Synthesis and biological activities of phenylahistin derivatives

Article abstract of DOI:10.1016/S0968-0896(99)00059-0

X-ray crystallographic analysis was performed and several phenylahistin derivatives were synthesized to elucidate the structural components necessary for the anti-microtubule activity of phenylahistin. We primarily focused on the unique isoprenylated dehy

Full text of DOI:10.1016/S0968-0896(99)00059-0

Bioorganic & Medicinal Chemistry 7 (1999) 1451±1457
Synthesis and Biological Activities of Phenylahistin Derivatives
Kaneo Kanoh, Shinkichi Kohno, Jun Katada, Junko Takahashi, Isao Uno and
Yoshio Hayashi*
Life Science Research Center, Advanced Technology Research Laboratories, Nippon Steel Corporation, 3-35-1 Ida,
Nakahara-ku, Kawasaki 211-0035, Japan
Received 8 January 1999; accepted 25 February 1999
AbstractÐX-ray crystallographic analysis was performed and several phenylahistin derivatives were synthesized to elucidate the
structural components necessary for the anti-microtubule activity of phenylahistin. We primarily focused on the unique iso-
prenylated dehydrohistidine structure. Our results showed that a uniplanar pseudo-three-ring structure formed by the hydrogen
bonding of diketopiperazine and imidazole rings is important for the anti-microtubule activity of phenylahistin. # 1999 Elsevier
Science Ltd. All rights reserved.
Introduction
Chemistry
Phenylahistin [1], a fungal metabolite from Aspergillus
ustus NSC-F038, was discovered during screening for
new cell cycle inhibitors. It is a member of a new class of
colchicine-like microtubule binding agents that exhibit
cytotoxic activity against a wide variety of tumor cell
lines.1±3 ( )-Phenylahistin [( )-1], a diketopiperazine
derivative, consists of l-phenylalanine and a unique
isoprenylated dehydrohistidine residue with a qua-
ternary carbon at the 5-position of the imidazole ring.2
To develop more potent anti-tumor agents based on this
diketopiperazine derivative, it is important to elucidate
the structural components important in the anti-micro-
tubule activity of ( )-1.
Simple alkylation and reduction of 1 and modi®cation
of cyclo(Gly-Phe) with imidazole derivatives seem to be
necessary for the synthesis of derivatives of 1. However,
the complete synthesis and subsequent analogue devel-
opment of 1 has not yet been accomplished due to the
presence of a unique quaternary carbon at the imidazole
ring. Derivatives 2±5 were synthesized from 1 by alky-
lation or reduction. As shown in Scheme 1, 2 and 3 were
synthesized by hydrogenation of each enantiomer of 1
over 10% palladium on carbon in MeOH under atmo-
spheric hydrogen at room temperature. Two-hour
hydrogenation yielded derivative 2, in which the 1,1-
dimethyl-2-propenyl group of 1 was reduced. Further
hydrogenation (24 h) with 2 yielded derivative 3, in
which the dehydrohistidine was reduced. The latter
reduction resulted in only one diastereomer, with an
optical rotation polarity similar to that of 2. Although
we did not determine the absolute con®guration at the
a-position of the substituted histidine residue of 3, this
diastereoselective hydrogenation may be due to the
steric hindrance of the phenyl ring of the Phe residue.
This conclusion is based on the results of (+)-1 X-ray
crystallography which indicates that this phenyl ring is
positioned above and overlays the diketopiperazine ring
(see Results and Discussion).
In the present study, we used X-ray crystallography to
analyze (+)-1 and the synthesis of its derivatives, and
comprehensively investigate the structural components
of phenylahistin that are necessary for anti-microtubule
activity. In particular, we focused on the isoprenylated
dehydrohistidine structure and found that it forms a
rigid uniplanar pseudo-three-ring structure, composed
of diketopiperazine and imidazole rings. The rings are
attached through a hydrogen bond between N8-H and
N3 and an a, b-unsaturated bond (C6±C7). The struc-
ture of this component is particularly important for the
anti-microtubule activity of phenylahistin.
As shown in Scheme 2, methylation of 1 with MeI and
NaH in DMF yielded mono- and tri-methylated com-
pounds. The extent of methylation could be controlled
by manipulating the reaction temperature rather than
the amount of reagents. Mono-methylated derivative 4
was predominantly obtained in a reaction with 10
Key words: Amino acids and derivatives; antitumor compounds; X-
ray crystal structure; natural products.
* Corresponding author. Present address: Department of Medicinal
Chemistry, Kyoto Pharmaceutical University, Yamashina-Ku, Kyoto
607-8414, Japan. Tel.: +81-75-595-4636; fax: +81-75-591-9900; e-mail:
yhayashi@mb.kyoto-phu.ac.jp
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00059-0

1452
K. Kanoh et al. / Bioorg. Med. Chem. 7 (1999) 1451±1457
Scheme 1. (a) H2/10% Pd-C, MeOH, rt, 2 h; (b) H2/10% Pd-C, MeOH, rt, 24 h.
Scheme 2. (a) MeI, NaH, DMF, 30ꢀC, 2 h; (b) MeI, NaH, DMF, rt, 2 h; (c) chiral HPLC.
Scheme 3. (a) MeOH, re¯ux, 14 h; (b) Ac2O, AcONa, 80ꢀC, 14 h; (c) 8 LDA, HMPA, DMF, 60ꢀC, 0.5 h; (d) Tf2O, pyridine, rt, 10 min; (e)
NH4OH, rt, overnight.
equivalent of the reagents at 30ꢀC. Methylation at the
Results and Discussion
t nitrogen of the imidazole ring, was detected by NMR
analysis.4 On the other hand, the tri-methylated deriva-
tive 5 was obtained in a reaction with 30 equivalent of
X-ray crystallographic analysis
the reagents at room temperature. Compounds 4 and 5
with an l-phenylalanine residue were separated by
HPLC with a chiral column.5
X-ray analysis of (+)-1 is shown in Figure 1 and
Table 2. The stereochemistry of the C6±C7 double-bond
was con®rmed to be Z, and the presence of a hydrogen
bond between N8-H and N3 was noted. These ®ndings
were in agreement with those established in NMR stu-
dies (low ®eld-shift of N8-H d 12.08 ppm).2,7 The results
Derivatives 9 and 10 were synthesized from cyclo (Gly-
Phe) 6, which was prepared by cyclization of H-Phe-
Gly-OMe. After acetylation of the two amide nitrogens
on the diketopiperazine ring of 6, 4(5)-imidazolecarbox-
aldehyde or 4-methyl-5-imidazolecarboxaldehyde (8) was
introduced in the presence of lithium diisopropylamide
(LDA) and hexamethylphosphoramide (HMPA).6 Sub-
sequent dehydration with tri¯ic anhydride-pyridine
and deacetylation with aqueous NH4OH gave com-
pounds 9 and 10, although less than 30% racemization
was observed in these steps. Compounds 9 and 10 with
an l-phenylalanine residue were puri®ed by chiral col-
umn HPLC.
Figure 1. Structure of ( )- phenylahistin [( )-1].

K. Kanoh et al. / Bioorg. Med. Chem. 7 (1999) 1451±1457
1453
Table 1. Biological activity of phenylahistin and its derivatives
IC50 (mM)
Compound
)-1
Structure
Microtubule proteina polymerization
P388 proliferation
0.21‹0.02b
(
25
(+)-1
>200 [15%]c
10‹1.5
(
)-2
30
0.23‹0.05
(+)-2
>200 [12%]
19‹4.2
(
)-3
>200
>200
>200
>200
(+)-3
4
5
9
100
0.95‹0.03
160‹5.5
>200
>200
>200 [14%]
10
>200 [28%]
7.5‹0.5
11d
>200
>200
colchicined
16
0.031‹0.01
aMicrotubule protein was prepared from bovine brain by two cycles of the assembly and disassembly method.9 The concentration of microtubule
protein used for the assay was 1.5 mg/mL.
bValues are mean‹SEM of three experiments.
cThe inhibition percentage at 200 mM is indicated within the square brackets when the inhibition was observed, since diketopiperazine derivatives are
insoluble at concentrations more than 200 mM.
dCyclo(His-Phe) 11 and colchicine were purchased from Sigma (St. Louis, MO).

1454
K. Kanoh et al. / Bioorg. Med. Chem. 7 (1999) 1451±1457
we synthesized derivatives of ( )-1 and examined its
e€ects on the polymerization of microtubule protein
prepared from the bovine brain9,10 and the proliferation
of P388 cells. We have recently demonstrated that ( )-
phenylahistin [( )-1] exhibited colchicine-like inhibition
of microtubule polymerization (IC50=25 mM).1 This
inhibitory activity of ( )-1 was similar to that of col-
chicine (IC50=16 mM), although the anti-proliferative
activity of ( )-1 (IC50=0.21 mM) was about seven times
less than that of colchicine (IC50=0.031 mM). The dif-
ferences in activity may be due to other biological
activities of colchicine, or lower cellular permeability of
( )-1 in anti-proliferative assays, since ( )-1 has a more
hydrophilic structure than colchicine. We also demon-
strated that ( )-1 with the l-Phe residue was 50 times
more active than its (+)-form in anti-proliferative
assays. (+)-1 also exhibited a weak inhibitory activity
against microtubule polymerization (15% inhibition at
200 mM) compared with that of ( )-1. These results
indicate that the spatial arrangement of the benzyl
group of the phenylalanine residue is important for the
potent biological activity.
Table 2. Results of X-ray crystallographic analysis of (+)-1
Crystal parameters
Empirical formula
Formula weight
Crystal system
C20H22N4O2
350.42
tetragonal
a=15.3509(7) A
c=8.309(2) A
V=1958.0(2) A3
Z=4
Lattice parameters:
Space group
Dcalc.
P42 (#77)
1.189 g/cm3
1
m (CuKa)
6.37 cm
Re®nement parameters
No. of re¯ections measured
Nonzero re¯ections (I>1.50s)
R-index Residuals: Ra
Residuals: RWb
2229
1724
0.057
0.067
4.31
Goodness of ®t indicatorc
aÆ k Foj jFc k =ÆjFoj.
2
1=2
‰ÆwꢁꢁjFoj jFcj† =ÆwFo2†Š
.
b
‰ÆwꢁjFoj jFcj†2=ꢁNo Nv†Š
.
1=2
c
In the present study, compound 2, in which the 1,1-
dimethyl-2-propenyl group of 1 was reduced (Table 1),
showed the same activity as 1, indicating that this dou-
ble bond is not important for the anti-microtubule
activity. However, compound ( )-3, in which the dehy-
drohistidine of ( )-2 was reduced, completely lost the
inhibitory activity. Modi®cation of the pseudo-three-
ring structure in the phenylahistin structure alters
activity, suggesting that inhibitory activity is dependent
upon a planar arrangement of the diketopiperazine and
imidazole rings.
Compound 4 was derived through methylation of the
nitrogen atoms of 1. When the t nitrogen of the imida-
zole ring of ( )-1 was methylated, the compound was
approximately ®ve times less active than ( )-1. This
®nding suggests that the imidazole nitrogen participates
in binding with the microtubule protein or that this
methylation a€ords structural hindrance to the con-
formation of the 1,1-dimethyl-2-propenyl group on the
imidazole ring. The tri-methylated compound 5 had a
severe reduction in inhibitory activity. This result also
suggests that the rigid plane conformation of ( )-1 is
important for anti-microtubule activity, since methyla-
tion of the diketopiperazine nitrogen (N8) disrupts the
formation of the hydrogen bond necessary for the rigid
pseudo-three-ring conformation.
Figure 2. Crystal structure of (+)-phenylahistin (ORTEP).
suggested that the two heterocycles, i.e., the diketopi-
perazine and imidazole rings, were ®xed in the same
plane by forming a pseudo-three-ring structure. The
benzyl group of the Phe residue was located out of this
plane and over the diketopiperazine ring, in a con-
formation which is reported as the most energetically
favorable for a diketopiperazine with an aromatic
amino acid residue.8 The movement of the 1,1-dimethyl-
2-propenyl group at the imidazole ring was restricted by
the steric hindrance of a hydrogen atom at the b-posi-
tion (C6) of the a,b-unsaturated His derivative. These
®ndings indicate that the conformation of (+)-pheny-
lahistin is highly restricted. Since the ( )-form of 1, the
biologically active enantiomer, only varies from the
(+)-form in conformation at the a-position of the Phe
residue, the rigid conformation of phenylahistin may be
important for binding to the microtubule protein.
To determine the importance of the alkyl group at the 5-
position on the imidazole ring, the 1,1-dimethyl-2-pro-
penyl group of ( )-1 was replaced with a hydrogen
atom or a methyl group, in compounds 9 and 10
respectively. Compound 9 containing a hydrogen atom
at position 5 lost most inhibitory activity. Compound 10
with a methyl group at the 5-position also exhibited
decreased activity in the anti-microtubule assay,
although a weak inhibitory e€ect on P388 cell pro-
liferation was observed. The marked decrease in biolo-
gical activity of 9 and 10 suggests that an alkyl group of
the proper length or a quaternary carbon at the 5-position
Biological activities of synthetic derivatives
To understand the relationship between the postulated
rigid plane structure of ( )-1 and its biological activity,

K. Kanoh et al. / Bioorg. Med. Chem. 7 (1999) 1451±1457
1455
of the imidazole ring is very important for the anti-
microtubule activity. Further SAR studies are necessary
to establish the importance of this component. Total
synthesis of phenylahistin, including the formation of a
chemical bond containing a quaternary carbon on the
imidazole ring, should provide this answer.
expanded using Fourier techniques.13 All calculations
were performed using the teXscan crystallographic soft-
ware package from Molecular Structure Corporation.
Chemistry
Melting points were determined on a Mettler FP62
apparatus and are uncorrected. 1H NMR and 13C-
NMR spectra were recorded on JEOL GSX270J spec-
trometer. The spectra were recorded with tetra-
methylsilane (d=0.0 for 1H); DMSO-d6 (d=39.5 for
13C); CDCl3 (d=77.0 for 13C) as internal reference.
Mass spectra (electrospray ionization, methanol as the
mobile phase) were analyzed with a Finnigan SSQ 7000
spectrometer. High-resolution fast atom bombardment
mass spectra were analyzed with a JEOL JMS-DX303
spectrometer. Infrared spectra were recorded on a JEOL
JIR-5500 infrared spectrophotometer in KBr pellets.
Silica-gel column chromatography was performed using
Merck 70±230 mesh silica gel 60. Optical rotations were
measured on a Horiba SEPA-200 polarimeter, and are
Conclusion
In the present study, we analyzed the X-ray crystal-
lography of (+)-phenylahistin and evaluated the biolo-
gical activity of phenylahistin derivatives. We determined
that the component of ( )-phenylahistin necessary for
anti-microtubule activity is the rigid uniplanar pseudo-
three-ring structure formed by a hydrogen bond. Our
results should be useful for the development of more
potent anti-microtubule agents based on the diketopi-
perazine structure.
Experimental
1
expressed in 10 deg cm2 g 1. Elemental analyses were
Microtubule protein polymerization assay
performed with a Fisons EA 1108 elemental analyzer.
Microtubule protein was prepared from bovine brain
tissue by two cycles of assembly and disassembly.9
Polymerization of microtubule protein was monitored
by an increase in turbidity at 37ꢀC in the microtubule
assembly bu€er10 containing 100 mM MES, 0.5 mM
MgCl2, 1 mM EGTA and 1 mM GTP. Polymerization
of microtubule protein was initiated by a temperature
shift from 0ꢀC to 37ꢀC. Turbidity was measured with a
thermo-controlled spectrophotometer (Beckman DU-
20, Fullerton, CA) at 360 nm. Compounds for all
experiments were dissolved in DMSO at a ®nal con-
centration of 2% (v/v).
( ) - Cyclo- [5 - (1,1 - dimethylpropyl)dehydrohistidinyl - L-
phenylalanine] ( )-2. 10 mg of 10% palladium on car-
bon was added to a solution of ( )-1 (30 mg,
0.086 mmol) in MeOH (10 mL) and the mixture was
stirred at room temperature for 2 h under hydrogen at
atmospheric pressure. After the catalyst was ®ltered o€
and the ®ltrate was concentrated under reduced pres-
sure, the residue was puri®ed by column chromato-
graphy on silica (5 g) using CHCl3:MeOH (50:1) as an
eluant. The desired fractions were collected and the sol-
vent was evaporated. The residual white powder was
recrystallized with EtOH±hexane to yield 12 mg (39%)
of ( )-2 as a colorless solid; mp 224±226 ꢀC; [a]d25 295
(c=0.15, MeOH); UV (MeOH) nm 322 (e 23300), 231 (e
Alamar Blue2 assay
1
8830), 203 (e 16800); IR (KBr) cm 3310, 3220, 2970,
1
P388 cells in exponential growth phase were seeded into
96-well tissue culture plates (5Â103 cells/100 ml/well) and
cultured in RPMI 1640 medium supplemented with
10% fetal bovine serum for 16 h. The compounds
(DMSO solution) were then added to each well at var-
ious concentrations, and the cells were cultured for an
additional 48 h. Live cells were counted using Alamar
Blue2 (BioSource International, Camarillo, CA).11
1670, 1440; H NMR (270 MHz, CDCl3) d 12.09 (br s,
1H), 8.98 (br s, 1H), 7.56 (s, 1H), 7.29±7.38 (m, 5H),
6.87 (s, 1H), 5.67 (s, 1H), 4.34 (ddd, J=2, 3, 10 Hz, 1H),
3.51 (dd, J=3, 14 Hz, 1H), 2.29 (dd, J=10, 14 Hz, 1H),
1.74 (q, J=7 Hz, 2H), 1.40 (s, 6H), 0.74 (t, J=7 Hz,
3H); 13C NMR (67.5 MHz, CDCl3) d 164.6, 159.9,
138.1, 135.5, 132.2, 132.1, 129.5 (2C), 129.1 (2C), 127.4,
123.6, 105.4, 57.2, 41.3, 36.2, 35.4, 27.9 (2C), 9.2;
HRMS m/z 352.1926 (M+) (calcd for C20H24N4O2:
352.1899). Anal. calcd for C20H24N4O2: C, 68.16; H,
6.86; N, 15.90, Found: C, 68.34, 6.78, 15.82.
Single-crystal X-ray di€raction analysis
A pale yellow crystal of (+)-1 with approximate
dimensions of 0.4Â0.4Â0.3 mm, which was crystallized
from EtOAc, was used for single-crystal X-ray di€rac-
tion analysis. All X-ray measurements were performed
on a Rigaku AFC7R di€ractometer with graphite
monochromated CuKa radiation and a rotating anode
generator. The crystal data and re®nement parameters
are summarized in Table 2. Of the 2229 collected re¯ec-
tions, 2064 were unique (Rint=0.019). The data were
corrected for Lorentz and polarization e€ects, and a
correction for secondary extinction was applied. The
structure was determined by direct methods12 and
(+)-Cyclo-[5-(1,1-dimethylpropyl)dehydrohistidinyl-D-
phenylalanine] (+)-2. This compound was prepared
using the same procedure applied for the preparation of
( )-2. 44% yield from (+)-1; colorless solid; mp 222±
223 ꢀC; [a]2d5 +284 (c=0.10, MeOH); UV (MeOH) nm
322 (e 22800), 231 (e 8720), 203 (e 16300); IR (KBr)
cm 1 3310, 3220, 2970, 1670, 1440; 1H NMR (270 MHz,
CDCl3) d 12.09 (br s, 1H), 8.98 (br s, 1H), 7.56 (s, 1H),
7.29±7.38 (m, 5H), 6.87 (s, 1H), 5.67 (s, 1H), 4.34 (ddd,
J=2, 3, 10 Hz, 1H), 3.51 (dd, J=3, 14 Hz, 1H), 2.29
(dd, J=10, 14 Hz, 1H), 1.74 (q, J=7 Hz, 2H), 1.40 (s,

1456
K. Kanoh et al. / Bioorg. Med. Chem. 7 (1999) 1451±1457
6H), 0.74 (t, J=7 Hz, 3H); 13C NMR (67.5 MHz,
CDCl3) d 164.6, 159.9, 138.1, 135.5, 132.2, 132.1, 129.5
(2C), 129.1 (2C), 127.4, 123.6, 105.4, 57.2, 41.3, 36.2,
35.4, 27.9 (2C), 9.2; HRMS m/z 352.1932 (M+) (calcd
for C20H24N4O2: 352.1899). Anal. calcd for C20H24
N4O2: C, 68.16; H, 6.86; N, 15.90. Found: C, 68.09,
6.87, 15.84.
using a chiral column (CHIRALCEL OD, 10Â250 mm)
eluted with a mixture of hexane and ethanol (3:1) at a
¯ow rate of 6.0 mL/min on a Waters system (600E ser-
ies). 19% yield from 1; white powder; mp 214±215 ꢀC;
[a]2d5 285 (c=0.30, MeOH); UV (MeOH) nm 317 (e
1
25400), 232 (sh, e 8640), 204 (e 16800); IR (KBr) cm
1
3200, 2980, 1680, 1440; H NMR (270 MHz, CDCl3) d
12.20 (br s, 1H), 7.39 (s, 1H), 7.38±7.24 (m, 5H), 7.09 (s,
1H), 6.01 (dd, J=18, 9 Hz, 1H), 5.68 (br s, 1H), 5.14 (d,
J=9 Hz, 1H), 5.00 (d, J=18 Hz, 1H), 4.33 (m, 1H),
3.66 (s, 3H), 3.50 (dd, J=14, 3 Hz, 1H), 2.92 (dd, J=14,
10 Hz, 1H), 1.59 (s, 6H); 13C NMR (67.5 MHz, CDCl3)
d 164.6, 160.1, 145.6, 138.5, 136.5, 135.6, 134.6, 129.5
(2C), 129.1 (2C), 127.4, 124.0, 112.7, 106.6, 57.1, 41.3,
39.2, 34.9, 28.9, 28.8; HRMS m/z 364.1909 (M+) (calcd
for C21H24 N4O2: 364.1899). Anal. calcd for
( )-Cyclo-[5-(1,1-dimethylpropyl)histidinyl-L-phenylalanine]
( )-3. This compound was prepared from ( )-2, using
the same procedure used for the preparation of ( )-2,
but with a 24 h reaction time. 60% yield from ( )-2;
white powder; mp 224±225 ꢀC; [a]d25
96 (c=0.16,
MeOH); UV (MeOH) nm 257 (e 194), 205 (e 18100); IR
1
(KBr) cm 3380, 3200, 2970, 1670, 1440; 1H NMR
(270 MHz, DMSO-d6) d 11.55 (br s, 1H), 8.25 (br s,
1H), 7.86 (br s, 1H), 7.41 (s, 1H), 7.29±7.14 (m, 5H),
4.24 (br s, 1H), 3.94 (br d, J=11 Hz, 1H), 3.11 (dd,
J=14, 4 Hz, 1H), 2.88 (dd, J=14, 5 Hz, 1H), 2.82 (dd,
J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H), 1.32 (dd,
J=15, 11 Hz, 1H), 1.13 (s, 3H), 1.12 (s, 3H), 0.60 (t,
J=7 Hz, 3H); 13C NMR (67.5 MHz, CDCl3) d 166.9,
165.4, 135.9, 132.5, 131.6, 131.2, 130.3 (2C), 127.9 (2C),
126.6, 55.7, 54.3, 38.2, 35.0, 34.3, 31.9, 27.6, 27.6, 9.1;
HRMS m/z 354.2098 (M+) (calcd for C20H26N4O2:
.
C21H24N4O2 1/4H2O: C, 68.36; H, 6.69; N, 15.19,
Found: C, 68.44, H, 6.67, N, 15.01.
( )-Cyclo-N,N0-dimethyl-[(Nim-methyl-5-(1,1-dimethyl-2-
propenyl))dehydrohistidinyl-L-phenylalanine] ( )-5. This
compound was prepared from 1 with 10 equivalents of
NaH and 30 equivalents of MeI at room temperature
using a procedure similar to that used for the prepara-
tion of ( )-4. For the puri®cation of ( )-5, a mixture of
hexane and ethanol (4:1) was used as an eluant in the
previously mentioned chiral column HPLC. 8% yield
.
354.2055). Anal. calcd for C20H26N4O2 1/3H2O: C,
66.64; H, 7.46; N, 15.54, Found: C, 66.75, 7.41, 15.52.
from 1; white powder; mp 95±98 ꢀC; [a]d25
(c=0.50, MeOH); UV (MeOH) nm 287 (e 11100), 203 (e
15900); IR (KBr) cm
632
(+)-Cyclo-[5-(1,1-dimethylpropyl)histidinyl-D-phenylalanine]
(+)-3. This compound was prepared from (+)-2
according to the procedure described for the prepara-
tion of ( )-3. 46% yield from (+)-2; white powder; mp
226±227 ꢀC; [a]2d5 +99 (c=0.10, MeOH); UV (MeOH)
nm 257 (e 174), 205 (e 18200); IR (KBr) cmj1 3380, 3200,
2970, 1670, 1440; 1H NMR (270 MHz, DMSO-d6) d
11.55 (br s, 1H), 8.25 (br s, 1H), 7.86 (br s, 1H), 7.41 (s,
1H), 7.29±7.14 (m, 5H), 4.24 (br s, 1H), 3.94 (br d,
J=11 Hz, 1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88 (dd,
J=14, 5 Hz, 1H), 2.82 (dd, J=15, 2 Hz, 1H), 1.45 (q,
J=7 Hz, 2H), 1.32 (dd, J=15, 11 Hz, 1H), 1.13 (s, 3H),
1.12 (s, 3H), 0.60 (t, J=7 Hz, 3H); 13C NMR
(67.5 MHz, CDCl3) d 166.9, 165.4, 135.9, 132.5, 131.6,
131.2, 130.3 (2C), 127.9 (2C), 126.6, 55.7, 54.3, 38.2,
35.0, 34.3, 31.9, 27.6, 27.6, 9.1; HRMS m/z 354.2110
(M+) (calcd for C20H26N4O2: 354.2055). Anal. calcd for
1
2900, 1690, 1640, 1380; 1H
NMR (270 MHz, CDCl3) d 8.76 (br s, 1H), 7.33±7.21
(m, 5H), 7.08 (s, 1H), 6.00 (dd, J=18, 11 Hz, 1H), 5.28
(d, J=11 Hz, 1H), 5.08 (d, J=18 Hz, 1H), 4.19 (dd,
J=10, 4 Hz, 1H), 3.86 (s, 3H), 3.38 (dd, J=14, 4 Hz,
1H), 3.13 (dd, J=14, 10 Hz, 1H), 2.92 (s, 3H), 2.55 (s,
3H), 1.60 (s, 3H), 1.57 (s, 3H); 13C NMR (67.5 MHz,
CDCl3) d 166.8, 160.2, 143.5, 137.8, 136.5, 134.8, 129.4
(2C), 128.8 (2C), 127.2, 124.6, 114.4, 105.6, 65.9, 40.6,
38.8, 36.6, 34.9, 33.1, 28.4, 28.1; HRMS m/z 392.2223
(M+) (calcd for C23H28N4O2: 392.2212). Anal. calcd for
C23H28N4O2: C, 70.38; H, 7.19; N, 14.27, Found: C,
70.26, H, 7.30, N, 14.27.
Cyclo-[glycinyl-L-phenylalanine] 6. A solution of H-Phe-
Gly-OMe, prepared from Boc-Phe-Gly-OMe with 4 N
HCl-dioxane (20 g, 59 mmol), in MeOH (100 mL), was
re¯uxed for 16 h. The white precipitate that appeared
during re¯ux was washed three times with 10 mL of
MeOH and collected to yield 7.5 g (62%) of 6 as a white
powder, mp 262±263 ꢀC (decomp.); [a]2d5 +60 (c=0.15,
.
C20H26N4O2 1/2H2O: C, 66.09; H, 7.49; N, 15.41,
Found: C, 65.80, 7.50, 15.30.
( )-Cyclo-[(Nim-methyl-5-(1,1-dimethyl-2-propenyl))de-
hydrohistidinyl-L-phenylalanine] ( )-4. 45 mg (1.88 mmol)
of sodium hydride (NaH) (60% in mineral oil) was
added in portions to a solution of 1 (200 mg, 0.57 mmol)
in DMF (15 mL) and the mixture was stirred at 30 ꢀC
for 10 min. To this mixture, 1.0 mL (17.1 mmol) of MeI
was added dropwise and stirred at 30 ꢀC for 2 h.
20 mL of saturated aqueous NH4Cl was added to the
reaction mixture and the mixture was extracted three
times with 50 mL of EtOAc. The combined organic lay-
ers were washed with saturated NaCl, dried over
Na2SO4 and concentrated in vacuo. Chromotography
of the residual white powder (150 mg) over 20 g of silica
gel with a CHCl3:MeOH eluant (50:1) yielded 130 mg
(63%) of 4: The ( )-form of 4 was isolated by HPLC
DMSO); UV (MeOH) nm 257 (e 101), 206 (e 5770); IR
1
(KBr) cm
3340, 3200, 3060, 1680, 1470, 1340; 1H
NMR (270 MHz, DMSO-d6) d 8.16 (br s, 1H), 7.90 (br s,
1H), 7.32±7.15 (m, 5H), 4.07 (br dd, J=7, 4 Hz, 1H), 3.35
(dd, J=18, 3 Hz, 1H), 3.10 (dd, J=14, 4 Hz, 1H), 2.88
(dd, J=14, 5 Hz, 1H), 2.75 (d, J=18 Hz, 1H); 13C NMR
(67.5 MHz, DMSO-d6) d 167.1, 165.7, 136.0, 130.1 (2C)
128.1 (2C), 126.8, 55.5, 43.7, 38.8; MS (ESI) m/z 205
+
.
(M+H) ; Anal. calcd for C11H12N2O2 1/5H2O: C, 63.57;
H, 6.01; N, 13.48, Found: C, 63.85, H, 5.86, N, 13.40.
Cyclo-N,N0-diacetyl-[glycinyl-L-phenylalanine] 7. The mix-
ture of 6 (0.5 g, 2.45 mmol) and fused sodium acetate

K. Kanoh et al. / Bioorg. Med. Chem. 7 (1999) 1451±1457
1457
(201 mg, 2.45 mmol) in acetic anhydride (10 mL) was
heated for 16 h at 100 ꢀC under nitrogen. After removal
of the acetic anhydride in vacuo at 45 ꢀC, the residue
was dissolved in EtOAc and the resulting organic layer
was washed with saturated NaCl, dried over Na2SO4. 7
was crystallized from EtOAc. 88% yield (0.62 g); color-
less solid; mp 84±85 ꢀC; [a]2d5 +7.8 (c=0.52, MeOH);
Cyclo-[(5-methyl)dehydrohistidinyl-L-phenylalanine] 10. This
compound was prepared from 7 with 4-methyl-5-imida-
zolecarboxaldehyde using the same procedure described
for the preparation of 9. Since HPLC puri®ed 10 con-
tained 21% of a racemized compound with d-phenyl-
alanine (58% ee), further puri®cation by HPLC with a
chiral column was performed using the same procedure
described for the puri®cation of ( )-4. 3% yield from 7;
colorless solid; mp 285±286 ꢀC (decomp); [a]d25 267
(c=0.21, DMSO); UV (MeOH) nm 319 (e 22800); IR
(KBr) cm 1 3400, 3180, 1680, 1450; 1H NMR (270 MHz,
DMSO-d6) d 11.50 (br s, 1H), 8.35 (br s, 1H), 7.74 (s,
1H), 7.24±7.14 (m, 5H), 6.20 (s, 1H), 4.48 (m, 1H), 3.33
(br s, 1H), 3.20 (dd, J=14, 4 Hz, 1H), 2.93 (dd, J=14,
5 Hz, 1H), 2.19 (s, 1H); 13C NMR (67.5 MHz, DMSO-
d6) d 164.2, 158.7, 135.6, 134.6, 132.3, 130.0 (2C), 128.0
(2C), 127.5, 126.6, 123.4, 101.7, 55.9, 38.7, 8.9; High-
resolution MS m/z 296.1261 (M+) (calcd for C16H16N4O2:
1
UV (MeOH) nm 209 (e 20400); IR (KBr) cm 1720,
1
1400, 1380, 1240; H NMR (270 MHz, CDCl3) d 7.33±
7.26 (m, 3H), 7.08±7.05 (m, 2H), 5.44 (t, J=5 Hz, 1H),
4.49 (d, J=19 Hz, 1H), 3.35 (dd, J=14, 5 Hz, 1H), 3.20
(dd, J=14, 5 Hz, 1H), 2.58 (s, 3H), 2.55 (s, 3H), 2.48 (d,
J=19 Hz, 1H); 13C NMR (67.5 MHz, CDCl3) d 171.2,
171.0, 167.9, 166.0, 134.3, 129.7 (2C), 129.1 (2C), 128.2,
59.0, 46.0, 38.7, 27.1, 26.8; MS (ESI) m/z 311
(M+Na)+; Anal. calcd for C15H16N2O4: C, 62.49; H,
5.59; N, 9.72, Found: C, 62.50, H, 5.50, N, 9.67.
.
Cyclo-[dehydrohistidinyl-L-phenylalanine] 9. To a solu-
tion of 7 (311 mg, 0.93 mmol) in dimethoxyethanol
(DME) (10 mL) was added 0.6 mL of 2 M lithium
diisopropylamide (LDA, 1.2 mmol) and the mixture was
stirred at 70 ꢀC for 10 min. To this solution was then
added a solution of 4(5)-formylimidazole (120 mg,
1.25 mmol, Maybridge Chemical Co., Cornwall, UK) in
HMPA (4 mL)-DME (6 mL) at 70 ꢀC. The solution
was allowed to warm to 30 ꢀC. After stirring for
30 min at this temperature, tri¯ic anhydride (370 mL,
2.2 mmol) and pyridine (180 mL, 2.2 mmol) were added
and the solution was allowed to warm to room tem-
perature. After an additional hour of stirring, aqueous
ammonia (3 mL) was added and stirred for 14 h at room
temperature. The reaction mixture was extracted with
CHCl3 three times, dried over Na2SO4 and concentrated
in vacuo. The resultant residue was puri®ed by HPLC
with a reverse phase column (Waters, mBondasphere
19Â150 mm, 10 mm, C-18) employing a gradient from 60
to 80% CH3CN in 0.1% TFA at a ¯ow rate of 17 mL/
min on a Waters system (600E series). However, since
puri®ed 9 contained 28% of a racemized compound
with d-phenylalanine (44% ee), further puri®cation by
HPLC with a chiral column was performed using the
same procedure described for the puri®cation of ( )-4.
8% yield from 7; white powder; mp 208±209 ꢀC; [a]d25
296.1273). Anal. calcd for C16H16N4O2 1/5H2O: C,
64.07; H, 5.51; N, 18.68. Found: C, 64.39, H, 5.65, N,
18.29.
Acknowledgements
The authors thank Dr. Hiroshi Kajiro for the helpful
suggestions and discussions. The authors also thank Mr.
Kiyoshi Morita for his support in analytical chemistry.
References
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4. Low ®eld shift of 8(N)-H (d 12.20 ppm) in H NMR was
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257 (c=0.21, DMSO); UV (MeOH) nm 307 (e 16700);
1
IR (KBr) cm
3400, 3120, 1680, 1440; 1H NMR
(270 MHz, DMSO-d6) d 12.88 (br s, 1H), 11.09 (br s,
1H), 8.44 (s, 1H), 8.13 (br s, 1H), 7.48 (s, 1H), 7.24±7.11
(m, 5H), 6.25 (s, 1H), 4.48 (m, 1H), 3.19 (dd, J=14,
4 Hz, 1H), 2.95 (dd, J=14, 5 Hz, 1H); 13C NMR
(67.5 MHz, DMSO-d6) d 164.8, 158.6, 135.8, 135.5,
132.3, 130.0 (2C), 128.0 (2C), 126.7, 125.6, 118.3, 101.8,
55.9; 38.9 (overlapping DMSO-d6); HRMS m/z
282.1084 (M+) (calcd for C15H14N4O2: 282.1117). Anal.
.
calcd for C15H14N4O2 CF3COOH: C, 51.52; H, 3.81; N,
14.14, Found: C, 51.15, H, 3.62, N, 13.84.