Effects of heterocyclic aromatic substituents on binding affinities at two distinct sites of somatostatin receptors. Correlation with the electrostatic potential of the substituents

Article abstract of DOI:10.1021/jm0205088

In our continuing program exploring glucose-based peptidomimetics of somatostatin (SRIF-14), we sought to improve the water solubility of our glycosides. This led to insights into the nature of the ligand binding sites at the SRIF receptor. Replacement of the C4 benzyl substituent in glucoside (+)-2 with pyridinylmethyl or pyrazin-2-ylmethyl congeners increased water solubility and enhanced affinity for the human SRIF subtype receptor 4 (sst4). We attribute this effect to hydrogen bond formation. The pyridin-3-ylmethyl substituent at C4, when combined with the imidazol-4-ylmethyl group at C2, generated (-)-19, which has the highest affinity of a glucose-based peptidomimetic at a human SRIF receptor to date (K_i 53 ± 23 nM, n = 6 at sst4). The C4 heterocyclic congeners of glucosides bearing a 1-methoxy substituent rather than an indole side chain at the anomeric carbon, such as (+)-16, also provided information about the Trp⁸ binding pocket. We correlated the SARs at both the C4 and the Trp⁸ binding pockets with calculations of the electrostatic potentials of the diverse C4 aromatic substituents using Spartan 3-21G(*) MO analysis. These calculations provide an approximate analysis of a molecule's ability to interact within a receptor binding site. Our binding studies show that benzene and indole rings, but not pyridinylmethyl nor pyrazin-2-ylmethyl rings, can bind the hydrophobic Trp⁸ binding pocket of sst4. The Spartan 3-21G(*) MO analysis reveals significant negative electrostatic potential in the region of the π-clouds for the benzene and indole rings but not for the pyridinylmethyl or pyrazin-2-ylmethyl congeners. Our data further demonstrate that the replacement of benzene or indole side chains by heterocyclic aromatic rings typified by pyridine and pyrazine not only enhances water solubility and hydrogen bonding capacity as expected, but can also profoundly diminish the ability of the π-cloud of the aromatic substituent to interact with side chains of an aromatic binding pocket such as that for Trp⁸ of SRIF-14. Conversely, these calculations accommodate the experimental findings that pyrazin-2-ylmethyl and pyridinylmethyl substituents at C4- of C1-indole-substituted glycosides afford higher affinities at sst4 than the C4-benzyl group of (+)-2. This result is consistent with the high electron density in the plane of the heterocycle depicted in Figure 6 which can accept hydrogen bonds from the C4 binding pocket of the receptor. Unexpectedly, we found that the 2-fluoropyridin-5-ylmethyl analogue (+)-14 more closely resembles the binding affinity of (+)-8 than that of (+)-2, thus suggesting that (+)-14 represents a rare example of a carbon linked fluorine atom acting as a hydrogen bond acceptor. We attribute this result to the ability of the proton to bind the nitrogen and fluorine atoms simultaneously in a bifurcated arrangement. At the NK1 receptor of substance P (SP), the free hydroxyl at C4 optimizes affinity.

Full text of DOI:10.1021/jm0205088

1858
J . Med. Chem. 2003, 46, 1858-1869
Effects of Heter ocyclic Ar om a tic Su bstitu en ts on Bin d in g Affin ities a t Tw o
Distin ct Sites of Som a tosta tin Recep tor s. Cor r ela tion w ith th e Electr osta tic
P oten tia l of th e Su bstitu en ts
Vidya Prasad,†,‡ Elizabeth T. Birzin,| Cheryl T. McVaugh,† Rachel D. van Rijn,†,§ Susan P. Rohrer,*,|
Gary Chicchi,| Dennis J . Underwood,*, Edward R. Thornton,*,† Amos B. Smith, III,*,† and Ralph Hirschmann*,†
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, Merck Research Laboratories,
Rahway, New J ersey 07065, and DuPont Pharmaceutical Company, Wilmington, Delaware 19880
Received November 8, 2002
In our continuing program exploring glucose-based peptidomimetics of somatostatin (SRIF-
14), we sought to improve the water solubility of our glycosides. This led to insights into the
nature of the ligand binding sites at the SRIF receptor. Replacement of the C4 benzyl
substituent in glucoside (+)-2 with pyridinylmethyl or pyrazin-2-ylmethyl congeners increased
water solubility and enhanced affinity for the human SRIF subtype receptor 4 (sst4). We
attribute this effect to hydrogen bond formation. The pyridin-3-ylmethyl substituent at C4,
when combined with the imidazol-4-ylmethyl group at C2, generated (-)-19, which has the
highest affinity of a glucose-based peptidomimetic at a human SRIF receptor to date (Ki 53 (
23 nM, n ) 6 at sst4). The C4 heterocyclic congeners of glucosides bearing a 1-methoxy
substituent rather than an indole side chain at the anomeric carbon, such as (+)-16, also
provided information about the Trp8 binding pocket. We correlated the SARs at both the C4
and the Trp8 binding pockets with calculations of the electrostatic potentials of the diverse C4
aromatic substituents using Spartan 3-21G(*) MO analysis. These calculations provide an
approximate analysis of a molecule’s ability to interact within a receptor binding site. Our
binding studies show that benzene and indole rings, but not pyridinylmethyl nor pyrazin-2-
ylmethyl rings, can bind the hydrophobic Trp8 binding pocket of sst4. The Spartan 3-21G(*)
MO analysis reveals significant negative electrostatic potential in the region of the π-clouds
for the benzene and indole rings but not for the pyridinylmethyl or pyrazin-2-ylmethyl
congeners. Our data further demonstrate that the replacement of benzene or indole side chains
by heterocyclic aromatic rings typified by pyridine and pyrazine not only enhances water
solubility and hydrogen bonding capacity as expected, but can also profoundly diminish the
ability of the π-cloud of the aromatic substituent to interact with side chains of an aromatic
binding pocket such as that for Trp8 of SRIF-14. Conversely, these calculations accommodate
the experimental findings that pyrazin-2-ylmethyl and pyridinylmethyl substituents at C4- of
C1-indole-substituted glycosides afford higher affinities at sst4 than the C4-benzyl group of
(+)-2. This result is consistent with the high electron density in the plane of the heterocycle
depicted in Figure 6 which can accept hydrogen bonds from the C4 binding pocket of the
receptor. Unexpectedly, we found that the 2-fluoropyridin-5-ylmethyl analogue (+)-14 more
closely resembles the binding affinity of (+)-8 than that of (+)-2, thus suggesting that (+)-14
represents a rare example of a carbon linked fluorine atom acting as a hydrogen bond acceptor.
We attribute this result to the ability of the proton to bind the nitrogen and fluorine atoms
simultaneously in a bifurcated arrangement. At the NK1 receptor of substance P (SP), the
free hydroxyl at C4 optimizes affinity.
In tr od u ction
for the binding of peptides to all somatostatin (SRIF)
receptor subtypes is the presence of adjacent indole and
alkylamine side chains in the i+1 and i+2 positions of
â-turns as in the tetradecapeptide SRIF-14, its crystal-
line congener octreotide and in 1 (Figure 1). The
glycosides (+)-2 and (-)-3 designed to mimic this
structural feature did indeed bind SRIF receptors on
AtT-20 cells, albeit weakly (Ki ca. 15 µM) (see Figure 1
for structures); a glycoside mimic (-)-3 was found to be
an SRIF agonist.1a,b Later (+)-2 and (-)-3 were found
to bind also human SRIF receptor subtypes, displaying
the highest affinity at subtype 4 (sst4).2 The SAR of
(-)-3 strikingly resembles that of the cyclic hexapeptide
1.1a For example, replacement of the axial Phe7 of SRIF-
In 1987 we initiated studies to test the proposition
that nonpeptidal mimetics of â-turns can be generated
via the attachment of appropriate side chains to monosac-
charide scaffolds typified first by glucose1 and later by
mannose.2 The minimal pharmacophoric requirement
* Corresponding author: R. Hirschmann, Department of Chemistry,
University of Pennsylvania, 231 South 34th St., Philadelphia, PA
19104-6323. Phone: (215) 898-7398. Fax: (215) 573-9711. E-mail:
rfh@sas.upenn.edu.
† University of Pennsylvania.
‡ Present address: Gilead Sciences, 333 Lakeside Dr., Foster City,
CA 94404.
| Merck Research Laboratories.
§ Present address: N.V. Organon, P.O. Box 20, The Netherlands.
Bristol-Myers Squibb Co. (formerly DuPont Pharmaceutical Co.).
10.1021/jm0205088 CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/15/2003

Binding Affinities at Somatostatin Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1859
and (-)-3 at C1 and C6) is supported by the parallelism
in SARs between the peptide 1 and the glycoside (-)-
3.1a Thus, the Trp8 and Lys9 side chains of SRIF-14, of
1, like their mimics in (+)-2 and (-)-3, anchor these
ligands to SRIF receptors. On the other hand, the
receptor subtype profileswhich depends on other side
chains of the ligands, perhaps even their scaffolds and
on other regions of the binding domain of the receptorss
differs between SRIF-14, octreotide, and the c-hexapep-
tides, as well as the glycosides.4,5
Unexpectedly, (+)-2 was also found to bind the NK1
receptor of substance P (SP) as an antagonist, with a
Ki of 150 nM.1c Compound (-)-6, lacking a C4 benzyl
substituent, has a Ki of 27 nM2 at the NK1 receptor,
but does not bind sst4, and the corresponding free amine
has a comparable affinity (Ki 66 nM) at the NK1
receptor. Conversely, (+)-4 binds sst4,2 but not the NK1
receptor.6 Taken together these results show that we
have synthesized analogues of (+)-2 which are selective
for either SRIF or NK1 receptors.
Str u ctu r e-Activity Rela tion sh ip s (SARs) of C4
Heter ocyclic Su bstitu en ts. (a ) At th e NK1 Recep -
tor . Herein we report the syntheses of the three
isomeric C4 pyridinylmethyl congeners (+)-7, (+)-8, and
(+)-9 as well as other heterocyclic substituents that
replace the 4-benzyl group of (+)-2 (see Table 1 for
structures). Binding affinities at the NK1 receptor were
generally enhanced relative to (+)-2, but not signifi-
cantly.7,8,9 Representative results are given in the Sup-
porting Information. The high affinity of (-)-6 (Figure
1) at the NK1 receptor implies that aromatic C4
substituents, even water soluble ones, such as the C4
pyridinylmethyl isomers, generate a less favorable
interaction with this receptor. It is also possible that
the C4 hydroxyl generates a favorable interaction with
the NK1 receptor. Affinities were generally greater for
the N-acetyl derivatives of the primary amines (not
shown) than for the free amines at the NK1 receptor;
the differences between diverse C4 heterocyclic substit-
uents were small.
(b) Hyd r ogen -Bon d Med ia ted Affin ity En h a n ce-
m en t a t sst4 via C4 P yr id in ylm eth yl or P yr a zin -
2-ylm eth yl Su bstitu tion . The SARs of the above-
mentioned heterocyclic congeners at sst4 (see Table 1)
differ markedly from those at the NK1 receptor. The
pyridin-3-ylmethyl derivative (+)-8 and especially the
pyrazin-2-ylmethyl congener (+)-10 displayed signifi-
cantly enhanced affinities relative to (+)-2 at sst4. The
isomeric pyridinylmethyl congeners (+)-7 and (+)-9 also
produced affinity enhancement, but to a lesser degree.
F igu r e 1. Potent SRIF agonist 1 and previously reported
monosaccharide-based mimetics (+)-2 and (-)-3. Congener
(+)-4 binds sst4, but not the NK1 receptor. Conversely, the
4-unsubstituted glucoside (-)-6 is a potent substance P recep-
tor ligand, but does not bind sst4. Analogues (+)-2 and (-)-3
bind both receptors. The L-mannose analogue (+)-5 has a Ki
of 100 nM at sst4,2 an affinity enhancement by more than a
factor of 15 over that of (+)-2.
Th e Ba sis F or Affin ity En h a n cem en t. The 5-fold
affinity enhancement at sst4 of the neutral C4 pyrazin-
2-ylmethyl analogue (+)-10 over the 4-benzyl congener
(+)-2 is too small to be attributed to salt bridge forma-
tion between this heterocycle and the receptor. Direc-
tional hydrogen bond formation appears the most
plausible explanation for the affinity enhancement of
(+)-10 as well as that of (+)-7, (+)-8, and (+)-9. The
affinity enhancement is greatest for (+)-8 and (+)-10,
less so for (+)-9, and least for (+)-7; these results are
also consistent with the directional nature of hydrogen
bonds.10 We also considered an alternate explanation
for the enhanced affinity of (+)-8 and (+)-10 over that
of (+)-2: the C4 heterocyclic aromatic rings might act
14 or the Phe7 of 1 (SRIF-14 numbering) by His7 was
known to enhance potency.3 We observed a correspond-
ing affinity enhancement when the 2-benzyl side chain
of (-)-3 is replaced by the imidazol-4-ylmethyl moiety
(+)-4.1a,2 Subsequently we achieved a Ki of 100 nM at
sst4 with the congener (+)-5.2 The underlying rationale
(that the â-turn of 1, defined by Trp8 and Lys9 (SRIF-
14 numbering), is mimicked by the side chains of (+)-2

1860 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Prasad et al.
Ta ble 1. SARs of C4 Heterocycle-Containing Analogues at the sst4 Receptor of SRIF
a
pKa values are for the NH+ protonated heterocycles. PKHB values represent hydrogen bond basicity of the heterocycles (pyridine,
etc.), obtained by FTIR spectrometry.14 All data points are shown; the center of the gray area represents the calculated Ki and the box
graph depicts the standard deviation.65 Data points within the 90% confidence limits (Q-test) were retained in the calculation of average
Ki whether they lie within the standard deviation area (grey zone).65 (For optical rotation signs of the compounds cited, see text.)
as facial π electron donors in edge-to-face aromatic-
aromatic interactions within the C4 binding pocket.11
The computed electrostatic potential surfaces, however,
are inconsistent with this explanation (see below).
The ortho-substituted pyridinylmethyls (+)-11 and
(+)-12 both had binding affinities comparable to that
of (+)-2 despite the striking difference of the pKa’s of
these two heterocycles. These results suggest unfavor-
able steric effects from the isosteric chloro and methyl
substituents. The inductive effect should favor the
methyl substituent, but only to the extent that hydrogen
bonding is still possible. The imidazole nitrogen of (+)-
13 is more basic than the nitrogen of (+)-8: the
diminished affinity may therefore be due in part to
greater solvation, causing the increased binding energy
to be more than offset by the desolvation energy
required for the compound to enter the transmembrane
domain. The decrease in binding affinity might also be
partially due to the change in ring size, therefore
affecting the directional nature of the hydrogen bonding
of the imidazole compared with pyrazin-2-ylmethyl or
pyridin-3-ylmethyl.12,13 In addition, the data show that
changes in pKa and in pKHB have different effects on
binding affinities.14
consistent with a hydrogen bond rather than a salt-
bridge.10,17 Steiner and Koellner indicate that the ener-
gies of hydrogen bonds cover a wide range (0.5 to over
30 kcal/mol).18,19 We conclude that hydrogen bonding
capabilities, rather than the pKa values are consistent
with the SARs.14
Showing that the pyridinylmethyl and pyrazin-2-
ylmethyl congeners (+)-7, (+)-8, (+)-9, and (+)-10
display higher binding affinities than the 4-benzyl
analogue (+)-2, did not allow us to predict the effect of
the fluorine substituent of (+)-14. The electronegativity
of the fluorine atom should markedly reduce the pres-
ence of the lone electron pair around the nitrogen atom.
Several investigators have examined the question
whether a fluorine substituent attached to a carbon
atom can act as a hydrogen bond acceptor, providing
contradictory conclusions. These are briefly summarized
below.
Dunitz and Taylor20,21 examined the crystal structures
from the Cambridge Structural Database and the
Brookhaven Protein Data Bank. They expressed the
observed short H bond formation as a percentage of the
opportunities for such H bonding and found this to be
42% for carbonyl oxygens, 32% for aryl amino nitrogens,
but only 0.6% for carbon-linked fluorine atoms. Recent
reports by Kool and co-workers22 are consistent with this
analysis. Rozovsky, McDermott, and their co-workers
reported crystallographic studies which revealed that
fluorinated triosephosphate isomerase is indistinguish-
able from the wild type, and that the fluorine atom
accepts a hydrogen bond from water and not from a
protein residue.23,24 Barberich et al., however, provided
examples for significant inter- and intramolecular hy-
Pyrazine has two types of proton-acceptor sites: the
nitrogen lone pairs and the ring π-cloud. Hydrogen
bonding occurs only with the former, via the lone pairs
of a nitrogen, analogous to that of pyridine.15 The
conserved directionality of hydrogen bonding involving
both pyridine and pyrazine nitrogens is consistent with
their similar binding affinities.16 Both the pyridine and
pyrazine are presumed to be largely unprotonated in
the receptor, and their affinity enhancements are

Binding Affinities at Somatostatin Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1861
drogen bonding between hydroxyl groups and C-F
bonds, citing a H-F distance (2.01 Å) obtained by
neutron diffraction, and an OH-F distance of 2.97 Å,
obtained by X-ray diffraction.25 The authors conclude
that “many questions about the nature and strength of
these dipole-dipole interactions and their biological
importance remain unanswered.” Finally, Krueger and
Mettee reported H bond formation constants (Kassoc) in
CCl4 solution at ∼ 28 °C with methanol of 1.5 for n-amyl
fluoride and 0.11 for fluorobenzene.26,27
In the event, the 2-fluoropyridin-5-ylmethyl derivative
(+)-14 (Table 1) displayed a Ki which more closely
resembles that of the pyridin-3-ylmethyl congener (+)-8
than that of the 4-benzyl analogue (+)-2. As mentioned
above, the electronegativity of the fluorine atom makes
it unlikely that the nitrogen atom of (+)-14 per se can
act as a hydrogen bond acceptor.28 It is therefore of
interest that anhydrous hydrogen fluoride is said to
form nonlinear, zigzag hydrogen bonds, even in the gas
phase,29 supporting the proposition that fluorine can act
as an hydrogen bond acceptor. This nonlinearity leads
us to speculate that dipole interaction of the proposed
hydrogen bond donor may involve both the pyridine
nitrogen and the fluorine of (+)-14. Such nonlinearity
might allow for a bifurcated hydrogen bond in which
the proton is simultaneously bound to both the pyridine
nitrogen and to the fluorine atom of (+)-14. Bifurcated
arrangements have been observed in several crystal
structures involving organic fluorine as a hydrogen bond
acceptor.30,31 There is one reported case of three-center,
bifurcated hydrogen bonding, in ammonium fluoroac-
etate.32
P r obin g th e Bin d in g Mod e of P yr id in -3-ylm eth yl
An alogu e (+)-8. We previously showed that the 1-meth-
oxyglycoside (+)-15, which lacks the Trp8 mimicking C1
indole substituent, binds SRIF receptors via an alter-
nate binding mode,2 made possible by the radial sym-
metry of the glucose as7-9 depicted in Figure 2b. This
raised the interesting question whether the C1 indole
containing C4 pyridinylmethyl and pyrazin-2-ylmethyl
analogues show enhanced affinity over the C4-benzyl
congener because they, too, bind via this alternative
binding mode as implied by part e in Figure 2 (i.e., the
pyridinylmethyl/pyrazin-2-ylmethyl rings serve as im-
proved indole surrogates). We therefore synthesized the
C1-methoxy pyridinylmethyl analogues (+)-16a through
(+)-16c as well as the C1-methoxy pyrazin-2-ylmethyl
congener (+)-17 and 2-fluoropyridin-5-ylmethyl conge-
ner (+)-18 (see Figure 3). None of these congeners bind
sst4 at concentrations of 10 µM. We therefore conclude
that (+)-8 and, by analogy, (+)-10 bind as shown in
Figure 2c and not as shown in Figure 2e.
Rela tion sh ip betw een th e C4 Su bstitu en t of
(+)-2 a n d P ep tid a l SRIF Recep tor Liga n d s. Molec-
ular modeling reveals33 that the Trp8 and Lys9 side
chains, the functionalities of SRIF-14 that are essential
for binding, overlay well with the corresponding side
chains of octreotide, the only crystalline peptidal or
glycosidic SRIF receptor ligand.34 This is reassuring
because these and the related side chains of (+)-2 and
of (-)-3 fit this same pocket in the receptor. It is possible
that Thr10 and Thr12 of SRIF map to the same positions
as does the C4-benzyl of the sugar (see Figure 4). It is
known that the Thr10 side chain is not required for
F igu r e 2. Probing the binding mode for pyridinylmethyl
glycosides at sst4. Presentations d and e are inconsistent with
the data. (a) Binding mode of (+)-2. (b) The established binding
mode for ligand (+)-15, lacking an indole side chain.2 (c) The
binding mode proposed herein for the pyridin-3-ylmethyl-
substituted analogue (+)-8. (d) Congeners of (+)-15 with a C4
heterocyclic substituent do not bind. (e) Conclusion: the
alternate binding mode typified by (+)-15 does not apply to
(+)-8, and therefore the pyridinylmethyl and the pyrazin-2-
ylmethyl substituents do not serve as indole replacements.

1862 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Prasad et al.
F igu r e 3. Pyrazinyl- and 2-fluoropyridin-5-ylmethyl-contain-
ing methyl glycosides (+)-17 and (+)-18.
F igu r e 5. A stereo representation of the averaged NMR
structural data of octreotide and of a representative sugar (+)-
2. There is no equivalent for Phe11 of SRIF-14 in octreotide,
which, however, has a counterpart for the C3-benzyl of the
sugar. Interestingly, the C-terminal residue of octreotide
(threoninol) overlaps the C4-benzyl of the sugar.
of SRIF-14 have been shown to stabilize the bioactive
conformation of this peptide via a herringbone-type
interaction.36 Nevertheless our binding data for the C4
heterocycles allow us to conclude that sst4 tolerates
hydrophilic aromatic groups at the C4 position of the
sugar.37
Wh y Do th e C1-Meth oxy-C4-h eter ocycles F a il To
Bin d sst4? Compounds (+)-16a -c (Figure 2) and (+)-
17 and (+)-18 (Figure 3), all of which are congeners of
the ligand (+)-15, fail to bind sst4. This indicates that
the C4 heterocycles, unlike the C4-benzyl, cannot be
accommodated in the Trp8 binding pocket (Figure 2).
We considered two possible explanations:
(1) An Un fa vor a ble Desolva tion Requ ir em en t?
If the C4 heterocyclic substituents are solvated, the
energy required for desolvation might interfere with
binding in the Trp8 binding pocket. We therefore
computed the electrostatic partial charges on the NH
of indole and piperidine and found them to be identical
(+0.37 and +0.36, respectively). This suggests that the
desolvation energies of the indole and the piperidine
NH’s would not differ significantly. Since Trp binds the
Trp8 binding pocket by definition, we reject this inter-
pretation, preferring the rationalization discussed be-
low.
(2) Th e Im p or ta n ce of th e π-Clou d s of Ar om a tic
r in gs for Bin d in g of th e Tr p 8 Recep tor P ock et.
Ap p lica tion of Sp a r ta n 3-21G(*) MO An a lysis. Fig-
ure 6 shows the calculations of aromatic electrostatic
potentials via Spartan 3-21G MO(*) analysis. It reveals
that the benzene or indole rings unlike the pyridine or
pyrazine rings found in (+)-16 through (+)-18 (Figures
3 and 4) can provide for effective π-donor interactions
with, for example, an edge of an aromatic amino acid
in the Trp8 binding pocket. Aromatic-aromatic interac-
tions make an important contribution to nonbonded
F igu r e 4. Comparison of SRIF-14 and a sugar (+)-2 (yellow
carbons). This alignment reveals the overlap of the two
essential side chains (Trp8 and Lys9) of SRIF-14 and their
mimics in the glucoside. Phe6 and Phe11 of SRIF-14, which are
thought to stabilize its bioactive conformation, are shown in
purple. The figure also reveals that the C4-benzyl of the sugar
is positioned close to the Thr10/Thr12 side chains of SRIF-14,
but it is also close to Phe11
.
binding, since Gly10-containing peptides are highly
potent.35 Moreover, the C4-benzyl group of (+)-2 also
overlaps well with the C-terminal threoninol of oct-
reotide (see Figure 5). These observations suggest a
hydrophilic environment for the C4 region of the glu-
coside. Nevertheless, the large structural differences
between the glycoside and the peptidal ligands, and
even between SRIF-14 and octreotide, do not allow us
to eliminate the possibility that the C4 position of the
sugar more closely overlaps Phe11 of SRIF. This issue
is further complicated by the fact that Phe6 and Phe11

Binding Affinities at Somatostatin Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1863
binding potential and play a significant role in protein
stabilization (0.6-1.3 kcal/mol).38 Hunter et al.11 have
proposed a model for aromatic interactions.39-42
Both the pyridine and pyrazine heterocycles have
electron deficientπ-systems and differ from the rela-
tively electron-rich π-systems of the indole and the
benzene rings. If such electron-rich π systems are
important for binding in the Trp8 binding pocket of the
sst4 receptor, this difference would explain the observed
loss of binding of analogues (+)-16a -c through (+)-18.
Ab initio calculations by Samanta et al.,43 on the other
hand, suggested that the interaction energies with the
π-face are of comparable magnitude for benzene and
pyridine although there is a greater variation over the
pyridine ring, thus making certain orientations more
stabilizing than others. Our data are consistent with
the views of Cozzi et al.41 and of Doyan and J ain42 as
detailed below.
Significantly, the electron distributions of the hetero-
cyclic aromatics at C4 also explain why the C1-methoxy-
C4-heterocyclics typified by (+)-16, lacking a significant
negative potential in the region of the π-cloud, fail to
bind the aromatic Trp8 binding pocket.
Models generated of the interaction of SRIF with
receptor subtypes 1 and 2 by Kaupmann et al.45 suggest
that Trp8 of SRIF binds in an aromatic cavity involving
the residues Phe232, Trp284, and Tyr288. In addition, they
provided a sequence alignment for all receptor subtypes
which allows one to conclude that in the Trp8 binding
pocket, Phe232 (human sst1 numbering) and Trp284 are
conserved among the five human receptor subtypes and
Tyr288 is conserved among sst1-4 (but is replaced by
Phe at sst5). We conclude that the Trp8 binding pocket
requires a ligand that contains electron-rich π-clouds.
This is not the case in the C4 binding pocket where a
hydrogen bond donor increases binding with the het-
erocyclic substituents of (+)-7 through (+)-10 and (+)-
14.
F u r th er Im p r ovem en t in Affin ity. Replacement of
Phe7 by His7 in both SRIF-14 and in the cyclic hexapep-
tide 146 enhances affinity. In the sugar series, replace-
ment of the C2 benzyl of (-)-3 by the corresponding
imidazol-4-ylmethyl similarly led to comparable en-
hancement in affinity.1a To test further the presumption
(Figure 2c) that the pyridin-3-ylmethylindole-containing
sugar binds the receptor via the C1 indole- and the C6
lysine-mimicking side chains, we introduced a imidazol-
4-ylmethyl side chain at C2 to afford (-)-19 (Figure 7).
Sp a r ta n 3-21G(*) Molecu la r Or bita l An a lysis of
Ar om a tic Electr osta tic P oten tia ls. We computed the
electrostatic potentials of the relevant aromatic sub-
stituents at C4 of the glycosides, using Spartan 3-21G(*)
molecular orbital analysis.44 The electrostatic potential
is defined as the energy of interaction of a point positive
charge with the nuclei and the electrons of the molecule
of interest. The potentials for the benzene, pyridine,
2-fluoropyridine, pyrazine, and indole rings are shown
in Figure 6. The Spartan 3-21G(*) molecular modeling
F igu r e 7. Incorporating a imidazol-4-ylmethyl at C2 of a C4
pyridin-3ylmethyl ligand; enhanced binding affinity at sst2 and
sst4.
F igu r e 6. Spartan 3-21G(*) MO analysis of aromatic electro-
static potentials which involve interactions of a positive charge
not only with the π-cloud but also with all other electrons and
nuclei of the molecule. The mesh surfaces depicted are the
surfaces upon which the electrostatic potential (i.e., the
attraction of the molecule for a positive point charge) equals
This congener indeed showed a further enhancement
in affinity (sst4 Ki 53 ( 23 nM, n ) 6) providing a 31-
fold affinity enhancement over (+)-2 while retaining
specificity for sst4.
-10 kcal mol-1
.
Prior to the synthesis of (-)-19, our highest affinity
ligand for sst4 was the diastereomeric L-mannose
derivative (+)-5 (Ki 100 nM)2 which displays the C2
substituent in an axial disposition, resembling Phe7 of
SRIF in this regard.47 Hoping to increase affinity
further, we replaced the C4 benzyl substituent of (+)-5
by the pyridin-3-ylmethyl congener, affording (+)-20; we
were disappointed that this chemically challenging
synthesis was found to diminish affinity (Ki 800 nM, see
Figure 8).
analysis explains why in the case of the glycosides
containing the C1 indole substituent, typified by (+)-2,
the C4 heterocyclic aromatics (+)-7 through (+)-10 and
(+)-14 reveal enhanced affinities compared with (+)-2.
Hydrogen bonding of the unshared electron pairs of the
C4 heterocyclic ring with a presumed hydrogen bond
donor of an amino acid in the binding pocket offers the
best explanation for the affinity enhancement.
The measurement of the electrostatic potential sur-
faces (Figure 6) convincingly argues against an alternate
explanation, that the C4 heterocyclic aromatic rings act
as facial π-electron donors in edge-to-face aromatic-
aromatic interactions within the C4 binding pocket.
Exp lor a tor y P h a r m a cok in etic Stu d ies. Repre-
sentative substituted glycosides were subjected to hy-
drolysis conditions with both gastric acid and glucosi-
dases and were found to be stable.48 These results,

1864 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Sch em e 1. Synthesis of Common Intermediate (-)-21a
Prasad et al.
a
Reagents: (a) p-methoxybenzaldehyde dimethyl acetal, pTSA, CHCl3; (b) BnBr, NaH, TBAI, THF; (c) DiBAl-H, CH2Cl2; (d) NaH,
TfO(CH ) N3, THF; (e) DDQ, CH2Cl2:H2O (20:1).
5
2
Sch em e 2. Synthesis of C4 Heterocycle-Substituted
Analoguesa
F igu r e 8. Binding affinities of imidazole-containing L-man-
nosides. (+)-5 reveals the affinity-enhancing effect of the
imidazol-4-ylmethyl. In (+)-20 an unfavorable interaction
apparently results from the presence of both the equatorial
pyridin-3-ylmethyl and the axial imidazol-4-ylmethyl side
chains.
together with those reported by others,49,50 demonstrate
the stability of substituted glycosides to both enzymatic
and gastric acid hydrolysis. The calculated logP of (-)-
a
Reagents: (a) NaH, R-Cl, TBAI, THF:DMF (4:1); (b) PPh3,
THF, H2O; (c) NaOH, MeOH.
19 (clogP 5.35) is significantly lower than that of our
first designed mimetic (+)-2 (clogP 9.14).51 Indeed, (-)-
19 displays improved aqueous solubility, which allowed
us to assess bioavailability via parenteral administra-
tion. Not surprisingly, (-)-19 was extensively metabo-
lized by the cytochrome P-450 superfamily of isozymes.52
We are, therefore, exploring the reduction in the number
of aromatic side chains53 in order to identify and block
potential oxidation sites.54 The use of carbohydrates as
orally available therapeutics has been precedented.55,56
Ch em istr y. Preparation of analogues (+)-7 through
(+)-14, containing a C1 indole substituent, all began
with the alcohol (-)-21.57 This alcohol was prepared in
five steps from known triol (-)-22 as shown in Scheme
1.2 Subsequent alkylation of (-)-21 in a THF:DMF (4:
1) mixed solvent system with 2-picolyl, 3-picolyl or
4-picolyl chloride isomers afforded analogues 24a -c,
respectively, in moderate yields (Scheme 2). The mixed
solvent system was required since the pyridinylmethyl
chlorides, obtained as their hydrochloride salts, were
only slightly soluble in THF. Attempts to obtain the free
pyridinylmethyl chlorides or to use pyridinylmethyl
triflates resulted only in polymerization. We attribute
the moderate yields to the low reactivity of the pyridi-
nylmethyl chloride electrophiles and to the instability
of the phenyl sulfonamide in the presence of strong
alkali base, particularly in the ion-stabilizing solvent
DMF. Introduction of the pyrazin-2-ylmethyl,58 2-chlo-
ropyridin-5-ylmethyl, and 2-methylpyridin-5-ylmethyl
groups also provided the desired products (24d -f,
respectively).
Staudinger reduction59,60 of the azides then gave the
corresponding amines in good yield. Removal of the
sulfone moiety by basic hydrolysis completed construc-
tion of the analogues (+)-7 through (+)-12, all contain-
ing a C4 heterocyclic substituent.

Binding Affinities at Somatostatin Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1865
Sch em e 3. Synthesis of the 2-Fluoropyridin-5-ylmethyl Analoguea
a
Reagents: (a) NaH, 5-chloromethyl-2-fluoropyridine, TBAI, THF; (b) PPh3, THF, H2O; (c) NaOH, MeOH; (d) KOH, MeCN.
Sch em e 4. Synthesis of Imidazole-Bearing Analogue (+)-13a
a
Reagents: (a) NaH, MMTr-protected 4-chloromethylimidazole, TBAI, THF:DMF (4:1); (b) PPh3, THF, H2O; (c) NaOH, MeOH; (d)
TFA, CH2Cl2.
Sch em e 5. Synthesis of an Imidazol-4-ylmethyl- and Pyridin-3-ylmethyl-Containing D-Glucosidea
a
Reagents: (a) TIPSOTf, 2,6-lutidine; (b) DiBAl-H, CH2Cl2; (c); (c) NaH, TfO(CH2) 5N3, THF; (d) DDQ, CH2Cl2:H2O (20:1); (e) NaH,
3-pyridinylmethyl chloride, TBAI, THF:DMF (4:1); (f) TBAF, THF; (g) NaH, MMTr-protected 4-chloromethylimidazole, TBAI, THF:DMF
(4:1); (h) PPh3, THF, H2O; (i) NaOH, MeOH; (j) TFA, HS(CH2)2SH, CH2Cl2.
Synthesis of the 2-fluoropyridin-5-ylmethyl analogue
(+)-14 (Scheme 3) required modification of the proce-
dure for the removal of the indole protecting group.
Treatment of (-)-21 with NaH in THF followed by
5-chloromethyl-2-fluoropyridine provided the C4-alky-
lated product. The azide was then reduced to furnish
amine (-)-25. Subsequent treatment of (-)-25 with
methanolic NaOH removed the indole protecting group
and also displaced the fluorine on the pyridyl ring to
generate the 2-methoxypyridin-5-ylmethyl analogue (+)-
26. On the other hand, treatment with 8 N KOH in
acetonitrile afforded the desired 2-fluoropyridin-5-yl-
methyl (+)-14, albeit in low yield.
The synthesis of the imidazole-bearing analogue (+)-
13 (Scheme 4) began with alkylation of (-)-21 employing
N-monomethoxytrityl (MMTr)-protected 4-chloromethyl

1866 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Sch em e 6. Synthesis of L-Mannoside Analogue (+)-20a
Prasad et al.
a
Reagents: (a) p-methoxybenzaldehyde dimethyl acetal, CSA, CHCl3; (b) Ac2O, DMSO; (c) L-selectride, THF; (d) TIPSOTf, 2,6-lutidine,
CH2Cl2; (e) DiBAl-H, CH2Cl2; (f) TfO(CH2) 5N3; (g) TBAF, THF; (h) NaH, Tr-protected 4-chloromethylimidazole, TBAI, THF:DMF (4:1);
(i) DDQ, CH2Cl2, pH 6 buffer; (j) NaH, 3-pyridinylmethyl chloride, TBAI, THF:DMF (4:1); (k) DTT, TEA, MeOH; (l) TFA, HS(CH2) 2 SH,
CH2Cl2; (m) NaOH, EtOH.
imidazole61 in a THF:DMF mixed solvent. Unmasking
of the azide again by Staudinger reduction,59,60 provided
the amine in excellent yield. Basic solvolysis (NaOH,
MeOH) at reflux accomplished removal of the indole
protecting group; subsequent removal of the MMTr
protecting group with TFA yielded the C4-imidazol-4-
ylmethyl analogue (+)-13.
with (+)-22 (Scheme 6), the enantiomer of the triol
employed in Scheme 1. Conversion to the corresponding
p-methoxybenzylidene acetal, followed by oxidation and
reduction with L-selectride, afforded the axial alcohol
(+)-31. Protection of the C2 alcohol as a TIPS-ether,
selective opening of the p-methoxybenzylidene acetal
with DiBAl-H, and alkylation of resultant C6 alcohol
via acid-catalyzed etherification then furnished (+)-32
in a 53% yield for the three steps. Removal of the axially
oriented TIPS protecting group with TBAF proceeded
in good yield. Etherification of the C2 alcohol with
MMTr-protected 4-chloromethylimidazole62 then fur-
nished the imidazole-bearing mannoside. Unfortunately
treatment of the latter with DDQ resulted in complete
removal of the MMTr protecting group of the imidazole,
with only partial removal of the PMB protecting group.
Thus, in this instance the MMTr protecting group
proved to be too labile relative to the p-methoxybenzyl
ether functionality.
To overcome this difficulty, the more robust trityl
group was selected as the protecting group for the
imidazole ring. Alkylation with trityl-protected 4-chlo-
romethylimidazole (66%), followed by DDQ-mediated
removal of PMB-protecting group at 0-5 °C in a pH 6.0
buffer, afforded alcohol (+)-33. Subsequent reaction of
(+)-33 with 3-picolyl chloride hydrochloride afforded the
fully elaborated (+)-34 in 76% yield. Staudinger reduc-
tion of azide (+)-34 using triphenylphosphine afforded
the desired amine, albeit in low yield. Unmasking of the
amine with dithiothreitol62 in the presence of triethy-
lamine, however, proceeded smoothly. Removal of the
trityl protecting group under acidic conditions was then
followed by deprotection of the indole to afford (+)-20
in 71% yield (two steps).
The synthesis of C2-imidazol-4-ylmethyl,C4-pyridin-
3-ylmethyl â-D-glucose (-)-19 was accomplished by first
introducing the pyridinylmethyl substituent at C4,
followed by the more labile imidazol-4-ylmethyl sub-
stituent at C2. The previously prepared p-methoxyben-
zylidene alcohol (-)-27 (Scheme 5) was protected at C2
by the triisopropylsilyl (TIPS) group; selective opening
of the p-methoxybenzylidene acetal with DiBAl-H yielded
the C6 primary alcohol. Subsequent treatment with
NaH in diethyl ether followed by addition of the crude
5-azidopentyl triflate installed the azide precursor of the
lysine side chain mimic. Oxidative-removal of the C4
p-methoxybenzyl group with DDQ yielded the C4 alco-
hol (-)-28, which upon etherification in a mixed solvent
system [THF:DMF (4:1)] with 3-picolyl chloride and
desilylation with tetrabutylammonium fluoride (TBAF)
in THF furnished the C2 alcohol (-)-29. Subsequent
alkylation with (N-MMTr)-4-chloromethylimidazole pro-
ceeded smoothly to afford the fully substituted glycoside
(Scheme 5).
Three operations were then required to arrive at
desired analogue (-)-19: (1) Staudinger reduction59,60
of the azide to afford the amine; (2) removal of the indole
sulfonamide; and (3) acidic removal of the monomethox-
ytrityl moiety in the presence of ethane dithiol as a
scavenger (67%, three steps).
The synthesis of the C2-imidazol-4-ylmethyl,C4-py-
ridin-3-ylmethyl â-L-mannose analogue (+)-20 began
Synthesis of methyl glucosides (+)-16a -c, (+)-17, and

Binding Affinities at Somatostatin Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1867
Sch em e 7. Synthesis of Various C4-Substituted Methyl
ethyl ring can replace a benzyl ring in the C4 position
of indole-containing glucosides at the NK-1 and SRIF
receptors but not in the Trp8 binding pocket of the SRIF
receptor. These results are explained by our calculations
of the electrostatic potentials of the diverse C4 substit-
uents, showing that only the benzene ring, and not the
pyridine and pyrazine congeners, display a π-electron
cloud resembling that of the indole ring of SRIF-14, of
1, and of (+)-2. Thus replacement of benzene or indole
side chains by heterocyclic aromatic rings typified by
pyridine and pyrazine produce two effects: they en-
hance water solubility and hydrogen bonding capacity
as expected, but they also diminish the ability of the
π-cloud associated with the heterocyclic ring to interact
with side chains of an aromatic binding pocket such as
that for Trp8 of SRIF-14.
Glycosidesa
a
Reagents: (a) NaH, THF, TfO(CH2) 5N3; (b) DDQ, CH2Cl2:
H2O (20:1); (c) NaH, R-CH2Cl, TBAI, DMF; (d) PPh3, THF, H2O.
(-)-18 (Scheme 7) began with etherifiation of the known
C6-alcohol (+)-35.63 Oxidative removal of the p-meth-
oxybenzyl group furnished the common C4-alcohol
intermediate. Etherification with the appropriate picolyl
or pyrazin-2-ylmethyl chlorides, followed by unmasking
of the primary amine (Staudinger reduction) yielded
analogues (+)-16 through (+)-18.
Biologica l Meth od s. Binding assays at the human
sst4 receptor were performed using (3-125I-Tyr11)-SRIF-
14 as the radioligand. The Packard Unifilter assay for
SRIF subtype receptor binding was utilized for these
experiments as described by Birzin and Rohrer.64
Ack n ow led gm en t. Financial support was provided
by the National Institutes of Health through Grant GM-
41821. We also thank Merck Research Laboratories for
an unrestricted grant. It is a pleasure to acknowledge
the significant contribution of Dr. Maria Cichy-Knight
who carried out our initial attempts to the install the
pyridinylmethyl side chains into the C3 and C4 positions
of the glucoside. It is also satisfying to thank Dr.
Margaret A. Cascieri for her profound contributions to
our research prior to her recent career change. We thank
Mr. Adam Charnley for proofreading this manu-
script. The authors are also appreciative of Mr. Paul
Marchesano for his expert assistance in the preparation
of this manuscript. Finally, we thank the three referees
for their valuable suggestions.
Con clu sion
Modifications to improve the water solubility of our
glycosides provided us with insights into the nature of
the ligand binding sites at both the NK1 and SRIF
receptors. For the former we showed that the free C4
hydroxyl congener (-)-6 provided optimal affinity (Ki
27 nM) since the C4 benzyl and even the three isomeric
water-solubilizing C4 pyridinylmethyl analogues cre-
ated less favorable interactions with the NK1 receptor.
On the other hand, at the SRIF receptor, the pyrazin-
2-ylmethyl and the three isomeric C4 pyridinylmethyl
substituents enhance affinity. The pyridin-3-ylmethyl
congener (+)-8 and the pyrazin-2-ylmethyl (+)-10 proved
most effective. We varied both the electronic and steric
properties of the C4 substituent and concluded that
affinity enhancement can be attributed to a directed
hydrogen bond. The high affinity of the C4 pyrazin-2-
ylmethyl congener, a neutral heterocyclic substituent,
also suggests formation of a hydrogen bond. It is
noteworthy that the fluoro-substituted pyridin-3-yl-
methyl congener (+)-14 (Table 1) can act as a hydrogen
bond acceptor possibly because of the bifurcation that
would allow the hydrogen bond to interact both with
the nitrogen and the neighboring fluorine.
Introduction of a pyridinylmethyl group also improved
aqueous solubility, facilitating pharmacokinetic studies
and providing affinity enhancement specific for sst4.
This substitution, when combined with the affinity
enhancing imidazol-4-ylmethyl group at C2, generated
a more water-soluble ligand (-)-19 with a Ki of 53 nM.
Through incorporation of the imidazol-4-ylmethyl af-
finity-enhancing substituent into the pyridin-3-ylm-
ethyl-containing glycoside (-)-19, we were able to
provide additional evidence that (+)-8 binds in the same
manner as other indole-containing ligands.
Su p p or tin g In for m a tion Ava ila ble: Biological methods
and binding data for the hNK1 receptor; experimental details
of the hydrolytic stability studies; complete characterization
of all analogues and synthetic intermediates, including 1H
NMR, 13C NMR, HRMS, optical rotation, and IR. This material
is available free of charge via the Internet at http://pubs.ac-
s.org.
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(6) Unpublished results from these laboratories.
(7) The SARs of 2 at the NK1 and SRIF receptors illustrate the role
of radial symmetry in the glucoside scaffold.8,9 Compound 2 binds
not only the SRIF receptor for which it was designed, but also
the NK1 and â2 adrenergic receptors. These activities were
discovered by serendipity, without systematic screening; fur-
thermore, a close analogue of 2 blocks the interaction of IkB with
its kinase. [The authors are indebted to Dr. Carl DeCicco,
Bristol-Myers Squibb Co. (formerly DuPont Pharmaceutical
Company) for this information.] We have attributed this pro-
miscuity to the radial symmetry of the glucose scaffold which is
exemplified by the fact that 2 binds the sst4 and the NK1
receptors via side chains in positions 1,6 and 1,2, respectively.
Cyclic hexapeptides, however, which lack such symmetry, bind
both receptors via side chains in the i+1 and i+2 positions.8,9 It
is important to appreciate that this promiscuity can be overcome
because, although 2 binds diverse receptors, excellent specificity
can be incorporated.
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