Synthesis and biology of new thyrotropin-releasing hormone (TRH) analogues

Article abstract of DOI:10.1016/S0968-0896(01)00265-6

We report synthesis and biological activities of several thyrotropin-releasing hormone (TRH) analogues in which the N-terminal pyroglutamic acid residue has been replaced with various carboxylic acids and the central histidine is modified with substituted-imidazole derivatives. Copyright

Full text of DOI:10.1016/S0968-0896(01)00265-6

Bioorganic & Medicinal Chemistry 10 (2002) 189–194
Synthesis and Biology of New Thyrotropin-Releasing
Hormone (TRH) Analoguesy
Rahul Jain,a,* Jatinder Singh,a Jeffery H. Perlmanb and Marvin C. Gershengornb
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67,
S.A.S. Nagar, Punjab 160 062, India
bDivision of Molecular Medicine, Department of Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA
Received 7 May 2001; accepted 18 July 2001
Abstract—We report synthesis and biological activities of several thyrotropin-releasing hormone (TRH) analogues in which the N-
terminal pyroglutamic acid residue has been replaced with various carboxylic acids and the central histidine is modified with sub-
stituted-imidazole derivatives. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
by Perlman et al.5 Removal of the carbonyl group of the
five-membered ring (replacement with proline) results in
loss of 100,000-fold in binding affinity (based on loss of
activity). An equivalent loss in affinity is observed on
replacement of Tyr-106 by Phe in helix 3 of the receptor.
It is possible, therefore, that a strong H-bond exists
between the hydroxyl of Tyr-106 and the ring carbonyl
group. Parallel manipulations show that the ring nitro-
gen of pyroglutamic acid forms a critical H-bond with
Asparagine-110 of helix 3.6 However, Tashjian et al.7
have concluded by single residue mutations at the
other amino acids that the role of Tyr106 in TRH
binding is indirect, and proposed a hydrogen bonding
interaction of Asn-289 in the third extracellular loop
(EL3) with lactam moiety of the pyroglutamyl ring of
TRH.
Thyrotropin-releasing hormone (TRH, l-pGlu-l-His-l-
Pro-NH2), a tripeptide synthesized in the hypothala-
mus,1 operates in the anterior pituitary to control levels
of TSH (thyroid-stimulating hormone) and prolactin.2
This peptide was the first hypothalamic releasing factor
characterized, establishing the fundamental proof for
the existence of a neuroendocrine regulation of pituitary
function by hypothalamic neuronal structures.1,3 The
same peptide is found in many other tissues and appears
to be involved in a wide variety of physiological activ-
ities.4 Elucidation of its mechanism of action, identifi-
cation of critical features of the molecule, separation of
its multiple activities through design of selective analo-
gues and affinity labels, and stabilization to enzymatic
degradation have been elusive goals for 30 years.
In a paper published recently,8 we find that retention of
the carbonyl, but replacement of the ring N–H group of
pGlu by a methylene group, provides an analogue (RJ-
601) whose binding affinity and signal-transducing
potency are approximately 100-fold less than those of
TRH itself. Hence, we concluded that the H-bond
interaction to Tyr-106 is considerably more important
than that to Asn-110, and the loss of NH binding is
partially compensated by the strong H-bonding ability
of the ketonic carbonyl group. To further validate this
hypothesis, and in continuation of our earlier work, this
paper describes synthesis and biological activities of
several TRH analogues, where pGlu has been replaced
with carboxylic acids (2–5) of varying hydrogen-bond
donating abilities (Fig. 1).
Results and Discussion
Isolation and cloning of the pituitary receptor, as well
as replacement by point mutation of predicted key
amino acids, have permitted Perlman et al.5 to for-
mulate the first model of the TRH peptide–receptor
complex. Since the first residue of TRH, pyroglutamic
acid, is known to be responsible for at least half of the
peptide’s binding energy, its role was first investigated
*Corresponding author. Tel.: +91-172-214682; fax: +91-172-214692;
e-mail: rahuljain@mailmetoday.com
yNIPER communication no. 106
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00265-6

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R. Jain et al. / Bioorg. Med. Chem. 10 (2002) 189–194
Development of synthetic methodologies for the ring-
substituted histidines enabled Cohen et al. to prepare
first highly selective cardiovascular analogue which was
nearly devoid of endocrine effects.9 Furthermore, 1-
CH3-Im-TRH is found to bind 7–10 times as tightly as
TRH to its receptor.10 This encouraged us to synthesize
substituted-imidazole TRH derivatives (RJ-640 and RJ-
641) where central histidine residue is replaced with its
1-methyl counterpart,11 and pGlu is replaced with its
(1S)- and (1R)-desaza counterpart. On the other hand,
easy availability to previously inaccessible 2-iodo-l-his-
tidine12 enabled us to synthesize TRH analogue RJ-504.
l-histidine methyl ester (6) and 2-iodo-l-histidine
methyl ester (7). Carboxylic acids (1–4) on reaction with
2,4,5-trichlorophenol in the presence of DCC in DMF
at ꢀ10 ꢁC gave 2,4,5-trichlorophenyl active esters (8–11)
in excellent yield (Schemes 1 and 2).
All tripeptides were assembled using the solution phase
peptide chemistry methodologies. Thus, 2,4,5-tri-
chlorophenyl esters (8–11) on reaction with histidine
methyl ester derivatives in anhydrous ethyl acetate for
24 h at ambient temperature afforded crude protected
dipeptides, which were purified by column chromato-
graphy on silica gel using EtOAc/CH3OH (94:6).
Hydrolysis of the methyl ester group in dipeptides with
0.5 N NaOH in methanol (40 min at room temperature)
gave crude dipeptides (12–20). The solution of latter
compounds on pH adjustment to 6.0 with Dowex ion
exchange resin (50ꢂ2-200, H+ form) provided free
dipeptides (12–20). Finally, TRH analogues were con-
veniently prepared by the treatment of l-ProNH2 with
the dipeptides (12–20) in the presence of DCC and
HOBt in DMF. All tripeptides were purified by flash
column chromatography on silica gel with CHCl3/
CH3OH (1:9) as eluant (Scheme 1). O-Benzyl protection
of the 3-oxoiminocyclopentyl ring in tripeptides was
removed by catalytic hydrogenation with 10% Pd on
carbon and H2 in ethanol for 2h at room temperature
and pressure to produce TRH analogues (RJ-644 and
RJ-645). However, an alternative synthetic route was
followed for the synthesis of RJ-643. Thus, l-His-l-Pro-
NH2 (21)17 on reaction with N-hydroxy-l-proline (5) in
the presence of DCC and HOBt in DMF afforded RJ-
643, which was purified by flash column chromato-
graphy on silica gel with CHCl3/CH3OH/NH4OH
(5:4:1) as eluant.
Synthesis
Cyclopentanecarboxylic acid (2) was purchased from
Aldrich Chemicals Ltd (Milwaukee, WI, USA). 3-Oxo-
cyclopentane-1-carboxylic acid (3) was prepared in four
step from 4-vinylcyclohexene following previously pub-
lished procedure.13 Resolution of racemic carboxylic acid
(3) with brucine hydrate readily afforded (1R)- and (1S)-
3-oxocyclopentane-1-carboxylic acids (3) as described
earlier (Fig. 1).14 O-Benzyl protected derivatives of oxime
(4) were synthesized by the treatment of (1R)- and (1S)-3-
oxocylopentane-1-carboxylic acids (3) with O-benzylhydr-
oxylamine and pyridine in ethanol by method previously
described.15 Both enantiomers of 3-oxocyclopentane-1-
carboxylic acid (3) and its oxime derivative (4) were
incorporated into the peptide. Whereas, N-hydroxy-l-
proline (5) was synthesized from l-proline in three steps
according to published procedure.16 Esterification of 1-
methyl-l-histidine11 and 2-iodo-l-histidine12 using
anhydrous methanolic hydrogen chloride gave methyl ester
derivatives. The latter compounds on treatment with 2M
solutions of ammonia in chloroform afforded 1-methyl-
Figure 1.
Scheme 1. (a) 2,4,5-trichlorophenol, DCC, DMF; (b) i. H-His(R1,R2)-OMe, EtOAc; ii. 0.5N NaOH, MeOH, ion-exchange (H+); (c) Pro-NH2,
DCC, HOBt, DMF.

R. Jain et al. / Bioorg. Med. Chem. 10 (2002) 189–194
191
Biological activity
data clearly indicated that the maximal extent of stimu-
lation of inositol phosphate formation and binding
affinities for peptides RJ-641 and RJ-640 were approxi-
mately 10-fold higher than those of RJ-600 and RJ-601.
These observations are consistent with earlier results
that the affinity and potency of 1-CH3-Im-TRH are
approximately 10-fold higher than those of TRH.10
Lastly; the analogue with iodine at the R2-position of
the imidazole ring (RJ-504) exhibited a markedly low-
ered affinity and potency. These findings are consistent
with previous observations that radioiodinated TRH
was a poor ligand for the receptor.
All TRH analogues were tested for their ability to acti-
vate thyrotropin-releasing hormone receptor (TRH-R
subtype 1) and were compared to TRH (Table 1). The
EC50s (activation potencies) for the second messenger
inositol phosphate formation, and the IC50s (binding
affinities) were obtained according to previously repor-
ted procedures.5,18 The data revealed that the affinities
and potencies of the analogues RJ-644 and RJ-645
[replacement of pGlu with (1S)- and (1R)-(3-oximino-
cyclopentyl)] were markedly lower than that of the
TRH. Similarly, peptide analogue RJ-643, synthesized
to check the hydrogen bonding interaction capabilities
within the receptor, where pGlu has been replaced with
N-hydroxy-l-proline exhibited markedly lowered affi-
nity and potency (Table 1). Furthermore, the cyclo-
pentyl derivative (RJ-592), where lactam group of pGlu
has been replaced with methylene groups to further
confirm the importance of the lactam group in binding
to the TRH receptor, exhibited an even greater loss in
affinity and potency (Table 1).
Conclusions
In conclusion, we have demonstrated that substitution
of the pGlu with other hydrogen-bond donating moi-
eties in the peptide does not stop the peptides from
binding and activating TRH-R. We believe that our
continuing efforts to accumulate data on the size, geo-
metry and flexibility of the pyroglutamate-binding
pocket will permit us to design irreversible affinity labels
to form covalent bonds to Tyr-106 and Asn-110. At the
same time, we are trying to rationalize the requirements
for the H-bonding to histidine and its analogues. Ulti-
mately, we hope to settle the controversy between the
divergent formulations of TRH-receptor binding, as
well as to design selective analogues, which may be
more useful clinically than TRH itself.
As reported earlier,8 tripeptide RJ-601 exhibited binding
affinity and signal-transducing potency, which are
approximately 100-fold less than those of TRH itself.
The incorporation of methyl group at the N-1 position
of imidazole ring (RJ-641, RJ-640, R1=CH3) caused a
10-fold increase in binding affinity and potency that was
independent of the changes at the pGlu residue. The
Experimental
Melting points were determined with a capillary appa-
ratus and are uncorrected. 1H NMR spectra were
obtained on a Bruker Avance DPX (300 MHz) spectro-
meter using TMS as internal reference. Mass spectra
were recorded on Shimadsu GCMS-QP 5000 spectro-
Scheme 2. (a) N-hydroxy-l-Pro-OH, DCC, HOBt, DMF.
Table 1. Binding affinities IC50 (nM) and activation potencies EC50 (nM) of TRH-R receptors by TRH analogues
a
b
Entry
R
R1
H
R2
I
IC50
EC50
950
RJ-504
(2S)-PyroGlu
14,000
>106
RJ-592Cyclopentyl
RJ-600
RJ-601
RJ-640
RJ-641
RJ-643
RJ-644
RJ-645
TRH
H
H
>106
980ꢃ440
100ꢃ40
(1S)-(3-Oxocyclopentyl)
(1R)-(3-Oxocyclopentyl)
(1S)-(3-Oxocyclopentyl)
(1R)-(3-Oxocyclopentyl)
(2S)-1-Hydroxyproline
(1S)-(3-Oximinocyclopentyl)
(1R)-(3-Oximinocyclopentyl)
(2S)-PyroGlu
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
13,500ꢃ5000
1950ꢃ300
3100ꢃ460
170ꢃ30
250ꢃ300
16ꢃ1
>300,000
160,000
70,000ꢃ35,000
50,000
46,000ꢃ9500
6300ꢃ4400
16ꢃ3
0.50ꢃ0.10
All data are means ꢃSD of duplicate determinations in two or three experiments.aFor binding, cells were incubated with 1 nM [3H]1-CH3-Im-TRH
in the absence or presence of various doses of unlabeled TRH analogues for 1 h at 37 ꢁC.
bFor activation, cells prelabeled with myo-[3H]inositol were incubated with various doses of TRH analogues for 1 h at 37 ꢁC, and inositol phosphate
formation was measured. Maximal extents of stimulation were similar for all analogues. Experiments were performed with intact AtT-20 mouse
pituitary tumor cells stably expressing TRH receptors.

192
R. Jain et al. / Bioorg. Med. Chem. 10 (2002) 189–194
meter, whereas ESI (Electron Spray Ionization) spectra
were recorded on a LCMS Finnigan Mat LCQ spectro-
meter. Optical rotations were recorded on a Perkin–
Elmer 241 MC Polarimeter. Elemental analyses were
performed by Atlantic Microlab Inc., Norcross, GA,
USA. All chromatographic purification was performed
with silica gel 60 (230–400 mesh), whereas all TLC
(silica gel) development was performed on silica gel
coated (Merck Kiesel 60 F254, 0.2 mm thickness)
sheets. Following abbreviations are used: pGlu, pyr-
oglutamic acid; DCC, 1,3-dicyclohexylcarbodiimide;
DCU, 1,3-dicyclohexylurea; DMF, N,N-dimethylfor-
mamide; Boc, tert-butyloxycarbonyl; HOBt, 1-hydro-
xybenzotriazole; EtOAc, ethyl acetate; DIEA, N,N-
diisopropylethylamine; Im, imidazole; MeOH, metha-
nol; OTCP, 2,4,5-trichlorophenyl ester.
8 [R=(2S)-pyroGlu]. Yield: 90%; mp: 71–72 ꢁC; 1H
NMR (CDCl3) d 2.5 (m, 4H, 2ꢂCH2), 4.55 (m, 1H,
CH), 7.30 (s, 1H, Ar–H), 7.56 (s, 1H, Ar–H); CIMS
(NH3) m/z 310 (M+1); analysis for C11H8Cl3NO3
(308.5); calcd C, 42.82; H, 2.61; N, 4.54; found C, 42.88;
H, 2.50; N, 4.33.
9 [R=cyclopentyl]. Yield: 88%; mp: 66 ꢁC; H NMR
1
(CDCl3) 1.94 (m, 4H, 2ꢂCH2), 2.10 (m, 4H, 2ꢂCH2),
2.51 (m, 1H, CH), 7.24 (s, 1H, Ar–H), 7.27 (s, 1H, Ar–
H), CIMS (NH3) m/z 295 (M+1); analysis for
C12H11Cl3O2 (293.6), calcd C, 49.09; H, 3.78; found C,
48.90; H, 4.00.
10a [R=(1S)-(3-oxocyclopentyl)]. Yield: 81%; mp 55–
ꢁ
1
56 C; H NMR (CDCl3) d 2.3–2.73 (m, 6H, 3ꢂCH2),
4.44 (m, 1H, CH), 7.29 (s, 1H, Ar–H), 7.51 (s, 1H, Ar–
H); CIMS (NH3) m/z 309 (M+1); analysis for
C12H9Cl3O3 (307.6), calcd C, 46.86; H, 2.95; found C,
46.99; H, 3.23; [a]2D5 ꢀ19.0ꢁ (c 1.2, CHCl3).
General procedure for the synthesis of substituted L-
histidine methyl esters (6–7)
To a solution of ring-substituted histidine derivative
(100 mmol) in methanol (100 mL) cooled to 0 ꢁC was
bubbled HCl gas for 45 min. The solution was allowed
to stand overnight at room temperature. The solvent
was evaporated under reduced pressure to afford the
salts of the substituted l-histidine methyl ester as semi-
10b [R=(1R)-(3-oxocyclopentyl)]. Yield: 63%; mp 56–
57 ꢁC; analysis for C12H9Cl3O3 (307.56), calcd C, 46.86;
H, 2.95; found C, 47.13; H, 2.88; [a]2D5 +18.8ꢁ (c 1.95,
CHCl3).
solid. A 2M solution of ammonia in CHCl (10 mL)
11a [R=O-benzyl oxime of (1S)-3-oxocyclopentyl].
Yield: 73%; oil; 1H NMR (CDCl3) d 2.12 (m, 2H,
CH2), 2.40 (m, 4H, 2ꢂCH2), 3.99 (m, 1H, CH), 5.20 (s,
1H, CH2), 7.30 (m, 3H, Ar–H), 7.50 (m, 2H, Ar–H);
CIMS (NH3) m/z 414 (M+1); analysis for
C19H16Cl3NO3 (412.7), calcd C, 55.30; H, 3.91; N, 3.39;
found C, 55.63; H, 3.77; N, 3.55.
3
was then added to the dihydrochloride salt, and the
reaction mixture was allowed to stand for 30 min at
room temperature. Separated solid filtered and com-
plete evaporation of the filtrate under reduced pressure
gave the substituted l-histidine methyl esters.
1-Methyl-L-histidine methyl ester (6). Yield: 95%; oil;
1H NMR (CDCl3) d 2.60–2.81 (m, 2H, CH2), 3.53 (s,
3H, N–CH3), 3.66 (s, 3H, OCH3), 4.10 (m, 1H, CH),
7.16 (s, 1H, 5-H), 7.37 (s, 1H, 2-H); CIMS (NH3) m/z
184 (M+1); analysis for C8H13N3O2 (183.2), calcd C,
52.45; H, 7.15; N, 22.94; found C, 52.44; H, 7.09; N,
23.07.
11b [R=O-benzyl oxime of (1R)-3-oxocyclopentyl].
Yield: 37%; oil; analysis for C19H16Cl3NO3 (412.7),
calcd C, 55.30; H, 3.91; N, 3.39; found C, 55.29; H, 3.98;
N, 3.44.
General method for the synthesis of dipeptides (12–20)
1
2-Iodo-L-histidine methyl ester (7). Yield 90%; oil; H
A solution of 2,4,5-trichlorophenyl ester (8–11, 1 mmol)
in EtOAc (10 mL) was added drop wise to substituted
l-histidine methyl ester (6–7) or l-histidine methyl ester
(1 mmol) in EtOAc (50 mL) during 15 min, and reaction
mixture was stirred at ambient temperature for 24 h.
Complete removal of the solvent in vacuo gave crude
product. Flash column chromatography on silica gel
using a solvent system of EtOAc/CH3OH (94:6) pro-
vided dipeptide methyl ester. Pure dipeptide methyl
ester (1 mmol) was then dissolved in a mixture of MeOH
(50 mL) and 0.5 N NaOH (40 mL). The solution was
stirred at ambient temperature for 30 min, and water
(35 mL) was added to the solution. The pH of the solu-
tion was then adjusted to 6.0 with Dowex (50ꢂ2–200,
H+ form) ion exchanger. The resin was removed by fil-
tration, and the filtrate was evaporated under vacuum
to afford crude product. Crystallized from methanol/
ethanol/ether (1:1:1).
NMR (CDCl3) d 2.74–2.90 (m, 2H, CH2), 3.66 (s, 3H,
OCH3), 4.13 (m, 1H, CH), 7.55 (s, 1H, 5-H); CIMS
(NH3) m/z 296 (M+1); analysis for C7H10IN3O2
(295.1), calcd C, 28.49; H, 3.42; N, 14.24; found C,
28.67; H, 3.11; N, 14.33.
General procedure for the synthesis of 2,4,5-
trichlorophenyl esters (8–11)
A mixture of DCC (1 mmol) and 2,4,5-trichlorophenol
(1 mmol) in DMF (50 mL) was cooled to ꢀ10 ꢁC in an
ice-salt bath and treated dropwise with a solution of
carboxylic acid (1–4, 1 mmol) in DMF (10 mL). The
reaction mixture was allowed to warm to room tem-
perature and was stirred overnight. Removal of the sol-
vent under reduced pressure afforded a white solid. The
solid was triturated with ethyl acetate (50 mL), and the
dicyclohexylurea was removed by filtration. Filtrate was
evaporated under reduced pressure to afford oil. Flash
chromatography on silica gel using EtOAc/hexanes (1:9)
gave active esters (8–11) in excellent yield.
12 [R=(2S)-pyroGlu, R1=R2=H]. Yield: 72%; mp
218 ꢁC (dec); 1H NMR (D2O) d 1.83 (m, 2H, CH2), 2.31
(m, 2H, CH2), 3.1 (m, 2H, CH2), 4.18 (m, 1H, CH), 4.41

R. Jain et al. / Bioorg. Med. Chem. 10 (2002) 189–194
193
(m, 1H, CH), 7.08 (s, 1H, 5-H), 8.30 (s, 1H, 2-H);
ESIMS m/z 267 (M+1); analysis for C11H14N4O4
(266.3), calcd C, 42.62; H, 5.30; N, 21.04; found C,
42.44; H, 5.72; N, 20.88; [a]2D5 +4.2ꢁ (c 1, H2O).
(M+1); analysis for C19H22N4O4 (370.4); calcd C,
61.61; H, 5.99; N, 15.13; found C, 61.66; H, 6.22; N,
14.97.
20 [ꢁR=pyroGlu, R1=H, R2=I]. Yield: 99%; mp 225–
1
13 [R=cyclopentyl, R1=R2=H]. Yield: 80%; mp 176–
227 C; H NMR (D2O) d 1.74 (m, 2H, CH2), 2.29 (m,
178 ꢁC (dec); H NMR (D2O) d 1.76 (m, 4H, 2ꢂCH2),
4H, 2ꢂCH2), 2.82 (m, 1H, CH), 3.05 (m, 1H, CH), 4.13
(m, 1H, CH), 4.36 (m, 1H, CH), 6.82(s, 1H, 5-H);
ESIMS m/z 393 (M+1); analysis for C11H13IN4O4
(392.2); calcd C, 33.69; H, 3.34; N, 14.29; found C,
33.77; H, 3.36; N, 14.34; [a]2D5 +7.8ꢁ (c 0.4, MeOH).
1
2.31 (m, 4H, 2ꢂCH2), 3.01 (m, 2H, CH2), 4.10 (m, 2H,
2ꢂCH), 7.03 (s, 1H, 5-H), 8.26 (s, 1H, 2-H); ESIMS m/z
252 (M+1); analysis for C12H17N3O3 (251.3); calcd C,
57.36; H, 6.82; N, 16.72; found C, 57.49; H, 6.84; N,
16.98.
General procedure for the synthesis of TRH analogues
14 [R=(1S)-(3-oxocyclopentyl), R1=R2=H]. Yield:
77%; mp: 185–186 ꢁC (dec); H NMR (D2O) d 1.75–
Dipeptide (12–20, 1 mmol) was dissolved in DMF
(25 mL), and HOBt (1 mmol) was added to the solution
followed by addition of DCC (1 mmol) under cooling at
0 ꢁC. Stirring of the reaction mixture continued for
another 30 min at 0 ꢁC. l-Prolineamide (1.2mmol) was
then added to this solution in one portion. The reaction
mixture was allowed to warm to room temperature and
was stirred for 48 h. Separated DCU was removed by
filtration, and filtrate evaporated in vacuo to afford
crude product. Flash column chromatography on silica
gel using CHCl3/CH3OH (1:9) as the solvent system
gave pure TRH analogue as white solid. The homo-
geneity of peptide analogues on TLC was assessed using
CHCl3/CH3OH/NH4OH (4:5:1) as mobile phase.
1
2.61 (m, 6H, 3ꢂCH2), 3.11 (m, 2H, CH2), 4.01 (m, 2H,
2ꢂCH), 7.13 (s, 1H, 5-H), 8.36 (s, 1H, 5-H); ESIMS m/z
266 (M+1); analysis for C12H15N3O4 (265.3); calcd C,
54.33; H, 5.70; N, 15.84; found C, 54.21; H, 5.89; N,
15.88.
15 [R=(1R)-(3-oxocyclopentyl), R1=R2=H]. Yield:
ꢁ
1
65%; mp: 192–193 C (dec); H NMR (D2O) d 1.80–
2.61 (m, 6H, 3ꢂCH2), 3.04 (m, 2H, CH2), 4.13 (m, 2H,
2ꢂCH), 7.03 (s, 1H, 5-H), 8.29 (s, 1H, 5-H); ESIMS m/z
266 (M+1); analysis for C12H15N3O4 (265.3); calcd C,
54.33; H, 5.70; N, 15.84; found C, 54.49; H, 5.73; N,
15.79.
16 [R=(1S)-(3-oxocyclopentyl), R1=CH3, R2=H].
Spectral data for RJ-504. Yield: 40%; 1H NMR
(CD3OD) d 1.75 (m, 4H, 2ꢂCH2), 2.50 (m, 6H,
3ꢂCH2), 2.78 (m, 2H, CH), 3.10 (m, 2H, CH), 4.01 (m,
1H, CH), 6.88 (s, 1H, 5-H), ESIMS m/z 489 (M+1);
analysis for C16H21IN6O4 (488.3); calcd C, 39.36; H,
4.33; N, 17.21; found C, 39.66; H, 4.47; N, 17.04; [a]D25
ꢀ30.4ꢁ (c 1.2, MeOH); Rf 0.50 (one spot).
Yield: 86%; mp: 201–202 ꢁC (dec); H NMR (D2O) d
1
1.70 (m, 2H, CH2), 2.28 (m, 4H, 2ꢂCH2), 2.89 (m, 1H,
CH), 3.09 (m, 1H, CH), 3.71 (s, 3H, N–CH3), 4.40 (m,
1H, CH), 7.08 (s, 1H, 5-H), 8.27 (s, 1H, 2-H); ESIMS
m/z 280 (M+1); analysis for C13H17N3O4 (279.3), calcd
C, 55.91; H, 6.14; N, 15.05; found C, 56.21; H, 6.03; N,
15.21.
Spectra data for RJ-592. Yield: 44%; 1H NMR
(CD3OD) d 1.18 (m, 8H, 3ꢂCH2), 2.37 (m, 8H,
4ꢂCH2), 2.56 (m, 1H, CH), 2.80 (m, 1H, CH), 2.90 (m,
1H, CH), 3.50 (m, 1H, CH), 4.40 (m, 1H, CH), 6.76 (s,
1H, 5-H), 7.53 (s, 1H, 2-H); ESIMS m/z 348 (M+1);
analysis for C17H25N5O3 (347.4); calcd C, 58.77; H,
7.25; N, 20.16; found C, 58.59; H, 6.89; N, 20.43; [a]D25
ꢀ25.4ꢁ (c 1.2, MeOH); Rf 0.63 (one spot).
17 [R=(1R)-(3-oxocyclopentyl), R1=CH3, R2=H].
Yield: 79%; mp: 196–198 ꢁC (dec); H NMR (D2O) d
1
1.73 (m, 2H, CH2), 2.33 (m, 4H, 2ꢂCH2), 2.92 (m, 1H,
CH), 3.11 (m, 1H, CH), 3.76 (s, 3H, N–CH3), 4.38 (m,
1H, CH), 7.03 (s, 1H, 5-H), 8.20 (s, 1H, 2-H); ESIMS
m/z 280 (M+1); analysis for C13H17N3O4 (279.3), calcd
C, 55.91, H, 6.14, N, 15.05, found C, 56.07; H, 6.34; N,
15.09.
Spectral data for RJ-600. Yield: 50%; 1H NMR
(CD3OD) d 1.86 (m, 6H, 3ꢂCH2), 2.17 (m, 6H,
3ꢂCH2), 2.94 (m, 2H, CH2), 3.27 (m, 1H, CH), 3.67 (m,
1H, CH), 4.30 (m, 1H, CH), 6.86 (s, 1H, 5-H), 7.55 (s,
1H, 2-H); ESIMS m/z 362(M+1); analysis for
C17H23N5O4 (361.4); calcd, C, 56.50; H, 6.41; N, 19.38;
found, C, 56.34; H, 6.45; N, 19.65; [a]2D5 ꢀ46.1ꢁ (c 1.15,
MeOH); Rf 0.55 (one spot).
18 [R=O-benzyl oxime of (1S)-(3-oxocyclopentyl),
R1=R2=H]. Yield: 88%; mp: 212–214 ꢁC; 1H NMR
(D2O) d 1.69 (m, 2H, CH2), 2.31 (m, 4H, 2ꢂCH2), 2.93
(m, 1H, CH), 3.08 (m, 1H, CH), 4.30 (m, 1H, CH), 5.17
(s, 2H, CH2), 7.05 (s, 1H, 5-H), 7.27 (m, 3H, Ar–H),
7.59 (m, 2H, Ar–H), 8.30 (s, 1H, 2-H); ESIMS m/z 371
(M+1); analysis for C19H22N4O4 (370.4); calcd C,
61.61; H, 5.99; N, 15.13; found C, 61.33; H, 5.76; N,
15.14.
Spectral data for RJ-601. Yield: 28%; 1H NMR
(CD3OD) d 1.85 (m, 6H, 3ꢂCH2), 2.15 (m, 6H,
3ꢂCH2), 2.94 (m, 2H, CH2), 3.26 (m, 1H, CH), 3.65 (m,
1H, CH), 4.31 (m, 1H, CH), 6.85 (s, 1H, 5-H), 7.51 (s,
1H, 2-H); ESIMS m/z 362(M+1); analysis for
C17H23N5O4 (361.4); calcd, C, 56.50; H, 6.41; N, 19.38;
found, C, 56.76; H, 6.76; N, 19.34; [a]2D5 ꢀ26.5ꢁ (c 1.14,
MeOH); Rf 0.55 (one spot).
19 [R=O-benzyl oxime of (1R)-(3-oxocyclopentyl),
R1=R2=H]. Yield: 65%; mp 196–198 ꢁC; 1H NMR
(D2O) d 1.72(m, 2H, CH 2), 2.40 (m, 4H, 2ꢂCH2), 2.98
(m, 1H, CH), 3.12 (m, 1H, CH), 4.28 (m, 1H, CH), 5.20
(s, 2H, CH2), 7.10 (s, 1H, 5-H), 7.30 (m, 3H, Ar–H),
7.60 (m, 2H, Ar–H), 8.33 (s, 1H, 2-H); ESIMS m/z 371

194
R. Jain et al. / Bioorg. Med. Chem. 10 (2002) 189–194
Spectral data for RJ-640. Yield: 23%; 1H NMR
(CD3OD) d 1.88 (m, 6H, 3ꢂCH2), 2.17 (m, 6H,
3ꢂCH2), 2.91 (m, 2H, CH2), 3.30 (m, 1H, CH), 3.63 (m,
1H, CH), 3.77 (s, 3H, N–CH3), 4.28 (m, 1H, CH), 6.90
(s, 1H, 5-H), 7.59 (s, 1H, 2-H); ESIMS m/z 376 (M+1);
analysis for C18H25N5O4 (375.4); calcd C, 57.59; H,
6.71; N, 18.65; found C, 57.89; H, 6.93; N, 18.54; [a]D25
ꢀ52.3ꢁ (c 1.2, MeOH); Rf 0.60 (one spot).
Spectral data for RJ-641. Yield: 36%; 1H NMR
(CD3OD) d 1.89 (m, 6H, 3ꢂCH2), 2.14 (m, 6H,
3ꢂCH2), 2.99 (m, 2H, CH2), 3.36 (m, 1H, CH), 3.61 (m,
1H, CH), 3.80 (s, 3H, N–CH3), 4.21 (m, 1H, CH), 6.91
(s, 1H, 5-H), 7.58 (s, 1H, 2-H); ESIMS m/z 376 (M+1);
analysis for C18H25N5O4 (375.4); calcd C, 57.59; H,
6.71; N, 18.65; found C, 57.47; H, 6.68; N, 18.58; [a]D25
ꢀ33.5ꢁ (c 1.2, MeOH); Rf 0.60 (one spot).
Spectral data for RJ-644. Yield: 35%; 1H NMR
(CD3OD) d 1.87 (m, 6H, 3ꢂCH2), 2.11–2.98 (m, 8H,
4ꢂCH2), 3.38 (m, 1H, CH), 3.66 (m, 1H, CH), 4.33 (m,
1H, CH), 6.87 (s, 1H, 5-H), 7.56 (m, 1H, 2-H); ESIMS
m/z 377 (M+1); analysis for C17H24N6O4 (376.4); calcd,
C, 54.24; H, 6.43; N, 22.33; found, C, 54.03; H, 6.12; N,
22.57; [a]2D5 ꢀ57.8ꢁ (c 1.2, MeOH); Rf 0.37 (one spot).
Spectral data for RJ-645. Yield: 43%; 1H NMR
(CD3OD) d 1.79 (m, 6H, 3ꢂCH2), 2.10–2.96 (m, 8H,
4ꢂCH2), 3.35 (m, 1H, CH), 3.69 (m, 1H, CH), 4.29 (m,
1H, CH), 6.86 (s, 1H, 5-H), 7.57 (m, 1H, 2-H); ESIMS
m/z 377 (M+1); analysis for C17H24N6O4 (376.4); calcd
C, 54.24; H, 6.43; N, 22.33; found C, 53.97; H, 6.57; N,
22.64; [a]2D5 ꢀ50.3ꢁ (c 1.2, MeOH); Rf 0.55 (one spot).
References and Notes
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Procedure for the synthesis of tripeptide RJ-643. N-
Hydroxy-l-proline (1 mmol) was dissolved in DMF
(20 mL), and HOBt (1.2 mmol) was added to the solu-
tion followed by addition of DCC (1.2mmol) under
cooling at 0 ꢁC. The mixture was stirred at the same
temperature for 6 h. A solution of the l-histidine-l-
17
proline-NH2 (1.2mmol) in DMF (15 mL) was then
added at once, and the reaction mixture was stirred at
5 ꢁC overnight. After the resulting precipitate was fil-
tered off, the filtrate was evaporated to dryness under
reduced pressure. The residue was subjected to column
chromatography with chloroform/methanol/ammonia
(50:40:10) as eluant. The required fractions were pooled
and concentrated under reduced pressure to afford RJ-
1
643 as powder. Yield: 21%; H NMR (CD3OD) d 1.88
(m, 6H, 3ꢂCH2), 2.67 (m, 6H, 3ꢂCH2), 2.90 (m, 2H,
CH2), 3.29 (m, 1H, CH), 3.67 (m, 1H, CH), 4.34 (m, 1H,
CH), 6.84 (s, 1H, 5-H), 7.50 (s, 1H, 2-H); ESIMS m/z
365 (M+1); analysis for C16H24N6O4 (364.4); calcd, C,
52.74; H, 6.64; N, 23.06; found, C, 54.03; H, 6.56; N,
23.02; [a]2D5 ꢀ63.3ꢁ (c 1.2, MeOH); Rf 0.30 (one spot).