Novel peptidomimetic hematoregulatory compounds

Article abstract of DOI:10.1016/S0960-894X(00)00036-6

The activity of a novel series of peptidomimetic hematoregulatory compounds, designed based on a pharmacophore model inferred from the structure-activity relationships of a peptide SK and F 107647 (1), is reported. These compounds induce a hematopoietic synergistic factor (HSF) which in turn modulates host defense. The compounds may represent novel therapeutic agents in the area of hematoregulation. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00036-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 531±534
Novel Peptidomimetic Hematoregulatory Compounds
Dirk A. Heerding, a,* Melanie Abruzzese, b Doreen Alberts, a Joelle Burgess, a
James F. Callahan, a William F. Hu€man, a Andrew G. King, b Stephen LoCastro, a
Peter DeMarsh, b Louis M. Pelus, b Joanne S. Takata a and Pradip K. Bhatnagar a
aDepartment of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, 1250 S. Collegeville Road,
Collegeville, PA 19426, USA
bDepartment of Molecular Virology and Host Defense, SmithKline Beecham Pharmaceuticals, 1250 S. Collegeville Road,
Collegeville, PA 19426, USA
Received 1 November 1999; accepted 12 January 2000
AbstractÐThe activity of a novel series of peptidomimetic hematoregulatory compounds, designed based on a pharmacophore
model inferred from the structure±activity relationships of a peptide SK&F 107647 (1), is reported. These compounds induce a
hematopoietic synergistic factor (HSF) which in turn modulates host defense. The compounds may represent novel therapeutic
agents in the area of hematoregulation. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
present the pharmacophore to its putative receptor.
Implicit in this model is the fact that the sca€old in 1
should not be unique. This model has also been used by
Cuthbertson et al. to design an alternative peptidomi-
metic analogue.14 In this paper we describe the in vitro
activity of a series of analogues based on 7, as well as
the in vivo activity of a selected analogue 5a.
Hematopoietic growth factors such as G-CSF, M-CSF,
GM-CSF and EPO have received considerable attention
in recent years.1±3 These factors play key roles in reg-
ulating cellular host defense mechanisms involved in
combating bacterial, fungal and viral infections.4 They
stimulate host defense mechanisms by both increasing
immunocompetent cell numbers and enhancing e€ector
cell functions. Although these proteinaceous factors are
clinically e€ective,5±8 a small molecule capable of
mimicking the e€ects of these factors may complement
existing immunomodulatory therapy.
Chemistry
Compounds 5a±e, 7 and 9 (Table 1) were synthesized as
outlined in Scheme 1. 1,4-Phenylenediamine dihy-
drochloride was di-acylated with Boc-b-Ala using 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC) and hydroxybenzotriazole hydrate (HOBt) in the
presence of i-Pr2NEt. The Boc-protecting groups were
removed by exposure to tri¯uoroacetic acid (TFA) and
the resulting diamine was acylated with either Boc-
Ser(Bn) or Boc-Glu(OBn) using benzotriazol-1-yloxy-
tris(dimethylamino)-phosphonium hexa¯uorophosphate
(BOP reagent) and HOBt in the presence of i-Pr2NEt to
give 3 and 6, respectively. Compound 3 was treated with
TFA and the resulting diamine was acylated with the
acids shown to give 4a±e. Racemic azetidine-2-car-
boxylic acid was used to synthesize 4d giving diastereo-
meric products which were easily separated by ¯ash
chromatography. No attempt was made to assign the
absolute stereochemistry of the azetidine ring of these
products. Exposure of compounds 4a±e to anhydrous
We have recently reported the activity of a low mole-
cular weight peptide, SK&F 107647 (1) which stimulates
hematopoiesis (hematopoiesis is the process whereby
stem cells, proliferate and di€erentiate into mature
blood cells, for reviews, see refs 9 and 10) in vitro and in
vivo (Fig. 1).11,12 We have also formulated a pharma-
cophore model associated with 1 which was used to
design and synthesize a structurally simpler, biologically
active, peptidomimetic analogue, 7.13 The model states
that the pGlu-Glu subunits of 1 represent the pharma-
cophore of this compound and that the remaining struc-
tural elements of 1 function as a sca€old to correctly
*Corresponding author. Tel.: +1-610-917-7944; fax: +1-610-917-
4206; e-mail: dirk_a_heerding@sbphrd.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00036-6

532
D. A. Heerding et al. / Bioorg. Med. Chem. Lett. 10 (2000) 531±534
Biology
The test compounds were evaluated in vitro for their
ability to induce a hematopoietic synergistic factor
(HSF), from a murine stromal cell line (C6.4).15,16 The
induced HSF synergizes with endogenous myelopoietic
growth factors (G-, M-, GM-CSF) and augments the
di€erentiation and maturation of stem cells from bone
marrow, resulting in an increase in progenitor cells
(CFU-GM) in vitro. The amount of HSF activity
induced was determined using a murine granulocyte-
macrophage colony-forming unit (CFU-GM) assay.17
The HSF activity of the stromal cell supernatants
exhibited
a
distinct `bell-shaped' dose response
curve.12,13 Inactive compounds were de®ned by the lack
of induced HSF activity at the doses tested (0.10, 1.0
and 10.0 mg/mL of test compounds). Using this assay,
we determined which structural changes were tolerated
in this series of compounds. However, due to the lack of
a receptor binding assay, it is dicult to quantitatively
assess the e€ects of relatively modest structural changes
on the interaction of a given analogue with its putative
receptor. The previously described analogue 1, was
included in all the assays at a single dose (1.0 mg/mL) as
a positive control.
Figure 1. Structure of SK&F 107647 (1).
Table 1. In vitro hematopoietic synergistic factor (HSF) released by
0.1, 1.0 and 10.0 mg/mL of 1, 5a±e, 7, and 9
No.
R
0.10 mg/mL
2.5Â10e2‹4.4e1 2.1Â10e4‹1.1e4 1.4Â10e6‹8.7e5
1.5Â10e2‹1e3 2.1Â10e3‹4.5e2 1.0Â10e4‹8e2
1.0 mg/mL
10.0 mg/mL
1
Compound 5a was tested in vivo in a murine model of
infection.18 Mice were treated ip with either compound
5a or dilution bu€er control. Two hours after treatment,
the mice were challenged iv with a lethal dose of C.
albicans and followed for survival.
5a
5b
2.3Â10E2‹8.1e1 1.5Â10E3‹9e2 1.4Â10E4‹1.1e4
1.4Â10E2‹8.6e1 1.1Â10E3‹9e2 1.5Â10E4‹8.4e3
2.7Â10E2‹1.2e2 4.4Â10E3‹9e2 1.1Â10E4‹9.3e3
Results and Discussion
5c
The pyroglutamic acid and glutamic acid moieties of 7
could be replaced by picolinic acid and serine to give 5a.
This matches the selectivity seen for the peptide SAR
around 1. The picolinoyl groups of 5a could be replaced
by small nitrogen-containing heterocyclic carboxylic
acids (5b±e). Substituting dehydroproline (5b), proline
(5c) or azetidine carbonyl (5d) for the picolinoyl group
in 5a give compounds that are equipotent in the HSF
assay, whereas, substitution with the pyrrolyl group
results in a 10-fold loss in potency. Furyl and thiophe-
nyl groups were not acceptable substitutions for the
picolinoyl groups of 5a. The location of the nitrogen in
the heterocyclic ring with respect to the amide was also
found to be important. Only rings containing nitrogen
alpha to the carbonyl group of the amide were tolerated.
For example, replacing the picolinoyl groups of 5a with
nicotinoyl or isonicotinoyl groups resulted in inactive
analogues. The size of the nitrogen-containing heterocyclic
group which could be tolerated was limited to monocyclic
4-, 5- or 6-membered rings. Replacing the picolinoyl sub-
stituent of 5a with fused bicyclic moieties such as 2-quino-
linecarbonyl or 1-isoquinolinecarbonyl groups resulted in
inactive analogues. From these results, it is clear that
there is a very stringent requirement for a nitrogen
containing heterocyclic, but that the `nature' of the
nitrogen atom can be varied, namely, a secondary amide
nitrogen, an aryl sp2 nitrogen and a secondary amine
nitrogen. There is no obvious explanation for the
5d
5e
NDa
2.2Â10E2‹8.3e1 2.0Â10E3‹8.2e2
7
9
1.6Â10e2‹8.3e1 1.8Â10e3‹8e2 1.5Â10E4‹1.2e4
1.4Â10E2‹1.2e2 1.5Â10E3‹1e3 1.7Â10E4‹1.1e4
aND, not detected.
HF gave, after puri®cation, 5a±e, which were used for
biological evaluation. Compound 6 was treated with TFA
and then acylated with pGlu. Exposure to anhydrous HF
gave 7.
Compound 9 was prepared in a similar fashion to the
analogues described above except that Fmoc-N-Me-
Ser(Bn) was used instead of Boc-Ser(Bn) or Boc-Glu(OBn)
to give 8. The Fmoc-protecting group of 8 was removed
with piperidine and the resulting diamine was acylated
with picolinic acid using BOP reagent and HOBt in the
presence of i-Pr2NEt. Exposure of this compound to
anhydrous HF gave 9.

D. A. Heerding et al. / Bioorg. Med. Chem. Lett. 10 (2000) 531±534
533
Scheme 1. (a) Boc-b-Ala, EDC, HOBt, i-Pr2NEt, DMF; (b) TFA, CH2Cl2; (c) BocSer(Bn), BOP reagent, HOBt, i-Pr2NEt, DMF; (d) R-CO2H, BOP
reagent, HOBt, i-Pr2NEt, DMF; (e) HF, 78 ꢀC to 0 ꢀC; (f) BocGlu(OBn), BOP reagent, HOBt, i-Pr2NEt, DMF; (g) pGlu, BOP reagent, HOBt,
i-Pr2NEt, DMF; (h) Fmoc-N(Me)Ser(Bn), BOP reagent, HOBt, i-Pr2NEt, DMF; (i) piperidine; (j) picolinic acid, BOP reagent, HOBt, i-Pr2NEt,
DMF.
observed functional group selectivity seen here except to
note that this selectivity matches that observed for the
peptide SAR around 1.12 Also, only the antipode shown
of the heterocyclic rings containing stereogenic centers
were active (5b and 5c). Combined, these data suggest that
there is a very speci®c interaction of these compounds
with the putative receptor.
The Ser units of 5a were replaced with Thr (d and l),
d-Ser, Ala and Gly. None of these substitutions were
tolerated at this position. Substituting N-methyl-Ser for
Ser in 5a to give 9 was the only variation that was tol-
erated. This selectivity again mirrors that seen in the
pharmacophore region of the peptide 1, suggesting that
these peptidomimetic compounds are targeting the same
putative receptor as 1.
Figure 2. E€ect of 5a on the survival of mice lethally infected with C.
albicans.
Compound 5a was chosen for in vivo studies because of
its structural similarity to the most potent pharmaco-
phore pattern (picolinoyl-serine) found in the peptide
series based on 1.12 Compound 1 has been shown to
induce HSF from murine bone marrow stromal cells,
and that this in turn can enhance e€ector cell function
in mice and a€ord protection in a murine infection
model.19,20 Like 1, 5a enhances e€ector cell function in
mice21 and is e€ective in increasing the survival rate of
mice challenged with lethal doses of C. albicans (Fig. 2).
Mice pretreated with 1.0, 10 and 100 mg/kg of 5a resul-
ted in a statistically signi®cant increase in the survival
rate. There were no survivors by day 13 in the diluent
treated control group. Similarly, mice pretreated with
the lowest dose of 5a (0.1 mg/kg) failed to demonstrate
signi®cant protection. Compound 5a also demonstrated
a `bell-shaped' dose response that is characteristic of
this type of hematoregulatory compound18 with the
1000 mg/kg dose failing to signi®cantly protect.

534
D. A. Heerding et al. / Bioorg. Med. Chem. Lett. 10 (2000) 531±534
11. Pelus, L. M.; King, A. G.; Broxmeyer, H. E.; DeMarsh, P.
Conclusion
L.; Petteway, S. R.; Bhatnagar, P. K. Exp. Hematol. 1994, 22,
239±247.
We have successfully designed a series of simple, small
molecule analogues of a potent hematoregulatory peptide
1. These compounds retain all of the in vitro biological
activity (albeit less potent) of the parent peptide, further
validating a pharmacophore model for the interaction
of a putative receptor with 1, 5a±e, 7 and 9. One possi-
ble explanation for the lower potency may be our choice
of sca€old. It is possible that the optimal con®guration
of a structurally simple sca€old has not yet been
achieved. Studies to determine the solution phase struc-
ture of the sca€old of 1 are underway and may provide
further insight. Compound 5a was further pro®led and
shown to retain the ex vivo and in vivo biological activity
of the parent peptide.
12. Bhatnagar, P. K.; Agner, E. K.; Alberts, D.; Arbo, B. E.;
Callahan, J. F.; Cuthbertson, A. S.; Engelsen, S. J.; Fjerding-
stad, H.; Hartmann, M.; Heerding, D.; Hiebl, J.; Hu€man, W.
F.; Hysben, M.; King, A. G.; Kremminger, P.; Kwon, C.;
LoCastro, S.; Lovhaug, D.; Pelus, L. M.; Petteway, S.;
Takata, J. S. J. Med. Chem. 1996, 39, 3814±3819.
13. Bhatnagar, P. K.; Alberts, D.; Callahan, J. F.; Heerding,
D.; Hu€man, W. F.; King, A. G.; LoCastro, S.; Pelus, L. M.;
Takata, J. S. J. Am. Chem. Soc. 1996, 118, 12862±12863.
14. Cuthbertson, A. S.; Husbyn, M.; Engebretsen, M.; Hart-
mann, M.; Lange, M.; Sandosham, J.; Fischer, P. M.; Fjer-
dingstad, H.; Lovhaug, D. J. Med. Chem. 1997, 40, 2876±2882.
15. King, A. G.; Frey, C. L.; Arbo, B.; Scott, M.; Johansen,
K.; Bhatnagar, P. K.; Pelus, L. M. Blood 1995, 86 (Suppl. 1),
309a.
16. King, A. G.; Scott, R.; Wu, D. W.; Strickler, J.; McNulty,
D.; Scott, M.; Johansen, K.; McDevitt, D.; Bhatnagar, P. K.;
Balcarek, J.; Pelus, L. M. Blood 1995, 86 (Suppl. 1), 310a.
17. King, A. G.; Talmadge, J. E.; Badger, A. M.; Pelus, L. M.
Exp. Hematol. 1992, 20, 223±228.
References and Notes
1. Broxmeyer, H. E. Hum. Cytokines: Their Role Dis. Ther.
Aggarwal, B. B.; Puri, R. K. Eds.; Blackwell: Boston, 1995; pp
27±36.
2. Guillosson, J.-J. Ann. Pharm. Fr. 1996, 54, 145±150.
3. Lowry, P. A. J. Cell Biochem. 1995, 58, 410±415.
4. Metcalf, D. Cancer 1990, 65, 2185±2195.
5. Aviles, A.; Guzman, R.; Delgado, S.; Nambo, M. J.; Gar-
cia, E. L.; Diaz-Maqueo, J. C. Am. J. Hemtol. 1996, 52, 275±
280.
6. Aviles, A.; Guzman, R.; Garcia, E. L.; Talavera, A.; Diaz-
Maqueo, J. C. Anti Cancer Drugs 1996, 7, 392±397.
7. Chatta, G. S.; Dale, D. C. Drugs Aging 1996, 9, 37±47.
8. Bonilla, M. A.; Dale, D.; Zeidler, C.; Last, L.; Reiter, A.;
Ruggiero, M.; Davis, M.; Koci, B.; Hammond, W.; Gillio, A.;
Welte, K. Br. J. Hematol. 1994, 88, 723±730.
18. The in vivo model is described in detail in DeMarsh, P. L.;
Sucoloski, S. K.; Frey, C. L.; Bhatnagar, P. K.; Koltin, Y.;
Actor, P.; Petteway, S. R. Immunopharm. 1994, 27, 199±206.
19. Frey, C. L.; King, A. G.; Sucoloski, S.; Strickler, J.; Pelus,
L. M.; Bhatnagar, P. K.; Wu, D. W.; Scott. R. Abstracts of
Papers, 35th Interscience Conference on Antimicrobial Agents
and Chemotherapy, San Francisco, CA, USA, 17±20 Septem-
ber 1995; American Society for Microbiology; G17.
20. The induced HSF from murine bone marrow stromal cells
has subsequently been identi®ed as a truncated form of the mur-
ine KC gene product. Frey, C. L.; Balcarek, J.; Arbo, B. E.;
Johansen, K.; Sucoloski, S.; Schneider, M.; Takata, J. S.; Strick-
ler, J.; Bhatnagar, P. K.; Pelus, L. M.; Dillon, S. B. Abstract of
Papers, 35th Interscience Conference on Antimicrobial Agents
and Chemotherapy, San Francisco, CA, USA, 17±20 September,
1995; American Society for Microbiology; G50.
9. Sachs, L. Proc. Natl. Acad. Sci. USA 1996, 93, 4742±4749.
10. Nathan, D. G. Soc. Gen. Physiol. Ser. 1988, 43 (Cell Phy-
siol. Blood), 19±24.
21. Frey, C. Personal communication.