Optimization of a dihydropyrrolopyrazole series of transforming growth factor-β type I receptor kinase domain inhibitors: Discovery of an orally bioavailable transforming growth factor-β receptor type I inhibitor as antitumor agent

Article abstract of DOI:10.1021/jm701199p

In our continuing effort to expand the SAR of the quinoline domain of dihydropyrrolopyrazole series, we have discovered compound 15d, which demonstrated the antitumor efficacy with oral bioavailability. This effort also demonstrated that the PK/PD in vivo target inhibition paradigm is an effective approach to assess potential for antitumor efficacy. The dihydropyrrolopyrazole inhibitor 15d (LY2109761) is representative of a novel series of antitumor agents.

Full text of DOI:10.1021/jm701199p

2302
J. Med. Chem. 2008, 51, 2302–2306
Optimization of a Dihydropyrrolopyrazole Series of Transforming Growth Factor-ꢀ Type I
Receptor Kinase Domain Inhibitors: Discovery of an Orally Bioavailable Transforming Growth
Factor-ꢀ Receptor Type I Inhibitor as Antitumor Agent
Hong-yu Li,*,‡ William T. McMillen,‡ Charles R. Heap, Denis J. McCann,§ Lei Yan,4 Robert M. Campbell,
Sreenivasa R. Mundla,†,# Chi-Hsin R. King, Elizabeth A. Dierks,§ Bryan D. Anderson, Karen S. Britt, Karen L. Huss,
Matthew D. Voss,# Yan Wang,‡ David K. Clawson,‡ Jonathan M. Yingling,4 and J. Scott Sawyer‡
DiscoVery Chemistry Research and Technology, Drug Disposition Research, Cancer Research, Lead Optimization Biology, and Chemical
Product Research and DeVelopment, Lilly Research Laboratories, A DiVision of Eli Lilly and Company, Lilly Corporate Center,
Indianapolis, Indiana 46285, and Medicinal Chemistry Department, Albany Molecular Research, Inc., Post Office Box 15098,
Albany, New York 12212
ReceiVed September 24, 2007
In our continuing effort to expand the SAR of the quinoline domain of dihydropyrrolopyrazole series, we
have discovered compound 15d, which demonstrated the antitumor efficacy with oral bioavailability. This
effort also demonstrated that the PK/PD in vivo target inhibition paradigm is an effective approach to assess
potential for antitumor efficacy. The dihydropyrrolopyrazole inhibitor 15d (LY2109761) is representative
of a novel series of antitumor agents.
Introduction
Cancer is currently second only to heart disease as a cause
of death and will become the primary cause in the next 10 to
20 years.1 Traditional cancer therapies make use of chemo-
therapy at the maximum tolerated dose, generally resulting in
significant toxicities and often with limited success. The so-
called “targeted therapies”, such as Gefitinib or Imatinib, are
considered less toxic and provide further ammunition in the fight
against cancer but often produce responses in only a limited
number of cancer patients. New, more universal, more effective,
and less toxic therapeutic modalities are therefore desirable.2
Figure 1
Angiogenesis plays an important role in the growth of most
processes. The role of TGF-ꢀ in cancer angiogenesis biology
solid tumors and progression to metastasis.3 Recently, it has
is complex and involves aspects of tumor suppression as well
been reported that the specific inhibition of tumor-induced
as tumor promotion.8 The ability of TGF-ꢀ to potentially inhibit
angiogenesis suppresses the growth of many types of solid
the proliferation of epithelial, endothelial, and hematopoietic
tumors. For this reason, it is conjectured that the inhibition of
cell lineages is central to the tumor suppressing mechanism.8–12
angiogenesis represents a novel therapeutic approach against
Therefore, this pathway presents an attractive target for the
tumors.4–7 The transforming growth factor-ꢀ (TGF-ꢀa) type I
development of cancer therapeutics that simultaneously attack
receptor is a member of a large family of growth factors
the tumor and its microenvironment.
involved in the regulation of a diverse array of angiogenetic
Over the past decade, the pursuit of TGF-ꢀ RI kinase small
molecule inhibitors has received a high level of attention in the
* Corresponding author. Phone: 317-433-3349. Fax: 317-276-7600.
pharmaceutical industry and in the medicinal chemistry
e-mail: li_hong-yu@lilly.com.
community.13–23 A unique combination of high-throughput
‡
Discovery Chemistry Research and Technology, Lilly Research
screening (HTS), X-ray crystal analysis of TGF-ꢀ inhibitors
bound to the active site, and structure–activity relationship
(SAR) studies has contributed to the discovery of some of the
more potent TGF-ꢀ RI inhibitors.17,18 Recently, we reported
on the discovery of a new class of small-molecule TGF-ꢀ RI
inhibitors, the dihydropyrrolopyrazoles, exemplified by com-
pounds 1 and 2 (Figure 1).17 Although potent TGF-ꢀ RI
inhibitors were identified and the solubility was improved with
the attachment of a solubilizing group through an amine linkage,
we found later that representative compound 1a17 did not exhibit
acceptable PK properties (rat, 10 mg/kg, p.o.; Auc, 398 ng·hr/
mL). Moreover, a mini-Ames test on suspected metabolite 1b
(also a potent TGF-ꢀ RI inhibitor) was positive, indicating a
likelihood of mutagenicity. During the continuing SAR effort
around quinoline ring substitutions, we have discovered that
after replacing the amine linker with an ether linkage, the oral
Laboratories.
Albany Molecular Research, Inc.
Drug Disposition Research, Lilly Research Laboratories.
Cancer Research, Lilly Research Laboratories.
§
4
Lead Optimization Biology, Lilly Research Laboratories.
Present Address: sreeni@sreenilabs.com. Sreeni Labs Private Limited,
†
Flat No. 504, Vijayasree Apartments, Behind Chermas, Ameerpet, Hydera-
bad 500 073, A.P., India.
#
Chemical Product Research and Development, Lilly Research
Laboratories.
a Abbreviations: TGF-ꢀ, transforming growth factor-ꢀ; TGF-ꢀ RI,
transforming growth factor-ꢀ type I receptor; HTS, high-throughput
screening; IVTI, in vivo target inhibition; BID, twice daily; Smad, a
composite term derived from Sma genes from Caenorhabditis elegans and
Mad gene (Drosophila melanogaster) involved as intracellular substrate
for TGF-ꢀ and BMP signaling; Lck, leukocyte-specific protein tyrosine
kinase; Sapk, stress-activated protein kinase; MKK, mitogen-activated
protein kinase kinase; Fyn, a molecule, present in the signaling pathway of
integrins; JNK, c-Jun N-terminal kinase.
10.1021/jm701199p CCC: $40.75
2008 American Chemical Society
Published on Web 03/04/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2303
Scheme 3a
Scheme 1a
a Reagents and conditions: (i) CO, Pd(OAc)2, PPh3, NaOAc, DMF/
MeOH, 80 °C, 66%; (ii) (Me)2NCH2CH2NH2, 100 °C, 74%.
Scheme 2a
a Reagents and conditions: (i) HBr, 99%; (ii) bromide/or mesylate,
Cs2CO3, for 14, 79%; for 15, 46%; for 16, 79%; for 17, 39%; for 18, 15%;
for 19, 35%; (iii) amine, DMF, for 14a, 75%; for 15a, 93%; for 15b, 64%;
for 15c, 71%; for 15d, 65%; for 16a, 49%; (iv) (a) LiOH/MeOH; (b) COCl2;
(c) N-Me-piperazine (overall yield, 48% for 17a).
a Reagents and conditions: (i) for 10: NaH, MeOH, 0 to 50 °C, 96 h,
88%; (ii) for 11: NaH, EtSH DMF, 0 °C to RT, 72 h, 87%; (iii) for 12:
pyrrolidine, 80 °C, 96 h, 91%.
Scheme 4a
exposure in rat was improved. Subsequently, activity in both
in vivo target inhibition (IVTI) and xenograft efficacy models
was demonstrated. In this paper, we report the discovery and
SAR of small molecule 15d, which demonstrated oral antitumor
efficacy.
Chemistry
The construction of the dihydropyrrolopyrazole ring with
differentially substituted quinolines by following procedures
published previously (7a-7f, Figure 1)17 is shown in Supporting
Information.
a Reagents and conditions: (i) (a) 2-(2-bromoethoxy)tetrahydro-2H-pyran,
Cs2CO3, DMF; (b) AcOH, THF, H2O; (ii) (a) MsCl, pyr; (b) morpholine;
for four steps, 81%.
Methyl benzoate 8 and amide analogue 9 were derived from
compound 7e, as depicted in Scheme 1. Palladium-catalyzed
carbonylation of bromide 7e with carbon monoxide in the
presence of Pd(OAc)2, PPh3, and NaOAc in DMF/MeOH gave
methyl benzoate 8. The ester structural moiety was then treated
with 2-N,N-dimethylaminoethylamine neat at 100 °C to afford
amide 9. The derivatization at the 2-position of the quinoline is
outlined in Scheme 2. The heating of chloride 7b in MeOH or
pyrrolidine gave compound 10 or 12, respectively. In the case
of the synthesis of compound 10, NaH was required. Similarly,
compound 11 was synthesized with EtSH in DMF catalyzed
by NaH.
Substitutions at the 7-position of the quinoline moiety are
shown in Schemes 3 and 4. Demethylation of the methoxy group
in compound 7a went smoothly with HBr and produced
compound 13 in 99% yield. Alkylation of phenol 13 catalyzed
by Cs2CO3 was straightforward (yields ∼ 15–70%), except for
compound 15. Due to the ꢀ-elimination of 1-bromo-2-chloro-
ethane (see Supporting Information), the yield for the synthesis
of compound 15 was low (six conditions, ∼10%) and purifica-
tion was not synthetically practical. However, by using the
corresponding mesylate instead of the bromide, alkylation went
well in 45% yield. The transformation of ester 17 to amide 17a
proceeded in moderate yield (48%) via hydrolysis and conver-
sion to the acyl chloride, followed by treatment with N-
methylpiperazine. The reaction of chlorides 14-16, either neat
or in DMF with various amines, furnished the final compounds
(14a, 15a-d, and 16a). Alternatively, the alcohol was synthe-
sized in a two-step sequence via alkylation with 2-bromoethoxy-
THP followed by deprotection of the THP group with AcOH.
Mesylation of the alcohol followed by displacement using
the appropriate amine quantitatively afforded compound 15d
(Scheme 4).
To explore the SAR at the 7-position of the quinoline ring,
the electron-deficient substrate 4-fluorobenzonitrile was reacted
with phenol 13 in the presence of 37% KF on alumina.24 This
reaction provided a 95% yield of the desired product 20a along
with 4% of amide 20b derived from the further hydrolysis of
20a (Scheme 5). Hydrolysis of nitrile 20a with HCl followed
by acid chloride formation and treatment with dimethyl amine
gave dimethylamide 22. Conversion of the amide to thioamide
with Lawesson reagent was followed by treatment with hydra-

2304 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Brief Articles
Scheme 5a
23 is apparently too large for extension of the amine tail
completely into solvent. In addition, the solvation effect on
potency was also evident because compounds with basic
amines at the terminal position of the side chain gave slightly
higher activity in comparison with other substituted analogues
(Table 1).
Inhibitors were then run through a diverse kinase panel to
examine selectivity profiles. Inhibitors 15a and 15d were highly
selective against ∼50 kinases, although weak activities were
observed for Lck, Sapk2R (p38R-MAPK), MKK6, Fyn, and
JNK3 (18–89% inhibition at the 20 µM, see Supporting
Information).
Compounds that showed cell-based activities under ∼0.2 µM
were evaluated for metabolism in vitro. Compounds bearing a
basic amine group at the terminal end of the side chains (14a,
15a, 15b, and 15d) have generally moderate to low metabolism
levels (<60% in both rat and human microsomes), indicating
that they are more likely to give adequate in vivo exposure (see
Supporting Information). Compound 15d was subjected to a
rat exposure study (10 mg/kg oral) and good exposure (Auc ≈
1000 ng ·hr/mL, 24 h) was observed. Importantly, acceptable
bioavailability (F, 21%, compound 15d) was also achieved.
a Reagents and conditions: (i) 4-fluorobenzonitrile, 18-crown-6, 37% KF
on alumina, DMF, for 20a, 95%; for 20b, 4%; (ii) HCl, 100%; (iii) (a)
ClCOCOCl, CH2Cl2; (b) dimethylamine in THF, 54%; (iv) (a) Lawesson’s
reagent, toluene; (b) NH2NH2, Raney nickel, MeOH, refluxed, 20%.
An IVTI assay based on inhibition of pSmad2 was performed
using three different protocols: single point screening at 50 mg/
kg oral, pharmacokinetic (PK) studies at multiple doses for
compounds having passed a 50% threshold target inhibition at
50 mg/kg oral, and a time-course pharmacodynamic (PD) study
at 75 mg/kg oral. In the event, all compounds that showed less
metabolism in vitro achieved more than 50% target inhibition
in vivo. This further confirmed that metabolism surrogates
provided useful and reliable data for predicting IVTI activity.
In the PK studies, the ED50 was analyzed as inhibition versus
plasma concentration (Supporting Information). As a result,
compound 15d was identified as the most potent TGF-ꢀ RI
inhibitor in vivo with an EC50 value of 0.268 µM. Inhibition
during the time-course experiment of optimal compound 15d
was measured at 0.5, 1, 2, 4, 8, and 24 h data points. As
expected, long duration of target inhibition (∼50% inhibition
after 4 h) was observed and correlated well with plasma
concentrations (Supporting Information).
zine and Raney nickel. Final compound 23 was purified by the
combination of normal phase and reverse-phase chromatography.
Results and Discussion
Although some of the SAR on the 7-position of the quinoline
has been reported,17,18 a detailed study of ether-linked side-
chains provided an opportunity for the further optimization of
physical properties and, subsequently, for in vivo activity. The
medicinal chemistry effort was initially focused on evaluating
the effect of substitutions in both biochemical and cellular
assays. LY364947 (4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-quino-
line)16 was used as a standard compound for calibration of assay
results. As we investigated the substituent requirement at the
2-position of quinoline, we found that chloride, along with
various other groups (-OMe, -SEt, and -pyrrolidine), exhibited
very weak activity with IC50 value >20 µM (Table 1). However,
substitution at the 6-position produced more active compounds.
Interestingly, both electron-deficient and relatively small groups
were tolerated at the 6-position and gave potent activities in
both the enzymatic assay (IC50 ∼100 nM) and cell-based assays
(IC50 50–300 nM). Substitutions through an ester or amide
combined with a solubilizing tail were detrimental. Compound
7f (6-OCF3) gave IC50 values of ∼0.2 µM in both cell-based
assays, while compound 9 (6-CONH(CH2)2N(CH3)3) gave an
IC50 of only 2.27 µM in the NIH 3T3 cell-proliferative assay.
Substitution at the 8-position with fluorine to give 7d
substantially diminished in vitro activity. Double substitutions
at the 6- and 8-positions with methoxy further supported the
observation that groups at the 8-position are not well tolerated
(Table 1).
Evaluation of efficacy was then performed in a nude mouse
tumor growth xenograft model bearing the human MX1-breast
carcinoma that has previously been demonstrated to be sensitive
to TGF-ꢀ inhibition. Compound 15d was administrated BID
via oral gavage in a saline solution starting on day 7 postim-
plantation and continued for 20 days. Tumors were allowed to
grow to a size of 2500 cm3 prior to study termination. Analysis
of the PK/PD data of compound 15d indicated that a dose of
75 mg/kg BID should be sufficient, and at this dose, a
statistically significant tumor growth delay in the MX1 model
was observed (Figure 3). No body weight loss was observed in
most of the treatment groups.
In conclusion, our expanded series of TGF-ꢀ RI inhibitors
has demonstrated antitumor efficacy and represents novel
antitumor agents. Traditional medicinal chemistry techniques
were used to quickly delineate the SAR of compounds having
new substitution on the quinoline ring. At the quinoline
6-position, small substituents were tolerated, while large
substitutions diminished the potency significantly. Although
substitution at the 7-position with a bulky phenoxy group was
detrimental, incorporating a basic amine side-chain through an
ether linkage was not only tolerable, but also provided com-
pounds with oral bioavailability such as 15d. The surrogate
It was reported that compounds with substituents at the
7-position comprised of electron-donating groups overall gave
good activity in vitro.17,18 Further expansion of the SAR at
the 7-position using novel aminoalkoxy side chains (14–19) gave
very potent inhibitors in the TGF-ꢀ RI assay with IC50 values
ranging from 6 to 129 nM. In contrast, the IC50 values of
compounds with phenyloxy side chains (20–23) were only
748–4820 nM, ∼100-fold less potent. Although the X-ray crystal
structure of 15d showed that the solubilizing group orients
toward solvent (Figure 2), the phenyl ether linker of compound

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2305
Table 1. Kinase and Cellular Activitya
a All IC50 values were 10 point determinations. Mean values ( SEM for a minimum of two determinations.
Experimental Section
3-[6-(2-Morpholin-4-yl-ethoxy)-naphthalen-1-yl]-2-pyridin-2-
yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole (15d). To a solution of
phenol (13, 376 mg, 1.15 mmol) and cesium carbonate (826 mg,
2.54 mmol) in DMF (5 mL) was added 2-(2-bromoehtoxy)tetrahy-
dro-2H-pyran (380 µL, 2.52 mmol). The resulting content was
stirred at 120 °C for 4 h, quenched with saturated sodium chloride,
and then extracted with chloroform. The organic layer was dried
over sodium sulfate and concentrated in vacuo. The residue was
purified by silica gel column chromatography (linear gradient from
dichloromethane to 10% methanol/dichloromethane over 20 min
and 10% methanol/dichloromethane for 20 min) to give a yellow
oil (424 mg). LRMS (ES+) m/z 457.0 (M + 1)+.
To a solution of AcOH/THF/H2O (4:2:1; 20 mL) was added THP
(tetrahydro-2H-pyran)-protected compound (421 mg, 0.92 mmol).
After stirring at 80 °C overnight, solvent was removed in vacuo.
The residue was dissolved in CHCl3/i-PrOH (3:1) and washed with
saturated Na2SO4. The organic layer was dried over sodium sulfate
and concentrated in vacuo. The resulting foam (13a) was pure
enough without further purification (425 mg). LRMS (ES+) m/z
373.1 (M + 1)+.
To a solution of alcohol (13a, 293 mg, 0.78 mmol) in dried
pyridine (5 mL) was added MeSO2Cl (68 µL, 0.81 mmol). After
stirring for 2 h, pyridine was removed in vacuo. The residue was
dissolved in CHCl3 and washed with saturated NaHCO3. The
Figure 2. X-ray crystal structure of 15d bound to the ATP binding
site of the TGF-ꢀ R1 kinase domain. The structure indicates the basic
amine tail orients outside of the binding pocket and within the solvent.
metabolism SAR was implemented to predict/select compounds
with better metabolism properties in vitro and helped to rapidly
identify appropriate compounds for testing in in vivo PK/PD
IVTI assays. A hallmark of this effort was also the successful
use of the PK/PD data to design long-term tumor xenograft
efficacy studies.

2306 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Brief Articles
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Figure 3. Effect of 15d (LY2109761) on the growth of the MX1
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a group of 10 Charles River nude mice at 106 cells subcutaneously.
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grow until a size of 2500 cm3 prior to study termination. Tumor volume
data were transformed to a log scale for analysis to equalize variance
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Acknowledgment. We are grateful to Dr. Philip W. Iversen
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suggestions.
Supporting Information Available: General experimental
procedure and procedures for preparation of 7c–7f, 8–13, 14a, 15,
15a–c, 16a, 17a, 18–19, 20a,b, 21–23, and their precursors; NMR
data of 13a and 15d. Scheme for synthesis of 7a–f; Enzymatic and
cell-based assay procedures; IVTI PK and PD data for key
compounds; Kinase selectivity (15d); Table for screening of
alkylation condition of phenol 13; ADME surrogate data for key
compounds; HPLC purities of all target compounds, and HPLC
tracings of 15d. This material is available free of charge via the
Internet at http://pubs.acs.org.
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