benzodiazepine derivatives as LXXLL peptide
mimetics that inhibit the interaction of vitamin D receptor with coactivators
Yusuke Mita, Kosuke Dodo, Tomomi Noguchi-Yachide, Hisayoshi Kobayashi,
Yuichi Hashimoto, Minoru Ishikawa
PII:
S0968-0896(12)00935-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2012.11.042
BMC 10449
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
24 October 2012
14 November 2012
20 November 2012
Please cite this article as: Mita, Y., Dodo, K., Noguchi-Yachide, T., Kobayashi, H., Hashimoto, Y., Ishikawa, M.,
Structure-activity relationship of benzodiazepine derivatives as LXXLL peptide mimetics that inhibit the interaction
of vitamin D receptor with coactivators, Bioorganic & Medicinal Chemistry (2012), doi: http://dx.doi.org/10.1016/
j.bmc.2012.11.042
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Structure-activity relationship of benzodiazepine derivatives as LXXLL peptide
mimetics that inhibit the interaction of vitamin D receptor with coactivators
Yusuke Mitaa, Kosuke Dodob, Tomomi Noguchi-Yachidea, Hisayoshi Kobayashia,
Yuichi Hashimotoa and Minoru Ishikawaa*
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0032, Japan
b
Synthetic Organic Chemistry Laboratory, RIKEN (The Institute of Physical and
Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Abstract
Suppression of vitamin D receptor (VDR)-mediated transcription is expected to be of
therapeutic value in Paget’s disease of bone. It is known that interaction between VDR
and coactivators is necessary for VDR transactivation, and the interaction occurs when
VDR recognizes an LXXLL peptide motif of coactivators. We previously reported that
benzodiazepine derivatives designed as LXXLL peptide mimetics inhibited the
interaction of VDR and coactivators, and reduced VDR transcription. Here, we
investigated the structure-activity relationship of 7- and 8-substituted benzodiazepine
derivatives, and established that the amino group at the 8-position is critical for the
inhibitory activity.
Keyword
Vitamin D; Antagonist; Co-factor; Nuclear receptor; Non-peptide; Structure-activity
relationship; Protein interaction inhibitor; LXXLL
1. Introduction
Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and is
associated with regulation of calcium homeostasis, bone mineralization, proliferation,
differentiation of various types of cells, and immune modulation. 1-3 The physiological
agonist of VDR is activated vitamin D3, 1,25(OH)2D3 (Fig. 1). Binding of 1,25(OH)2D3
to the VDR-ligand binding domain (LBD) allows VDR to interact with vitamin
D-responsive elements (VDREs), to change its conformation through folding of helix

12, and to recruit cofactors, including vitamin D-interacting protein (DRIP) 205.4,5
Paget’s disease of bone is characterized by an increased number of osteoclasts and
excessive bone resorption in focal areas.6 Osteoclast precursors from patients with
Paget’s disease show hypersensitivity to 1,25(OH)2D3,7 and therefore, VDR antagonists
are expected to be therapeutic drugs. However, extensive investigations to find VDR
antagonists have yielded only a restricted series of secosteroid VDR antagonists8-13 (Fig.
1), and no non-secosteroid VDR antagonist has been found, to our knowledge. This fact
suggests that substrate recognition by VDR-LBD is highly specific. Thus, as an
alternative approach to reduce VDR-mediated transcriptional activation, we14 and
others15 have attempted to inhibit the interactions between the VDR-LBD and
coactivators that are necessary for VDR transactivation. It is known that ligand-bound
VDR-LBD recognizes LXXLL peptide motifs of coactivator proteins, and hydrophobic
interactions of three leucine residues and hydrogen bonds, known as ‘‘charge clamps’’,
are important for recognition.16 Previously, we designed and synthesized several
benzodiazepine derivatives intended to mimic the pharmacophore of LXXLL peptide,
and we reported that these molecules inhibited VDR / coactivator interaction and
VDR-mediated transcription (Fig. 2).14 Here, we describe the design, synthesis, and
structure-inhibitory activity relationship of a series of benzodiazepine derivatives
modified at the 7- and 8-positions.
2. Results and discussion
2.1. Effect of deletion of the amino group at the 8-position
In the structure of our prototype inhibitors 1, the amino group at the 8-position was
designed to mimic the charge clamp of the LXXLL motif (Fig. 2). However, it was not
established whether the amino group actually contributed to the inhibitory effect of
these compounds on the VDR-coactivator interaction. To evaluate whether the amino
group is required for inhibition of VDR-mediated transcription, we synthesized
benzodiazepine compounds with a hydrogen atom at the 8-position (Schemes 1 and 2).
Introduction of L-leucine methyl ester into 2-bromobenzonitrile (2) by
Buchwald-Hartwig cross-coupling reaction gave 3. Reduction of nitrile 3 with Raney
nickel and hydrogen induced intramolecular cyclization,17 affording 4. N-Alkylation of
amide 4 with 1-bromo-3-methylbutane gave 5, and subsequent iodination of 5 with
ICl-pyridine complex17 gave 6. Suzuki coupling of 6 and commercially available

2-methyl-1-propenyl boronic acid pinacol ester (9) gave 7a. On the other hand, Suzuki
coupling of 6 and (E)-but-1-enylboronic acid pinacol ester (12), synthesized from
but-1-yne (10) according to a previous report (Scheme 2),18 afforded 7b and 7c. We
considered that 7b was formed by isomerization, as reported previously.19 Reduction of
the double bond of 7a with palladium charcoal and hydrogen gave 8. To generate more
potent inhibitors, we focused on the pocket of the VDR near Leu 630 of the LXXLL
peptide fragment. That pocket is composed of residues Ile234, Ile238, Leu259, Ala263
and Val417 (Fig. 3).16 We designed and synthesized benzodiazepine compounds bearing
an alkyl alcohol at the 7-position because we thought that the alkyl alcohol moiety
might form a hydrogen bond with the oxygen atom of Leu259 in the pocket (Fig. 3 and
Schemes 3 and 4). Boronic acids 14a-b were synthesized by hydroboration of the
corresponding alkynes 13a-b (Scheme 3).18 Suzuki coupling of 6 and 14a-b gave 15a-b,
then removal of the TBS group with TBAF gave 16a-b. Compounds 17a-b were
prepared by reduction of the double bond of 15a-b with palladium charcoal and
hydrogen. Removal of the TBS group of 17a-b with TBAF gave 18a-b (Scheme 4).
To investigate the cell-level inhibition of VDR-mediated transcriptional activity, we
utilized a VDR-responsive reporter gene assay14 with CMX-GAL4N-hVDR LBD as the
recombinant receptor gene, TK-MH100x4-LUC as the reporter gene, and CMX
β-galactosidase gene for normalization. Human embryonic kidney (HEK) 293 cells
were incubated with 1,25(OH)2D3 (3 nM) in the presence or absence of test compounds.
After incubation, cells were assayed for luciferase reporter gene and β-galactosidase
activity. The activities of the synthesized compounds are shown in Table 1.
Synthesized benzodiazepine analogs that had a hydrogen atom at the 8-position
showed weaker inhibition of VDR-mediated transcriptional activity than 1, which has
the amino group at the 8-position. In particular, 7a, corresponding to 1, showed no
inhibitory activity on VDR-mediated transcription, whereas the IC50 value of 1 is 26 μM.
These results suggest that the amino group at the 8-position of 1 is critical for the
inhibitory activity, and support the idea that the amino group at the 8-position mimics
the charge clamp of the LXXLL motif. Therefore, we decided to synthesize a series of
compounds having an amino group at the 8-position in order to generate more potent
inhibitors. On the other hand, the compounds with a hydroxyalkyl group, 16a-b and
18a-b, did not show increased inhibition of VDR-mediated transcription. These results
suggest that the hydroxyalkyl group at the 7-position does not contribute to the

inhibitory activity.
2.2. SAR at the 7-position of benzodiazepine derivatives bearing an amino group at
the 8-position
To evaluate the SAR at the 7-position of benzodiazepine, we synthesized a series of
benzodiazepine derivatives bearing an amino group at the 8-position (Scheme 5).
Suzuki coupling reaction of 1914 and boronic acid pinacol ester 12 or commercially
available (E)-pent-1-enylboronic acid pinacol ester gave 20a-b. Removal of the Boc
groups of 20a-b with TFA gave compounds 21a-b. To generate 22a-b, 21a-b were
cyclized by application of the Buchwald-Hartwig cross-coupling reaction. 23a-b were
prepared by reduction of 22a-b with palladium charcoal and hydrogen. However, this
scheme was not favorable to synthesize a series of compounds with various substituents
at the 7-position, because multiple steps were necessary, so we designed a new scheme
that would be more suitable to obtain compounds for evaluation of SAR at the
7-position of benzodiazepine (Scheme 6).
Nucleophilic
substitution
of
2-bromo-4-fluorobenzonitrile
(24)
with
4-methoxybenzylamine followed by removal of the PMB group, and protection of the
amino group with diBoc gave 27. Introduction of L-leucine methyl ester into 27 by
Buchwald-Hartwig cross-coupling reaction followed by reduction of the nitrile with
Raney nickel and hydrogen resulted in intramolecular cyclization to afford 29.
N-Alkenylation of the amide group of 29 with 1-bromo-3-methyl-2-butene gave 30.
Successive reduction and removal of the diBoc group with HCl afforded 32. Iodination
of 32 with ICl-pyridine gave 33, and then introduction of a phenyl group into 33 by
Suzuki coupling gave 34.
We used the VDR-responsive reporter gene assay to investigate the activity of these
compounds. Replacement of the isobutenyl group at the 7-position with a normal alkyl,
alkenyl, or phenyl group did not affect the inhibitory activity (Table 2). Replacement of
the isobutenyl group (1) with iodine (33) retained the inhibitory activity, while
replacement of the hydrogen atom (32) resulted in loss of the inhibitory activity. These
results suggested that a hydrophobic and moderately bulky group at the 7-position of
benzodiazepine compounds is favorable for inhibitory activity towards VDR-mediated
transcription.
According to the SAR, we designed new benzodiazepine compounds containing a

phenyl group with carboxylic acid, because we thought that carboxylic acid might form
a hydrogen bond with the nitrogen atom of Lys260 (Fig. 4 and Scheme 7). Suzuki
coupling reaction of 33 with 4-ethoxycarbonylphenylboronic acid pinacol ester afforded
35, then hydolysis of ethyl ester with KOH gave 36. We also synthesized 42 because we
thought that 42 might form a hydrogen bond between its hydroxyl group and the oxygen
atom of Leu259 in the pocket (Fig. 3). This compound was obtained by Suzuki coupling
reaction of 33 and the corresponding boronic acid pinacol ester 40, and reduction with
hydrogen and palladium charcoal (Scheme 8). Compound 40 was synthesized from
commercially available ethyl 3-hydroxybenzoate (37) through iodination, Mitsunobu
reaction, and introduction of the boronic acid pinacol ester group. The position of the
iodine atom of 38 was determined by two-dimensional heteronuclear multiple-bond
correlation spectroscopy (HMBC).
In VDR reporter gene assay, 35 showed a lower IC50 value than 1, while 36 showed
little inhibitory activity (Table 3). One possible explanation is that the introduced
carboxyl groups might be important to improve the inhibitory activity of VDR, but the
compound with the free carboxylic acid, 36, is likely to have poor membrane
permeability. In this case, ethyl benzoate 35 might work as a prodrug of 36 that would
be able to penetrate the cell membrane, and be hydrolyzed in the cytoplasm to give 36,
which could then form a hydrogen bond between the carboxyl group and the nitrogen
atom of Lys260. On the other hand, 42, which has both a carboxyl group and a hydroxyl
group, showed an intermediate IC50 value between 34 (with a non-substituted phenyl
group) and 35 (with only an ester group). This result indicates that the hydroxyl group
does not increase the inhibitory activity of VDR.
3. Conclusion
Suppression of VDR-mediated transcription is expected to be of therapeutic value in
Paget’s disease of bone. It is known that interaction between VDR and coactivators is
necessary for VDR transactivation, and the interaction occurs when VDR recognizes an
LXXLL peptide motif of coactivators. We previously reported that benzodiazepine
molecules designed as LXXLL peptide mimetics inhibited the interaction of VDR and
coactivators, and reduced VDR transcription. In this study, we investigated the SAR at
the 7- and 8-positions of our compounds. The results showed firstly that the amino
group at the 8-position is essential for the inhibitory activity, and support the idea that

this amino group works as a charge clamp, stabilizing the binding between the inhibitor
and the VDR-LBD. Secondly, replacement of the isobutenyl group at the 7-position
with various hydrophobic groups had no effect on the inhibitory activity. But,
interestingly, the benzodiazepine derivative bearing an ethyl benzoate group 35 showed
more potent inhibitory activity. We hypothesize that 35 works as
a
membrane-permeable prodrug that is hydrolyzed in the cytoplasm to afford 36, which
can form a hydrogen bond between its carboxyl group and the nitrogen atom of Lys260,
resulting in potent inhibitory activity.
4. Experimental
4.1. General
Melting points were determined by using a Yanagimoto hot-stage melting point
apparatus and are uncorrected. 1H-NMR spectra were recorded on a JEOL JNM-GX500
(500 MHz) spectrometer. Chemical shifts are expressed in parts per million relative to
tetramethylsilane. Mass spectra were recorded on a JEOL JMS-DX303 spectrometer.
4.1.1. General procedure of Suzuki coupling (GP-A)
Under an Ar atmosphere, K3PO4, Pd(dppf)Cl2 and the corresponding boronic acid
or boronic acid pinacol ester were added to a solution of halide in DMF. The reaction
o
mixture was stirred for an appropriate time at 80 C, then the reaction was quenched
with H2O and the mixture was extracted with AcOEt. The organic layer was dried over
MgSO4 and concentrated. The resulting residue was purified by silica gel
chromatography, PTLC and/or HPLC to afford the target molecule.
4.1.2. General procedure of hydroboration and hydrolysis (GP-B)
o
A bomb flask was charged with THF and BH3•SMe2 at 0 C, then (+)-α-pinene was
added dropwise. The solution was stirred at 0 °C for 10 minutes then allowed to warm
to rt and stirred at rt for 2-3 hours, during which time a white precipitate formed. The
solution was recooled to 0 °C and alkyne was added to it, resulting in a clear, colorless
solution. The flask was then sealed with a Teflon screw cap and the mixture was stirred
at 0 °C for 5-30 minutes, warmed to rt, and stirred at rt for 1.5-2.5 hours. The solution
was recooled to 0 °C and acetaldehyde was added. The bomb flask was resealed with
the Teflon screw cap and the reaction mixture was stirred at 40 °C for 4.5-5 hours. It

was allowed to cool to 0 oC and H2O was added. Stirring was continued for 3-7 hours at
rt, and then the solution was diluted with EtOAc. Purification was performed by
partition under basic conditions or by silica gel chromatography to afford the target
molecule.
4.1.3. General procedure of removal of TBS group (GP-C)
Under an Ar atmosphere, a 1.0 M solution of tetra-n-butylammonium fluoride (TBAF)
in THF was added to a stirred solution of a compound bearing a TBS group in THF. The
reaction mixture was stirred for an appropriate time, then the reaction was quenched
with H2O and brine, and the mixture was extracted with AcOEt. The organic layer was
dried over MgSO4 and concentrated. The resulting residue was purified by PTLC to
afford the target molecule.
4.1.4. (S)-1,2,4,5-Tetrahydro-2-(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (4)
Under an Ar atmosphere, Pd2(dba)3•CHCl3 (104 mg, 100 μmol), (R)-BINAP (139 mg,
223 μmol) and Cs2CO3 (3.61 g, 11. 1 mmol) were successively added to a solution of
2-bromobenzonitrile (2) (1.08 g, 5.95 mmol) and L-leucine methyl ester hydrochloride
(1.44 g, 7.97 mmol) in toluene (11 mL). The reaction mixture was stirred for 4 hours at
o
110 C, then the reaction was quenched with H2O and the mixture was extracted with
AcOEt. The organic layer was dried over MgSO4 and concentrated. The resulting
residue was purified by silica gel chromatography (hexane / AcOEt = 1 / 0 to 20 / 1) to
afford 3 (mixture, 306 mg) as a pale yellow oil. This mixture was used for the next step
without further purification. Under an Ar atmosphere, an H2O suspension of Raney
nickel (1.5 mL, TCI) was added to a solution of the above mixture in MeOH (12 ml) /
Et3N (1.2 mL). The Ar atmosphere was replaced with H2. The reaction mixture was
stirred for 24 hours at rt, then filtered through Celite and concentrated. The resulting
residue was purified by silica gel chromatography (hexane / AcOEt = 1 / 1 to 1 / 2) to
afford 4 (241 mg, 1.10 mmol, 19 % (2 steps)) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 7.07 (dd, J = 8.0, 7.3 Hz, 1H), 6.91 (d, J = 7.3 Hz, 1H),
6.67 (dd, J = 8.0, 7.3 Hz, 1H), 6.56 (d, J =8.0 Hz, 1H), 6.83-6.31 (m, 1H), 5.01 (dd, J =
16.0, 5.5 Hz, 1H), 4.50-4.41 (m, 1H), 3.90 (dd, J = 16.0, 6.7 Hz, 1H), 3.58-3.42 (m, 1H),
1.92-1.79 (m, 2H), 1.55-1.47 (m, 1H), 0.99 (d, J = 6.1 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H);
MS (FAB) m/z 218 (M)+, 219 (M+H)+.

4.1.5. (S)-1,2,4,5-Tetrahydro-4-(3-methylbutyl)-2-(2-methylpropyl)-3H-1,4-
benzodiazepin-3-one (5)
Under an Ar atmosphere, NaH (60 %, dispersion in paraffin liquid) (10.2 mg, 255
μmol) was added to a solution of 4 (36.6 mg, 168 μmol) in DMF (2.0 mL) at 0 oC. The
reaction mixture was stirred for 15 min at rt, then 1-bromo-3-methylbutane (23.2 μL,
o
184 μmol) was added to it at 0 C. Stirring was continued for 7 hours at rt, then the
reaction was quenched with H2O and the mixture was extracted with AcOEt. The
organic layer was dried over MgSO4 and concentrated. The resulting residue was
purified by silica gel chromatography (hexane / AcOEt = 1 : 0 to 5 : 1) to afford 5 (39.0
mg, 135 μmol, 80 %) as white needles.
mp 85 oC; 1H-NMR (500 MHz, CDCl3) δ 7.05 (dd, J = 7.3, 6.7 Hz, 1H), 6.91 (d, J =
6.7 Hz, 1H), 6.62 (dd, J = 8.0, 7.3 Hz, 1H), 6.49 (d, J = 8.0 Hz, 1H), 5.37 (d, J = 16.5
Hz, 1H), 4.53 (td, J = 6.8, 6.4 Hz, 1H), 3.76 (d, J = 16.5 Hz, 1H), 3.57-3.43 (m, 3H),
1.94-1.88 (m, 1H), 1.86-1.77 (m, 1H), 1.52-1.31 (m, 4H), 0.97 (d, J = 5.5 Hz, 3H), 0.96
(d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.1 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H); MS (FAB) m/z
288 (M)+, 289 (M+H)+; HMRS (FAB) m/z calcd for C18H28N2O 288.2202, found
288.2206 (M)+.
4.1.6. (S)-1,2,4,5-Tetrahydro-7-iodo-4-(3-methylbutyl)-2-(2-methylpropyl)-3H-1,4-
benzodiazepin-3-one (6)
ICl-pyridine complex (36.5 mg, 151 μmol) was added to a solution of 5 (39.0 mg,
135 μmol) in CH2Cl2 / H2O (CH2Cl2 3.6 mL and H2O 1.8 mL). The reaction mixture
was stirred 4.5 hours at rt, and then the reaction was quenched with sat. NaHCO3 aq.
(3.0 mL) and 21 mM Na2S2O3 aq. (3.0 mL). After the solution had changed color
(brown to pale yellow), it was extracted with CH2Cl2. The organic layer was dried over
MgSO4 and concentrated. The resulting residue was purified by silica gel
chromatography (hexane / AcOEt = 1 / 0 to 6 / 1) to afford 6 (51.4 mg, 124 μmol, 92 %)
as a white solid.
mp 94-96 oC; 1H-NMR (500 MHz, CDCl3) δ 7.28 (dd, J = 8.6, 1.8 Hz, 1H), 7.21 (d, J
= 1.8 Hz, 1H), 6.27 (d, J = 8.6 Hz, 1H), 5.29 (d, J = 16.8 Hz, 1H), 4.58-4.54 (m, 1H),
3.68 (d, J = 16.8 Hz, 1H), 3.55-3.53 (m, 1H), 3.51-3.47 (m, 2H), 1.93-1.87 (m, 1H),
1.83-1.74 (m, 1H), 1.51-1.31 (m, 4H), 0.96 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H),

0.90 (d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.1 Hz, 3H); MS (FAB) m/z 414 (M)+, 415
(M+H)+; HMRS (FAB) m/z calcd for C18H27IN2O 414.1168, found 414.1163 (M)+.
4.1.7. (S)-1,2,4,5-Tetrahydro-4-(3-methylbutyl)-7-(2-methylprop-1-enyl)-2-
(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (7a)
This compound was prepared by means of GP-A, with K3PO4 (55.2 mg, 260 μmol),
Pd(dppf)Cl2 (6.10 mg, 8.34 μmol), 9 (15.6 μL, 76.4 μmol), 6 (26.4 mg, 63.7 μmol) and
DMF (0.65 mL). Purification by silica gel chromatography (hexane / AcOEt = 1 / 0 to 6
/ 1) and PTLC (hexane / AcOEt = 6 / 1) afforded 7a (3.4 mg, 9.9 μmol, 15%) as a white
solid.
mp 112-113 oC; 1H-NMR (500 MHz, CDCl3) δ 6.94 (dd, J = 8.0, 1.9 Hz, 1H), 6.79 (d,
J = 1.9 Hz, 1H), 6.45 (d, J = 8.0 Hz, 1H), 6.11 (s, 1H), 5.36 (d, J = 16.8 Hz, 1H),
4.59-4.54 (m, 1H), 3.73 (d, J = 16.8 Hz, 1H), 3.57-3.42 (m, 3H), 1.93-1.86 (m, 1H),
1.86 (s, 3H), 1.83 (s, 3H), 1.83-1.76 (m, 1H), 1.52-1.32 (m, 4H), 0.97 (d, J = 6.8 Hz,
3H), 0.96 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H); MS
(FAB) m/z 342 (M)+, 343 (M+H)+; HMRS (FAB) m/z calcd for C22H34N2O 342.2671,
found 342.2666 (M)+.
4.1.8. (S,E)-7-(But-1-enyl)-1,2,4,5-tetrahydro-4-(3-methylbutyl)-2-(2-
methylpropyl)-3H-1,4-benzodiazepin-3-one (7b) and (S)-7-(but-1-en-2-yl)-1,2,4,5-
tetrahydro-4-(3-methylbutyl)-2-(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (7c)
These compounds were prepared by means of GP-A, with K3PO4 (40.7 mg, 145
μmol), Pd(dppf)Cl2 (6.10 mg, 8.34 μmol), 12 (39.4 mg, 216 μmol), 6 (21.3 mg, 51.4
μmol) and DMF (0.50 mL). Purification by silica gel chromatography (hexane / AcOEt
= 6 / 1), PTLC (NH plate, FUJI SILYSIA) (hexane / AcOEt = 2 / 1) and HPLC with
Sensyu Pak PEGASIL ODS (20φ x 250 mm) (CH3CN / H2O = 85 : 15) afforded 7b
(3.70 mg, 10.8 μmol, 21%) as a yellow oil and 7c (2.50 mg, 7.30 μmol, 14 %) as a
yellow oil.
7b: 1H-NMR (500 MHz, CDCl3) δ 7.07 (dd, J = 8.6, 1.9 Hz, 1H), 6.90 (d, J = 1.9 Hz,
1H), 6.43 (d, J = 8.6 Hz, 1H), 6.22 (d, J = 15.9 Hz, 1H), 6.05 (td, J = 15.9, 6.1 Hz, 1H)
5.34 (d, J = 16.5 Hz, 1H), 4.59-4.53 (m, 1H), 3.74 (d, J = 16.5 Hz, 1H), 3.54-3.44 (m,
3H), 2.23-2.15 (m, 2H), 1.94-1.87 (m, 1H), 1.85-1.76 (m, 1H), 1.57-1.33 (m, 4H), 1.07
(t, J = 7.9 Hz, 3H), 0.97 (d, J = 5.5 Hz, 3H), 0.96 (d, J = 6.1 Hz, 3H), 0.90 (d, J = 6.8

Hz, 3H), 0.83 (d, J = 6.1 Hz, 3H); MS (FAB) m/z 342 (M)+, 343 (M+H)+; HMRS (FAB)
m/z calcd for C22H34N2O 342.2671, found 342.2673 (M)+.
7c; 1H-NMR (500 MHz, CDCl3) δ 7.14 (dd, J = 8.2, 2.5 Hz, 1H), 7.00 (d, J = 2.5 Hz,
1H), 6.46 (d, J = 8.2 Hz, 1H), 5.38 (d, J = 16.5 Hz, 1H), 5.15 (s, 1H), 4.92-4.91 (m, 1H),
4.62-4.57 (m, 1H), 3.77 (d, J = 16.5 Hz, 1H), 3.59-3.44 (m, 3H), 2.48-2.42 (m, 2H),
1.94-1.88 (m, 1H), 1.85-1.75 (m, 1H), 1.50-1.35 (m, 4H), 1.09 (t, J = 7.4 Hz, 3H), 0.97
(d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.81 (d, J = 6.8
Hz, 3H); MS (FAB) m/z 342 (M)+, 343 (M+H)+; HMRS (FAB) m/z calcd for C22H34N2O
342.2671, found 342.2673 (M)+.
4.1.9. (S)-1,2,4,5-Tetrahydro-4-(3-methylbutyl)-2,7-di(2-methylpropyl)-3H-1,4-
benzodiazepin-3-one (8)
Under an Ar atmosphere in a pressure-tight flask, 10 % Pd/C (6.50 mg) was added to
a solution of 7a (2.00 mg, 5.80 μmol) in AcOEt (6 mL). The Ar atmosphere was
replaced with H2 (3.0 atm). The reaction mixture was stirred for 14 hours at 50 oC, then
filtered through Celite and concentrated. The resulting residue was purified by PTLC
(hexane / AcOEt = 6 / 1) to afford 8 as a white solid in quantitative yield.
mp 103-105 oC; 1H-NMR (500 MHz, CDCl3) δ 6.83 (dd, J = 8.0, 1.9 Hz, 1H), 6.69
(d, J = 1.9 Hz, 1H), 6.43 (d, J = 8.0 Hz, 1H), 5.35 (d, J = 16.2 Hz, 1H), 4.58-4.52 (m,
1H), 3.73 (d, J = 16.2 Hz, 1H), 3.62-3.56 (m, 1H), 3.44-3.37 (m, 2H), 2.36-2.28 (m, 2H),
1.93-1.86 (m, 1H), 1.84-1.73 (m, 2H), 1.49-1.32 (m, 4H), 0.96 (d, J = 6.7 Hz, 3H), 0.95
(d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.1 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H), 0.86 (d, J = 6.7
Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H); MS (FAB) m/z 344(M)+, 345 (M+H)+; HMRS (FAB)
m/z calcd for C22H36N2O 344.2828, found 344.2827 (M)+.
4.1.10. (E)-But-1-enylboronic acid (11)
This compound was prepared by means of GP-B, with THF (11 mL), BH3•SMe2
(1.80 mL, 19.4 mmol), (+)-α-pinene (6.80 mL, 42.9 mmol), 10 (excess), acetaldehyde
(10.4 mL, 185 mmol) and H2O (5.0 mL). Purification was performed by partition. The
mixture was diluted with AcOEt and extracted with 10% NaOH aq (2 x 10 mL). The
combined aqueous solutions were acidified to pH 2 with concentrated HCl aq. and
extracted with EtOAc (3 x 30 mL). The combined organic extracts were washed with
sat. NaHCO3 aq., dried over MgSO4, and concentrated to afford 11 (1.03 g, 10.3 mmol,

53%) as a colorless solid.
1H-NMR (500 MHz, DMSO-d6 / D2O = 95 / 5) δ 6.46 (td, J = 17.6, 6.1 Hz, 1H), 5.28
(td, J = 17.6, 1.8 Hz, 1H), 2.09-2.01 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H); MS (FAB) not
detected.
4.1.11. (E)-Buten-1-ylboronic acid pinacol ester (12)
Compound 11 (217 mg, 2.17 mmol) and MgSO4 (0.63 g, 5.23 mmol) were added to a
solution of pinacol (237 mg, 2.00 mmol) in CH2Cl2 (6 mL). The reaction mixture was
stirred 21 hours at rt, then filtered and the filtrate was concentrated to afford pure 12
(338 mg, 1.86 mmol, 93 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ 6.70 (td, J = 18.0, 6.1 Hz, 1H), 5.43 (td, J = 18.0, 1.9
Hz, 1H), 2.20-2.14 (m, 2H), 1.26 (s, 12 H), 1.02 (t, J = 7.3 Hz, 3H); MS (FAB) not
detected.
4.1.12. (E)-3-(tert-Butyldimethylsilyloxy)prop-1-enylboronic acid (14a)
This compound was prepared by means of GP-B, with THF (6.3 mL), BH3•SMe2
(1.10 mL, 11.6 mmol), (+)-α-pinene (3.50 mL, 22.1 mmol), 13a (2.00 mL, 9.86 mmol),
acetaldehyde (7.00 mL, 126 mmol) and H2O (2.5 mL). Purification was performed by
silica gel chromatography. The mixture was diluted with AcOEt, dried over MgSO4 and
concentrated. The resulting residue was purified by silica gel chromatography (hexane /
AcOEt = 3 / 1 to 2 / 1) to afford 14a (467 mg, 2.16 mmol. 22 %) as a colorless oil.
1H-NMR (500 MHz, CD3OD) δ 6.57 (td, J = 17.7, 3.7 Hz, 1H), 5.87 (td, J = 17.7, 1.8
Hz, 1H), 4.26 (dd, J = 3.7, 1.8 Hz, 2H), 0.94 (s, 9H), 0.04 (s, 6H); MS (FAB) not
detected.
4.1.13. (E)-4-(tert-Butyldimethylsilyloxy)but-1-enylboronic acid (14b)
This compound was prepared by means of GP-B, with THF (6.3 mL), BH3•SMe2
(1.10 mL, 11.6 mmol), (+)-α-pinene (3.50 mL, 22.1 mmol), 13b (2.10 mL, 10.1 mmol),
acetaldehyde (7.00 mL, 126 mmol) and H2O (2.5 mL). Purification was performed by
silica gel chromatography. The mixture was diluted with AcOEt, dried over MgSO4 and
concentrated. The resulting residue was purified by silica gel chromatography (hexane /
AcOEt = 3 / 1 to 2 / 1) to afford 14b (638 mg, 2.77 mmol. 27 %) as a colorless oil.
1H-NMR (500 MHz, CD3OD) δ 6.54 (td, J = 17.3, 3.7 Hz, 1H), 5.64 (d, J = 17.3 Hz,

1H), 3.71 (t, J = 6.1 Hz, 2H), 2.36 (td, J = 6.7, 6.1 Hz, 2H), 0.90 (s, 9H), 0.06 (s, 6H);
MS (FAB) not detected.
4.1.14. (S)-1,2,4,5-Tetrahydro-7-(3-hydroxyprop-1-enyl)-4-(3-methylbutyl)-2-
(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (16a)
Intermediate 15a was prepared by means of GP-A, with K3PO4 (69.9 mg, 329 μmol),
Pd(dppf)Cl2 (9.60 mg, 13.1 μmol), 14a (34.6 mg, 160 μmol), 6 (39.8 mg, 96.1 μmol)
and DMF (1.0 mL). Purification by silica gel chromatography (hexane / AcOEt = 1 / 0
to 6 / 1) and PTLC (hexane / AcOEt = 4 / 1) afforded almost pure 15a (mixture, 13.8
mg) as a yellow oil. This was used for the next reaction without further purification.
Compound 16a was prepared by means of GP-C, with 1.0 M TBAF in THF (18.0 μL,
18.0 μmol), almost pure 15a (mixture, 7.10 mg) and THF (0.50 mL). Purification by
silica gel chromatography (hexane / AcOEt = 1 : 1) afforded 16a (1.80 mg, 5.23 μmol,
11 %, 2 steps) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ 7.11 (dd, J = 7.9, 1.8 Hz, 1H), 6.95 (d, J = 1.8 Hz, 1H),
6.46 (d, J = 15.9 Hz, 1H), 6.44 (d, J = 7.9 Hz, 1H), 6.61 (td, J = 15.9, 6.1 Hz, 1H), 5.36
(d, J = 16.5 Hz, 1H), 4.62-4.57 (m, 1H), 4.30-4.26 (m, 2H), 3.75 (d, J = 16.5 Hz, 1H),
3.60-3.58 (m, 1H), 3.55-3.46 (m, 2H) 1.95-1.88 (m, 1H), 1.85-1.75 (m, 1H), 1.51-1.42
(m, 2H), 1.42-1.33 (m, 2H), 0.97 (d, J = 6.1 Hz, 3H), 0.96 (d, J = 6.1 Hz, 3H), 0.90 (d, J
= 6.1 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H); MS (FAB) m/z 344 (M)+, 345 (M+H)+; HMRS
(FAB) m/z calcd for C21H32N2O2 344.2464, found 344.2466 (M)+.
4.1.15. (S)-1,2,4,5-Tetrahydro-7-(4-hydroxybut-1-enyl)-4-(3-methylbutyl)-2-
(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (16b)
Intermediate 15b was prepared by means of GP-A, with K3PO4 (21.7 mg, 102 μmol),
Pd(dppf)Cl2 (6.20 mg, 8.47 μmol), 14a (26.5 mg, 115 μmol), 6 (12.2 mg, 29.4 μmol)
and DMF (300 μL). Purification by silica gel chromatography (hexane / AcOEt = 1 / 0
to 6 / 1) afforded 15b (mixture, 7.90 mg) as a yellow oil. This was used for the next
reaction without further purification. Compound 16b was prepared by means of GP-C,
with 1.0 M TBAF in THF (25.5 μL, 25.5 μmol), almost pure 15b (7.10 mg, mixture)
and THF (0.50 mL). Purification by silica gel chromatography (hexane / AcOEt = 1 : 1)
afforded 16b (2.90 mg, 8.09 μmol, 28 %, 2 steps) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ 7.09 (dd, J = 8.3, 1.9 Hz, 1H), 6.91 (d, J = 1.9 Hz, 1H),

6.43 (d, J = 8.3 Hz, 1H), 6.35 (d, J = 15.9 Hz, 1H), 5.97 (td, J = 7.3, 15.9 Hz, 1H), 5.34
(d, J = 16.5 Hz, 1H), 4.61-4.54 (m, 1H), 3.75-3.70 (m, 2H), 3.74 (d, J = 16.5 Hz, 1H),
3.56-3.53 (m, 1H), 3.52-3.46 (m, 2H) 2.45 (dd, J = 7.3, 6.7 Hz, 2H), 1.94-1.87 (m, 1H),
1.85-1.75 (m, 1H), 1.52-1.34 (m, 4H), 0.97 (d, J = 6.1 Hz, 3H), 0.96 (d, J = 6.1 Hz, 3H),
0.90 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.7 Hz, 3H); MS (FAB) m/z 358 (M)+, 359
(M+H)+; HMRS (FAB) m/z calcd for C22H34N2O2 358.2620 found 358.2623 (M)+.
4.1.16. (S)-1,2,4,5-Tetrahydro-7-(3-hydroxypropyl)-4-(3-methylbutyl)-2-
(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (18a)
Using a protocol similar to that in section 4.1.14, 10 % Pd/C (2.00 mg) was added to a
stirred solution of almost pure 15a (mixture, 7.90 mg) in 1,4-dioxane (0.70 mL). The
reaction mixture was stirred for 6 hours at rt, then filtered through Celite and
concentrated. The resulting residue was purified by PTLC (hexane / AcOEt = 4 : 1) to
afford almost pure 17a (mixture, 4.90 mg). This was used for the next reaction without
further purification. Compound 18a was prepared by means of GP-C, with 1.0 M TBAF
in THF (13.0 μL, 13.0 μmol), almost pure 17a (mixture, 4.90 mg) and THF (0.50 mL).
Purification by PTLC (hexane / AcOEt = 1 : 1) afforded 18a (1.20 mg, 3.46 μmol, 6%
(3 steps)) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ 6.89 (dd, J = 8.0, 1.8 Hz, 1H), 6.75 (d, J = 1.8 Hz, 1H),
6.45 (d, J = 8.0 Hz, 1H), 5.34 (d, J = 16.5 Hz, 1H), 4.56-4.53 (m, 1H), 3.73 (d, J = 16.5
Hz, 1H), 3.68-3.64 (m, 2H), 3.57-3.41 (m, 3H), 2.57 (t, J = 7.9 Hz, 2H), 1.92-1.85 (m,
1H), 1.84-1.76 (m, 3H), 1.50-1.42 (m, 2H), 1.41-1.34 (m, 2H), 0.97 (d, J = 6.1 Hz, 3H),
0.95 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 6.1 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H); MS (FAB)
m/z 346 (M)+, 347 (M+H)+; HMRS (FAB) m/z calcd for C21H34N2O2 346.2620, found
346.2620 (M)+.
4.1.17. (S)-1,2,4,5-Tetrahydro-7-{4-(tert-butyldimethylsilyloxy)butyl}-4-
(3-methylbutyl)-2-(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (17b)
Using a protocol similar to that in section 4.1.15, 10 % Pd/C (2.80 mg) was added to
a stirred solution of 16b (mixture, 22.3 mg) in 1,4-dioxane (2.0 mL). The reaction
mixture was stirred 22 hours at rt, then filtered through Celite and concentrated. The
resulting residue was purified by PTLC (hexane / AcOEt = 4 / 1) to afford 17b (15.2 mg,
32.0 μmol, 34 %, 2 steps) as a yellow oil.

1H-NMR (500 MHz, CDCl3) δ 6.87 (dd, J = 8.2, 1.9 Hz, 1H), 6.72 (d, J = 1.9 Hz, 1H),
6.44 (d, J = 8.2 Hz, 1H), 5.33 (d, J = 16.5 Hz, 1H), 4.56-4.51 (m, 1H), 3.73 (d, J = 16.5
Hz, 1H), 3.61 (t, J = 6.1 Hz, 2H), 3.56-3.43 (m, 2H), 3.41-3.39 (m, 1H), 2.48 (t, J = 7.4
Hz, 2H), 1.92-1.86 (m, 1H), 1.84-1.77 (m, 1H), 1.61-1.50 (m, 4H), 1.48-1.41 (m, 2H),
1.40-1.33 (m, 2H), 0.96 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 5.5 Hz, 3H), 0.89 (s, 9H), 0.89
(d, J = 6.1 Hz, 3H), 0.81 (d, J = 6.7 Hz, 3H), 0.04 (s, 6H); MS (FAB) m/z 474 (M)+, 475
(M+H)+.
4.1.18. (S)-1,2,4,5-Tetrahydro-7-(4-hydroxybutyl)-4-(3-methylbutyl)-2-
(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (18b)
This compound was prepared by means of GP-C, with 1.0 M TBAF in THF (18.0 μL,
18.0 μmol), 17b (5.60 mg, 11.8 μmol) and THF (0.51 mL). Purification by PTLC
(hexane / AcOEt = 1 : 1) afforded 18b (2.8 mg, 7.8 μmol, 66 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ 6.87 (dd, J = 7.9, 1.8 Hz, 1H), 6.73 (d, J = 1.8 Hz, 1H),
6.44 (d, J = 7.9 Hz, 1H), 5.34 (d, J = 16.5 Hz, 1H), 4.57-4.52 (m, 1H), 3.73 (d, J = 16.5
Hz, 1H), 3.67-3.64 (m, 2H), 3.56-3.44 (m, 2H), 3.42-3.38 (m, 1H), 2.50 (t, J = 6.7 Hz,
2H), 1.92-1.86 (m, 1H), 1.85-1.75 (m, 1H), 1.67-1.54 (m, 4H), 1.50-1.41 (m, 2H),
1.41-1.32 (m, 2H), 0.96 (d, J = 5.5 Hz, 3H), 0.95 (d, J = 6.1 Hz, 3H), 0.89 (d, J = 6.8 Hz,
3H), 0.82 (d, J = 6.7 Hz, 3H); MS (FAB) m/z 360 (M)+, 361 (M+H)+; HMRS (FAB) m/z
calcd for C22H36N2O2 360.2777, found 360.2773 (M)+.
4.1.19. (S,E)-8-Amino-7-(but-1-enyl)-4-(3-methylbutyl)-2-(2-methylpropyl)-
1,2,4,5-tetrahydrohydro-3H-1,4-benzodiazepin-3-one (22a)
Intermediate 20a was prepared by means of GP-A, with K3PO4 (208 mg, 980 μmol),
Pd(dppf)Cl2 (30.2 mg, 41.3 μmol), 12 (58.3 mg, 320 μmol), 19 (204 mg, 335 μmol) and
DMF (3.3 mL). Purification by silica gel chromatography (hexane / AcOEt = 4 / 1)
afforded 20a (mixture, 99.8 mg) as a brown oil. This was used for the next reaction
without further purification. Trifluoroacetic acid (2.00 mL, 26.9 mmol) was added to a
o
solution of 20a (mixture, 99.8 mg) in CH2Cl2 (2.0 mL) at 0 C. The reaction mixture
o
was stirred for 2 h at 0 C, then adjusted to pH 10 with 2 N NaOH aq. and extracted
with CH2Cl2. The organic layer was dried over MgSO4 and concentrated. The resulting
residue (78.3 mg) was used for the next reaction without purification. Under an Ar
atmosphere, Pd2(dba)3•CHCl3 (20.8 mg, 20.1 μmol) was added to a solution of the

above residue (78.3 mg), ( ) -BINAP (26.9 mg, 43.2 μmol), and Cs2CO3 (295 mg, 905
o
μmol) in toluene (6 mL). The reaction mixture was stirred for 10 hours at 110 C, then
the reaction was quenched with sat. NaHCO3 aq. and the mixture was extracted with
CH2Cl2. The organic layer was dried over MgSO4 and concentrated. The residue was
purified by silica gel chromatography (hexane / AcOEt = 2 / 1) to afford a yellow oil
(24.3 mg). A part of this oil (15.7 mg) was purified by HPLC with Sensyu Pak
PEGASIL ODS (20φ x 250 mm) (CH3CN / H2O = 80 : 20) to afford 22a (10.9 mg, 30.5
μmol, 9 % (3 steps)) as a yellow oil.
1H-NMR (500 MHz, CDCl3) δ 6.80 (s, 1H), 6.26 (td, J = 1.3, 15.9 Hz, 1H), 5.94 (td,
J = 6.7, 15.9 Hz, 1H), 5.82 (s, 1H), 5.24(d, J = 16.5 Hz, 1H), 4.54-4.49 (m, 1H), 3.68 (d,
J = 16.5 Hz, 1H), 3.65-3.55 (m, 2H), 3.55-3.47 (m, 1H), 3.47-3.40 (m, 1H), 3.40-3.38
(m, 1H), 2.24-2.16 (m, 4H), 1.91-1.85 (m, 1H), 1.81-1.76 (m, 1H), 1.52-1.36 (m, 4H),
1.08 (t, J = 7.3 Hz, 3H), 0.96 (d, J = 6.1 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3 H), 0.89 (d, J =
6.8 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H); MS (FAB) m/z 357 (M)+, 358 (M+H)+; HMRS
(FAB) m/z calcd for C22H35N3O 357.2780, found 357.2776 (M)+.
4.1.20. (S,E)-2-Amino-N-(4-amino-2-bromo-5-(pent-1-enyl)benzyl)-N-
(3-methylbutyl)-4-methylpentanamide (21b)
Intermediate 20b was prepared by means of GP-A, with K3PO4 (220 mg, 1036 μmol),
Pd(dppf)Cl2 (34 mg, 46.5 μmol), (E)-pent-1-enylboronic acid pinacol ester (641 mg,
327 μmol), 19 (206 mg, 338 μmol) and DMF (1.0 mL). Purification by silica gel
chromatography (hexane / AcOEt = 3 / 1) afforded 20b (mixture, 129 mg) as a brown
oil. This was used for the next reaction without further purification. Trifluoroacetic acid
(2.50 mL, 33.7 mmol) was added to a solution of the above oil (129 mg) in CH2Cl2 (2.5
mL) at 0 oC. The reaction mixture was stirred for 2 hours at 0 oC, then adjusted to pH 11
with 2 N NaOH aq. and extracted with CH2Cl2. The organic layer was dried over
MgSO4 and concentrated. The resulting residue was purified by silica gel
chromatography (CHCl3 / MeOH = 10 / 1) to afford 21b (73.0 mg, 161 μmol, 48 % (2
steps)) as a yellow oil. The product was a mixture of rotamers in a ratio of 0.5 : 0.5
(determined by 1H-NMR).
1H-NMR (500 MHz, CDCl3) δ 7.04 (s, 0.5H), 6.90 (s, 0.5H), 6.88 (s, 0.5H), 6.85 (s,
0.5H), 6.26 (d, J = 15.6 Hz, 1H), 6.00 (td, J = 6.7, 15.6 Hz, 1H), 4.98 (d, J = 15.0 Hz,
0.5H), 4.50 (d, J = 17.1 Hz, 0.5H), 4.37 (d, J = 17.1 Hz, 0.5H), 4.27 (d, J = 15.0 Hz,

0.5H), 3.78-3.68 (m, 3H), 2.20-2.15 (m, 2H), 1.49-1.34 (m, 8H), 0.96-0.89 (m, 12 H),
0.88 (d, J = 6.7 Hz, 1.5H), 0.84 (d, J = 6.7 Hz, 1.5 H); MS (FAB) m/z 452, 454 (M+H)+.
4.1.21. (S,E)-8-Amino-4-(3-methylbutyl)-2-(2-methylpropyl)-1,2,4,5-
tetrahydrohydro-7-(pent-1-enyl)-3H-1,4-benzodiazepin-3-one (22b)
Under an Ar atmosphere, Pd2(dba)3•CHCl3 (30.1 mg, 29.1 μmol) was added to a
solution of 21b (73 mg, 161 μmol), ( )-BINAP (24.6 mg, 39.5 μmol), and Cs2CO3 (300
mg, 923 μmol) in toluene (2.5 mL). The reaction mixture was stirred for 6 hours at 110
oC, then the reaction was quenched with H2O and the mixture was extracted with
CH2Cl2. The organic layer was dried over MgSO4 and concentrated. The residue was
purified by silica gel chromatography (hexane / AcOEt = 2 / 1), PTLC (hexane / AcOEt
= 3 / 2) and PTLC (hexane / AcOEt = 2 / 1) to afford 22b (11.3 mg, 30.4 μmol, 19 %) as
a yellow oil.
1H-NMR (500 MHz, CDCl3) δ 6.80 (s, 1H), 6.26 (d, J = 15.6 Hz, 1H), 5.89 (td, J =
15.6, 6.4 Hz, 1H), 5.82 (s, 1H), 5.24 (d, J = 16.2Hz, 1H), 4.53-4.50 (m, 1H), 3.65-3.55
(m, 2H), 3.55-3.47 (m, 1H), 3.47-3.40 (m, 2H), 2.16 (td, J = 7.4, 6,4 Hz, 2H), 1.90-1.85
(m, 1H), 1.82-1.74 (m, 1H), 1.53-1.34 (m, 6H), 0.96-0.93 (m, 9H), 0.90 (d, J = 6.8 Hz,
3H), 0.85 (d, J = 6.7 Hz, 3H); MS (FAB) m/z 371 (M)+, 372 (M+H)+; HMRS (FAB) m/z
calcd for C23H27N3O 371.2937, found 371.2937 (M)+.
4.1.22. (S,E)-8-Amino-7-butyl-4-(3-methylbutyl)-2-(2-methylpropyl)-1,2,4,5-
tetrahydrohydro-3H-1,4-benzodiazepin-3-one (23a)
Under an Ar atmosphere, 10 % Pd/C (7.70 mg) was added to a stirred solution of 22a
(7.60 mg, 21.3 μmol) in AcOEt (2.0 mL). The Ar atmosphere was replaced with H2. The
reaction mixture was strongly stirred for 18 hours at rt, then filtered through Celite and
concentrated to afford pure 23a in quantitative yield as a yellow solid.
o
1
mp 94-96 C; H-NMR (500 MHz, CDCl3) δ 6.57 (s, 1H), 5.87 (s, 1H), 5.24 (d, J =
16.5Hz, 1H), 4.52-4.48 (m, 1H), 3.65 (d, J = 16.5 Hz, 1H), 3.54-3.40 (m, 4H), 3.33-3.26
(m, 1H), 2.37 (t, J = 7.3 Hz, 2H), 1.90-1.82 (m, 1H), 1.82-1.74 (m, 1H), 1.54-1.32 (m,
8H), 0.97-0.91 (m, 9H), 0.89 (t, J = 6.7 Hz, 3H), 0.83 (d, J = 6.1 Hz, 3H); MS (FAB)
m/z 359 (M)+, 360 (M+H)+; HMRS (FAB) m/z calcd for C22H37N3O 359.2937, found
359.2934 (M)+.

4.1.23. (S,E)-8-Amino-4-(3-methylbutyl)-2-(2-methylpropyl)-1,2,4,5-
tetrahydrohydro-7-pentyl-3H-1,4-benzodiazepin-3-one (23b)
Under an Ar atmosphere, 10 % Pd/C (2.00 mg) was added to a stirred solution of 22b
(8.30 mg, 22.3 μmol) in AcOEt (2.0 mL). The Ar atmosphere was replaced with H2. The
reaction mixture was strongly stirred for 6 hours at rt, then filtered through Celite and
concentrated to afford pure 23b in quantitative yield as a yellow solid.
o
1
mp 86-87 C; H-NMR (500 MHz, CDCl3) δ 6.57 (s, 1H), 5.87 (s, 1H), 5.25 (d, J =
16.2Hz, 1H), 4.50 (t, J = 6.7 Hz, 1H), 3.65 (d, J = 16.2 Hz, 1H), 3.52-3.42 (m, 4H),
3.35-3.25 (m, 1H), 2.36 (t, J = 7.3 Hz, 2H), 1.89-1.84 (m, 1H), 1.81-1.75 (m, 1H),
1.56-1.30 (m, 10H), 0.96-0.93 (m, 6H), 0.91-0.88 (m, 6H), 0.83 (d, J = 6.7 Hz, 3H); MS
(FAB) m/z 373 (M)+, 374 (M+H)+; HMRS (FAB) m/z calcd for C23H39N3O 373.3093,
found 373.3089 (M)+.
4.1.24. 2-Bromo-4-(4-methoxybenzylamino)benzonitrile (25)
4-Methoxybenzylamine
(5.3
mL,
40.2
mmol)
was
added
to
2-bromo-4-fluorobenzonitrile (24) (5.36 g, 26.8 mmol). The reaction mixture was
stirred 5 hours at 140 oC in a sealed flask, then the reaction was quenched with H2O and
the mixture was extracted with CH2Cl2. The organic layer was dried over MgSO4 and
concentrated. The resulting residue was recrystallized (from hexane / AcOEt) to afford
25 (5.96 g, 18.8 mmol, 70 %) as a pale yellow solid. The mother liquor was
concentrated and the residue was purified by silica gel chromatography (hexane /
AcOEt = 4 / 1) to afford 25 (1.76 g, 5.56 mmol, 21 %) as a pale yellow solid.
1H-NMR (500 MHz, CDCl3) δ 7.37 (d, J = 8.5 Hz, 1H), 7.23 (d, J = 9.2 Hz, 2H), 6.90
(d, J = 9,2 Hz, 2H), 6.83 (d, J = 2.4 Hz, 1H), 6.52 (dd, J = 8.5, 2.4 Hz, 1H), 4.54 (m,
1H), 4.28 (d, J = 4.9 Hz, 2H), 3.81 (s, 3H); MS (FAB) m/z 317, 329 (M+H)+.
4.1.25. 4-Amino-2-bromobenzonitrile (26)
2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (2.90 g, 12.8 mmol) was added
to a stirred solution of 25 (4.03g, 12.8 mmol) in CH2Cl2 (85 mL) and H2O (43 mL) at rt.
The reaction mixture was stirred for 3.5 hours, and then filtered to remove precipitated
solids. The filtrate was added to sat. NaHCO3 aq., and extracted with CH2Cl2. The
organic layer was dried over MgSO4 and concentrated. The resulting residue was
purified by silica gel chromatography (hexane / AcOEt = 2 / 1) to afford 26 (2.13 g,

10.8 mmol, 85 %) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 7.39 (d, J = 7.9 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 6.58
(dd, J = 8.5, 2.4 Hz, 1H), 4.21 (m, 2H); MS (FAB) m/z 197, 199 (M+H)+.
4.1.26. N-(3-Bromo-4-cyanophenyl)imidodicarbonic acid bis(1,1-dimethylethyl)
ester (27)
Di-tert-butyl dicarbonate
(Boc2O) (24.6 g, 113 mmol) in THF (75 mL),
4-dimethylaminopyridine (DMAP) (532 mg, 4.35 mmol) and diisopropylethylamine
(DIPEA) (35.0 mL, 201 mmol) were added to a solution of 26 (7.70 g, 39.1 mmol) at 0
oC. The reaction mixture was refluxed for 2 hours, then cooled to rt, and concentrated to
about 30 mL. H2O and brine were added to it, and the mixture was extracted with
AcOEt. The organic layer was dried over MgSO4 and concentrated. The resulting
residue was recrystallized from hexane / AcOEt to afford 27 (8.40 g, 21.1 mmol, 54 %)
as white cubic crystals. The mother liquor was concentrated and the residue was
purified by silica gel chromatography (hexane / AcOEt = 6 / 1) and reprecipitation
(from EtOH) to afford 27 (899 mg, 2.26 mmol, 6 %) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 7.57 (d, J = 8.6 Hz, 1H), 7.52 (d, J = 1.9 Hz, 1H), 7.23
(dd, J = 8.6, 1.9 Hz, 1H), 1.45 (s, 18H); MS (FAB) m/z 397, 399 (M)+.
4.1.27. (S)-Methyl 2-(5-(bis(tert-butoxycarbonyl)amino)-2-cyanophenylamino)-4-
methylpentanoate (28)
Compound 27 (2.00 g, 5.03 mmol), L-leucine methyl ester hydrochloride (1.09 g,
6.04 mmol), Pd2(dba)3 (260 mg, 252 μmol), Xantphos (437 mg, 755 μmol) and Cs2CO3
(3.28 g, 10.1 mmol) were successively added to a flask. The atmosphere was replaced
with Ar, and toluene (50 mL) was added to the flask. The reaction mixture was stirred
14 hours at 110 oC, then H2O (20 μL) was added and stirring was continued for 19 hours
o
at 110 C. The reaction was quenched with brine and H2O, and the mixture was
extracted with AcOEt. The organic layer was dried over Na2SO4, and concentrated. The
resulting residue was purified by amino silica gel chromatography (NH-SiO2, FUJI
SILYSIA) (hexane / AcOEt = 5 / 1) to afford 28 (2.16 g, 4.68 mmol, 93 %) as a slightly
yellow syrup.
1H-NMR (500 MHz, CDCl3) δ 7.41 (d, J = 7.9 Hz, 1H), 6.54 (dd, J = 7.9, 1.9 Hz, 1H),
6.39 (d, J = 1.9 Hz, 1H), 4.82-4.79 (m, 1H), 4.10-4.04 (m, 1H), 3.73 (s, 3H), 1.82-1.71

(m, 3H), 1.43 (s, 18 H), 1.00 (d, J = 6.7 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H; MS (FAB) m/z
461 (M)+.
4.1.28. (S)-8-bis(tert-butoxycarbonyl)amino-1,2,4,5-tetrahydro-2-(2-methylpropyl)-
3H-1,4-benzodiazepin-3-one (29)
Under an Ar atmosphere, an H2O suspension of Raney nickel (4 mL, TCI) was added
to a solution of 28 (539 mg, 1.17 mmol) in MeOH (40 mL) and Et3N (4 mL). The Ar
atmosphere was replaced with H2. The reaction mixture was stirred for 13.5 hours at rt,
then filtered through Celite and concentrated. The resulting residue was purified by
silica gel chromatography (hexene / AcOEt = 1 / 1 to 1 / 2) to afford 29 (388 mg, 895
μmol, 76 %) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 6.89 (d, J = 8.0 Hz, 1H), 6.46 (dd, J = 8.0, 1.8 Hz, 1H),
6.38 (d, J = 1.8 Hz, 1H), 6.05 (dd, J = 6.7, 6.1 Hz, 1H), 4.99 (dd, J =16.5, 6.7 Hz, 1H),
4.48-4.43 (m, 1H), 3.89 (dd, J = 16.5, 6.7 Hz, 1H), 3.52-3.50 (m, 1H), 1.92-1.79 (m,
2H), 1.51-1.49 (m, 1H), 1.45 (s, 18H), 0.98 (d, J = 6.1 Hz, 3H), 0.97 (d, J = 6.7 Hz,
3H); MS (FAB) m/z 433 (M)+, 434 (M+H)+.
4.1.29. (S)-8-Bis(tert-butoxycarbonyl)amino-1,2,4,5-tetrahydro-4-(3-methylbut-
2-enyl)-2-(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (30)
Tetrabutylammonium iodide (331 mg, 895 μmol), 1-bromo-3-methyl-2-butene (103
μL, 895 μmol) and t-BuOK (108 mg, 964 μmol) were added to a solution of 29 (388 mg,
o
895 μmol) at -20 C. The reaction mixture was stirred for 5 minutes at -20 oC, then the
reaction was quenched with H2O and the mixture was extracted with AcOEt. The
organic layer was dried over Na2SO4 and concentrated. The resulting residue was
purified by silica gel chromatography (hexane / AcOEt = 3 / 1 to 3 / 2) to afford 30 (328
mg, 654 μmol, 76 %) as a colorless amorphous solid.
1H-NMR (500 MHz, CDCl3) δ 6.81 (d, J = 8.0 Hz, 1H), 6.40 (dd, J = 8.0, 1.9 Hz, 1H),
6.30 (d, J = 1.9 Hz, 1H), 5.21 (d, J = 16.5 Hz, 1H), 5.09 (dd, J = 7.9, 6.7 Hz, 1H),
4.65-4.57 (m, 1H), 4.20 (dd, J =14.7, 6.7 Hz, 1H), 3.99 (dd, J = 14.7, 7.3 Hz, 1H), 3.75
(d, J = 16.5 Hz, 1H), 3.57-3.47 (m, 1H), 1.94-1.87 (m, 1H), 1.85-1.75 (m, 1H), 1.69 (s,
6H), 1.47-1.42 (m, 1H), 1.44 (s, 18H), 0.97 (d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.7 Hz,
3H); MS (FAB) m/z 501 (M)+, 502 (M+H)+.

4.1.30. (S)-8-Bis(tert-butoxycarbonyl)amino-1,2,4,5-tetrahydro-4-(3-methylbutyl)
-2-(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (31)
Under an Ar atmosphere, 10 % Pd/C (33.0 mg) was added to a solution of 30 (328
mg, 654 μmol) in 1,4-dioxane (7.0 mL). The Ar atmosphere was replaced with H2. The
reaction mixture was stirred 16 hours at rt, then filtered through Celite and concentrated.
The resulting residue was purified by silica gel chromatography (hexane / AcOEt = 3 / 1
to 1 / 1) to afford 31 in quantitative yield as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ 6.89 (d, J = 8.0 Hz, 1H), 6.41 (dd, J = 8.0, 2.2 Hz, 1H),
6.29 (d, J = 2.2 Hz, 1H), 5.35 (d, J = 16.7 Hz, 1H), 4.61-4.56 (m, 1H), 3.77 (d, J =16.7
Hz, 1H), 3.65-3.57 (m, 1H), 3.53-3.48 (m, 1H), 3.42-3.33 (m, 1H), 1.95-1.87 (m, 1H),
1.84-1.75 (m, 1H), 1.50-1.45 (m, 1H), 1.43 (s, 18H), 1.42-1.30 (m, 2H), 0.96 (d, J = 6.7
Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.1 Hz, 3H), 0.83 (d, J = 6.1 Hz, 3H); MS
(FAB) m/z 503 (M)+, 504 (M+H)+.
4.1.31. (S)-8-Amino-1,2,4,5-tetrahydro-4-(3-methylbutyl)-2-(2-methylpropyl)-3H-
1,4-benzodiazepin-3-one (32)
4 N HCl in 1,4-dioxane (4.0 mL) was added to 31 (200 mg, 397 μmol). The reaction
mixture was stirred for 3 hours at rt, then the reaction was quenched with sat. NaHCO3
aq., and the mixture was extracted with CHCl3. The organic layer was dried over
Na2SO4 and concentrated. The resulting residue was purified by silica gel
chromatography (CHCl3 / MeOH = 20 / 1) to afford 32 (118 mg, 389 μmol, 98 %) as a
white solid.
mp 185-186 oC; 1H-NMR (500 MHz, CDCl3) δ 6.70 (d, J = 8.0 Hz, 1H), 5.99 (dd, J =
8.0, 1.9 Hz, 1H), 5.84 (d, J = 1.9 Hz, 1H), 5.25 (d, J = 16.5 Hz, 1H), 4.57-4.51 (m, 1H),
3.65 (d, J =16.7 Hz, 1H), 3.56-3.41 (m, 4H), 3.41-3.38 (m, 1H), 1.92-1.85 (m, 1H),
1.83-1.74 (m, 1H), 1.54-1.31 (m, 4H), 0.96 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H),
0.89 (d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H); MS (FAB) m/z 303 (M)+, 304
(M+H)+; HMRS (FAB) m/z calcd for C18H29N3O 303.2311, found 303.2306 (M)+.
4.1.32. (S)-8-Amino-1,2,4,5-tetrahydro-7-iodo-4-(3-methylbutyl)-2-
(2-methylpropyl)-3H-1,4-benzodiazepin-3-one (33)
ICl-pyridine complex (1.40 mg, 5.80 μmol) was added to a solution of 32 (2.30 mg,
7.58 μmol) in CH2Cl2 / H2O (CH2Cl2 202 μL and H2O 101 μL). The reaction mixture

was stirred 6.5 hours at rt, then the reaction was quenched with H2O and the mixture
was extracted with CH2Cl2. The organic layer was dried over MgSO4 and concentrated.
The resulting residue was purified by PTLC (hexane / AcOEt = 1 / 1) to afford 33 (2.40
mg, 5.59 μmol, 74 %) as a white solid.
o
1
mp 150 C; H-NMR (500 MHz, CDCl3) δ 7.14 (s, 1H), 5.93 (s, 1H), 5.20 (d, J =
16.2 Hz, 1H), 4.53-4.49 (m, 1H), 3.92-3.85 (m, 2H), 3.16 (d, J = 16.2 Hz, 1H),
3.52-3.41 (m, 3H), 1.91-1.85 (m, 1H), 1.82-1.72 (m, 1H), 1.53-1.33 (m, 4H), 0.96 (d, J
= 6.1 Hz, 3H), 0.95 (d, J = 5.5 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H), 0.85 (d, J = 6.1 Hz,
3H); MS (FAB) m/z 429 (M)+, 430 (M+H)+; HMRS (FAB) m/z calcd for C18H28N3O
429.1277, found 429.1272 (M)+.
4.1.33. (S)-8-Amino-1,2,4,5-tetrahydro-4-(3-methylbutyl)-2-(2-methylpropyl)-
7-phenyl-3H-1,4-benzodiazepin-3-one (34)
This compound was prepared by means of GP-A, with K3PO4 (34.4 mg, 162 μmol),
Pd(dppf)Cl2 (7.20 mg, 9.84 μmol), phenylboronic acid (7.50 mg, 61.5 μmol), 33 (21.8
mg, 50.8 μmol) and DMF (0.50 mL). Purification by silica gel chromatography (hexane
/ AcOEt = 2 / 1 to 3 / 2) and PTLC (hexane / AcOEt = 3 / 2) afforded 34 (4.90 mg, 12.9
μmol, 25 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ 7.43-7.38 (m, 4H), 7.32-7.28 (m, 1H), 6.71 (s, 1H),
5.92 (s, 1H), 5.29 (d, J = 16.2 Hz, 1H), 4.60-4.55 (m, 1H), 3.72-3.63 (m, 2H), 3.69 (d, J
= 16.2 Hz, 1H), 3.52-3.45 (m, 1H), 350 (t, J = 7.3 Hz, 2H), 1.93-1.87 (m, 1H),
1.84-1.76 (m, 1H), 1.52-1.44 (m, 2H), 1.43-1.34 (m, 2H), 0.98 (d, J = 6.2 Hz, 3H), 0.96
(d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.1 Hz, 3H); MS (FAB) m/z
379 (M)+, 380 (M+H)+; HMRS (FAB) m/z calcd for C24H33N3O 379.2624, found
379.2621 (M)+.
4.1.34. (S)-Ethyl 4-[8-amino-1,2,4,5-tetrahydro-2-(2-methylpropyl)-4-
(3-methylbutyl)-3-oxo-3H-1,4-benzodiazepin-7-yl]benzoate (35)
This compound was prepared by means of GP-A, with K3PO4 (73.2 mg, 345 μmol),
Pd(dppf)Cl2 (7.50 mg, 10.3 μmol), 4-ethoxycarbonylphenylboronic acid pinacol ester
(47.4 mg, 172 μmol), 33 (49.0 mg, 114 μmol) and DMF (1.0 mL). Purification by PTLC
(hexane / AcOEt = 3 / 2) afforded 35 (31.0 mg, 68.7 μmol, 60 %) as a yellow oil.
1H-NMR (500 MHz, CDCl3) δ 8.08 (d, J = 7.9 Hz, 2H), 7.49 (d, J = 7.9 Hz, 2H), 6.73

(s, 1H), 5.92 (s, 1H), 5.29 (d, J = 15.9 Hz, 1H), 4.61-4.56 (m, 1H), 4.40 (t, J = 6.7 Hz,
2H), 3.72-3.68 (m, 3H), 3.55-3.52 (m, 1H), 3.50 (t, J = 7.6 Hz, 2H), 1.94-1.87 (m, 1H),
1.84-1.78 (m, 1H), 1.52-1.43 (m, 2H), 1.43-1.35 (m, 2H), 1.41 (t, J = 6.7 Hz, 3H), 0.98
(d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.1 Hz, 3H), 0.90 (d, J = 6.2 Hz, 3H), 0.84 (d, J = 6.7
Hz, 3H); MS (FAB) m/z 451 (M)+, 452 (M+H)+; HMRS (FAB) m/z calcd for
C27H37N3O3 451.2835, found 451.2833 (M)+.
4.1.35. (S)-4-[8-Amino-1,2,4,5-tetrahydro-2-(2-methylpropyl)-4-(3-methylbutyl)-
3-oxo-3H-1,4-benzodiazepin-7-yl]benzoic acid (36)
KOH (3.00 mg, 53.5 μmol) in MeOH (0.50 mL) was added to a solution of 35 (12.7
mg, 28.1 μmol) in MeOH (0.50 mL). The reaction mixture was stirred for 1.5 h at 60 oC,
then H2O (0.20 mL) was added to it, because TLC monitoring indicated that the
o
reaction had not proceeded. Stirring was continued for 6 hours at 60 C, then the
reaction was quenched with 2 N HCl aq., and sat. NaHCO3 aq. was added to adjust the
o
pH to 7 at 0 C. Brine and H2O were further added and the mixture was extracted with
AcOEt and CHCl3. The organic layer was dried over MgSO4 and concentrated. The
residue was purified by PTLC (CHCl3 / MeOH = 10 / 1) to afford 36 (5.50 mg, 13.0
μmol, 46 %) as a yellow oil.
1H-NMR (500 MHz, CDCl3) δ 8.14 (d, J = 7.4 Hz, 2H), 7.52 (d, J = 7.4 Hz, 2H), 6.74
(s, 1H), 5.92 (s, 1H), 5.30 (d, J = 16.5 Hz, 1H), 4.61-4.56 (m, 1H), 3.71 (d J = 16.5 Hz,
1H), 3.53-3.47 (m, 2H), 1.95-1.88 (m, 1H), 1.84-1.77 (m, 1H), 1.52-1.44 (m, 2H),
1.44-1.35 (m, 2H), 0.98 (d, J = 6.1 Hz, 3H), 0.97 (d, J = 6.1 Hz, 3H), 0.90 (d, J = 6.7 Hz,
3H), 0.84 (d, J = 6.7 Hz, 3H); MS (FAB) m/z 423 (M)+, 424 (M+H)+; HMRS (FAB) m/z
calcd for C25H33N3O3 423.2522, found 423.2523 (M)+.
4.1.36. Ethyl 3-hydroxy-4-iodobenzoate (38)
N-Iodosuccinimide (1.40 g, 6.24 mmol) was added to a stirred solution of ethyl
3-hydroxybenzoate (37) (1.02 g, 6.13 mmol) in AcOH (30 mL) at 0 oC, then the reaction
mixture was stirred for 21 h at rt. At 0 oC, 10 N NaOH aq. was added to adjust the pH to
5, and a white solid precipitated. The solid was removed by filtration and the filtrate
was extracted with AcOEt. The white solid was added to the organic layer, and the
resulting solution was dried over MgSO4 and concentrated. The residue was purified by
silica gel chromatography (hexane / AcOEt = 4 / 1) and recrystallization (from CH2Cl2 /

hexane) to afford 38 (1.13 g, 3.88 mmol, 63 %) as a white amorphous solid.
1H-NMR (500 MHz, CDCl3) δ 7.75 (d, J = 8.6 Hz, 1H), 7.63 (d, J = 1.8 Hz, 1H), 7.34
(dd, J = 8.6, 1.8 Hz, 1H), 5.41 (s, 1H), 4.37 (q, J = 6.7 Hz, 2H), 1.39 (t, J = 6.7 Hz, 3H);
13C-NMR(125 MHz, CDCl3) δ 165.9, 155.0, 138.5, 132.57, 123.0, 115.7, 91.5, 61.4,
14.2; HMBC was used to determine the iodide position; MS (FAB) m/z 292 (M)+, 293
(M+H)+.
4.1.37. Ethyl 3-[2-(benzyloxy)ethoxy]-4-iodobenzoate (39)
Diisopropyl azodicarboxylate (DIAD) (590 μL, 3.00 mmol) was added dropwise to a
stirred solution of 38 (436 mg, 1.49 mmol), 2-(benzyloxy)ethanol (456 mg, 3.00 mmol)
and PPh3 (1.11 g, 1.81 mmol) in THF (15.0 mL). The reaction mixture was stirred for
27 hours at rt, then concentrated, quenched with H2O, and extracted with AcOEt. The
organic layer was dried over MgSO4, and concentrated. The resulting residue was
purified by silica gel chromatography (hexane / AcOEt = 4 / 1) to afford 39 (591 mg,
1.39 mmol, 93 %) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ 7.86 (d, J = 7.9 Hz, 1H), 7.46 (d, J = 1.8 Hz, 1H),
7.42-7.34 (m, 5H), 7.31-7.26 (m, 1H), 4.27 (s, 2H), 4.37 (q, J = 7.3 Hz, 2H), 4.28 (t, J =
4.9 Hz, 2H), 3.93 (t, J = 4.9 Hz, 2H), 1.39 (t, J = 7.3 Hz, 3H); MS (FAB) m/z 426 (M)+,
427 (M+H)+.
4.1.38. (S)-Ethyl 4-[8-amino-1,2,4,5-tetrahydro-2-(2-methylpropyl)-4-
(3-methylbutyl)-3-oxo-3H-1,4-benzodiazepin-7-yl]-3-[2-(benzyloxy)ethoxy]
benzoate (41)
DMSO (10.0 mL) was added to a mixture of 39 (224 mg, 526 μmol),
bis(pinacolato)diboran (280 mg, 1.10 mmol), KOAc (150 mg, 1.53 mmol) and
PdCl2(dppf) (30.3 mg, 41.4 μmol). The atmosphere was replaced with Ar. The reaction
mixture was stirred for 1.5 h at 80 oC, then the reaction was quenched with H2O and the
mixture was extracted with AcOEt. The organic layer was washed with H2O, dried over
MgSO4, and concentrated. The resulting residue was purified by silica gel
chromatography (hexane / AcOEt = 1 / 0 to 5 / 1) and PTLC (hexane / AcOEt = 5 / 2) to
afford 40 (mixture, 18.4 mg) as a colorless oil. This was used for the next reaction
without further purification. Compound 41 was prepared by means of GP-A, with
K3PO4 (21.8 mg, 103 μmol), Pd(dppf)Cl2 (2.80 mg, 3.80 μmol), 40 (mixture, 18.4 mg),

33 (14.5 mg, 33.8 μmol) and DMF (0.75 mL). Purification by silica gel chromatography
(hexane / AcOEt = 3 / 2) and PTLC (hexane / AcOEt = 3 / 2) afforded 41 (8.20 mg, 13.6
μmol, 3 % (2 steps)) as a yellow oil.
1H-NMR (500 MHz, CDCl3) δ 7.71 (dd, J = 7.9, 1.5 Hz, 1H) , 7.63 (d, J = 1.5 Hz,
1H), 7.35-7.25 (m, 6H), 6.69 (s, 1H), 5.84 (s, 1H), 5.29-5.18 (m, 1H), 4.60-4.52 (m, 1H),
4.53 (s, 2H), 4.39 (q, J = 7.0 Hz, 2H), 4.28-4.20 (m, 2H), 3.81-3.78 (m, 2H), 3.69-3.60
(m, 3H), 3.55-3.40 (m, 3H), 1.95-1.86 (m, 1H), 1.84-1.76 (m, 1H), 1.52-1.34 (m, 2H),
1.40 (t, J = 7.0 Hz, 3H), 1.28-1.23 (m, 2H), 0.99-0.94 (m, 6H), 0.90-0.86 (m, 3H),
0.85-0.80 (m, 3H); MS (FAB) m/z 601 (M)+, 602 (M+H)+.
4.1.39 (S)-Ethyl 4-[8-Amino-1,2,4,5-tetrahydro-2-(2-methylpropyl)-4-
(3-methylbutyl)-3-oxo-3H-1,4-benzodiazepin-7-yl]-3-(2-hydroxyethoxy) benzoate
(42)
Under an Ar atmosphere in a pressure-tight flask, 10 % Pd/C (8.20 mg) was added to a
solution of 41 (8.20 mg, 13.6 μmol) in 1,4-dioxane (3.5 mL). The atmosphere was
o
replaced with H2 (2.5 atm). The reaction mixture was stirred 4 hours at 50 C, then
filtered through Celite and concentrated. The resulting residue was purified by PTLC
(hexane / AcOEt = 1 / 1) to afford 42 (1.20 mg, 2.35 μmol, 17 %) as a colorless oil. The
product was a mixture of conformers in a ratio of 0.3 : 0.7 (determined by 1H-NMR).
1H-NMR (500 MHz, CDCl3) δ 7.75 (d, J = 8.5 Hz, 0.3H), 7.73 (d, J = 8.5 Hz, 0.7H),
7.62 (s, 0.3H), 7.58 (s, 0.7H), 7.30-7.24 (m, 1H), 6.70 (s, 0.7H), 6.66 (s, 0.3H), 6.07 (s,
0.3H), 5.99 (s, 0.7H), 5.34-5.27 (m, 1H), 4.65-4.45 (m, 2H), 4.44-4.37 (m, 2H),
4.24-4.19 (m, 2H), 4.00-3.35 (m, 8H), 1.94-1.86 (m, 1H), 1.86-1.74 (m, 1H), 1.54-1.30
(m, 7H), 1.05-0.95 (m, 6H), 0.94-0.76 (m, 6H); MS (FAB) m/z 511 (M)+, 512 (M+H)+;
HMRS (FAB) m/z calcd for C29H41N3O5 511.3046, found 511.3050 (M)+.
4.2. Reporter gene assay
Inhibitory activity towards VDR-mediated transcriptional activation was determined by
reporter gene assay as previously described.14, 20 Each experiment was performed in
triplicate and the normalized average values are presented.

Acknowledgement
The work described in this paper was partially supported by Grants-in-Aid for
Scientific Research from The Ministry of Education Culture, Sports, Science and
Technology, Japan, and the Japan Society for the Promotion of Science.

Reference and notes
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Blumgerg, B.; Kastner, P.; Mark, M.; Chambon, P.; Evans, R. M. Cell 1995, 83,
835-839.
3. Björklund, S.; Almouzni, G.; Davidson, I.; Nightingale, K. P.; Weiss, K. Cell 1999, 96,
759-767.
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Figure 1. Structures of 1,25(OH)2D3 and representative VDR antagonists
Figure 2. Illustration of the interaction between LXXLL peptide fragment and VDR,
and a representative benzodiazepine that we reported previously.14 Important structures
in the LXXLL peptide fragment, that is the isobutyl chains of leucine residues and the
charge clamp-related hydrogen bonds, are shown in green, blue and red. The
corresponding structures of benzodiazepine 1 are shown in the same color. The residues
of VDR are shown in gray. See also Figures 3 and 4.
Figure 3. The pocket composed of residues Ile234, Ile238, Leu259, Ala263, and Val417
of VDR (PBD ID 1RK3). The VDR surface is shown in gray and key residues of the
pocket are shown in white. The peptide is shown in green and magenta, and key leucine
residues in the peptide are shown in green. The image was drawn with PYMOL.
Figure 4. Spatial relationship between LXXLL peptide fragment and Lys260 of VDR
(PBD ID 1RK3). The VDR surface is shown in gray and Lys260 is shown in white. The
peptide is shown in green and magenta, and key leucine residues in the peptide are
shown in green. The image was drawn with PYMOL.
Scheme 1. Reagents and conditions: (a) L-Leucine methyl ester hydrochloride,
o
Pd2(dba)3･CHCl3, (R)-BINAP, Cs2CO3, toluene, 110 C; (b) Raney nickel, H2, MeOH /
o
Et3N = 10 / 1, rt; (c) 1-bromo-3-methylbutane, NaH, DMF, 0 C to rt; (d) ICl-pyridine,
CH2Cl2 / H2O = 2 / 1, rt, (e) 9 or 12, PdCl2(dppf), K3PO4, DMF, 80 oC; (f) Pd/C, H2 (3
atm), AcOEt, 50 oC
o
Scheme 2. Reagents and conditions: (a) BH3•SMe2, (+)-α-pinene, THF, 0 C to rt;
acetaldehyde, 40 oC; H2O, rt; (b) pinacol, MgSO4, CH2Cl2, rt
o
Scheme 3. Reagents and conditions: (a) BH3•SMe2, (+)-α-pinene, THF, 0 C to rt;
acetaldehyde, 40 oC; H2O, rt

o
Scheme 4. (a) 14a or 14b, PdCl2(dppf), K3PO4, DMF, 80 C; (b) TBAF, THF, rt; (c)
Pd/C, H2, 1,4-dioxane, rt; (d) TBAF, THF, rt
Scheme 5. Reagents and conditions: (a) boronic acid pinacol ester, PdCl2(dppf), K3PO4,
DMF, 80 oC; (b) TFA, CH2Cl2, 0 oC; (c) Pd2(dba)3･CHCl3, ( )-BINAP, Cs2CO3, toluene,
110 oC; (d) Pd/C, H2, AcOEt, rt
o
Scheme 6. Reagents and conditions: (a) 4-methoxybenzylamine, 140 C; (b) DDQ,
CH2Cl2 / H2O = 2 / 1, rt; (c) (Boc)2O, DMAP, DIPEA, THF, reflux; (d) L-leucine
o
methyl ester hydrochloride, Pd2(dba)3, Xantphos, Cs2CO3, H2O, toluene, 110 C; (e)
Raney nickel, H2, MeOH / Et3N = 10 / 1, rt; (f) 1-bromo-3-methylbut-2-ene, TBAI,
o
t-BuOK, THF, -20 C; (g) Pd/C, H2, 1,4-dioxane, rt; (h) HCl, 1,4-dioxane, rt; (i)
ICl-pyridine, CH2Cl2 / H2O = 2 / 1, rt, (j) phenylboronic acid, PdCl2(dppf), K3PO4,
DMF, 80 oC
Scheme 7. Reagents and conditions: (a) 4-ethoxycarbonylphenyl boronic acid pinacol
ester, PdCl2(dppf), K3PO4, DMF, 80 oC; (b) KOH, MeOH / H2O = 5 / 1, 60 oC
Scheme 8. Reagents and conditions: (a) NIS, AcOH, rt; (b) 2-phenoxyethanol, DIAD,
o
PPh3, THF, rt; (c) bis(pinacolate)diboron, PdCl2(dppf), KOAc, DMSO, 80 C; (d) 33,
PdCl2(dppf), K3PO4, DMF, 80 oC; (e) Pd/C, H2 (2.5 atm), 1,4-dioxane, 50 oC
Table 1. SAR at the 7-position of benzodiazepine derivatives without an amino group in
VDR reporter gene assaya
aHEK293 cells were treated with 1,25(OH)2D3 (3 nM) and test compounds
bInhibition ratio at 30 μM
cN.A. means no inhibitory activity at 30 μM
Table 2. SAR at the 7-position of benzodiazepine derivatives with an amino group in
VDR reporter gene assaya
aHEK293 cells were treated with 1,25(OH)2D3 (3 nM) and test compounds