Approaches towards the development of chimeric DPP4/ACE inhibitors for treating metabolic syndrome

Article abstract of DOI:10.1016/j.bmcl.2017.04.036

Designing drug candidates exhibiting polypharmacology is one of the strategies adopted by medicinal chemists to address multifactorial diseases. Metabolic disease is one such multifactorial disorder characterized by hyperglycaemia, hypertension and dyslip

Full text of DOI:10.1016/j.bmcl.2017.04.036

Accepted Manuscript
Approaches Towards the Development of Chimeric DPP4/ACE Inhibitors for
Treating Metabolic Syndrome
Jitendra A. Sattigeri, Sachin Sethi, Joseph A. Davis, Shahadat Ahmed, Geeta V.
Rayasam, Balasaheb G. Jadhav, Satya M Chilla, Dhrubajyoti Datta, A. Gadhave,
Vamshi K. Tulasi, Tarun Jain, Sreedhara Voleti, Biju Benjamin, Sunitha Udupa,
Garima Jain, Yogender Singh, Kona Srinivas, Vinay S. Bansal, Abhijit Ray,
Pradip K. Bhatnagar, Ian A. Cliffe
PII:
S0960-894X(17)30415-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.036
BMCL 24889
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 January 2017
24 March 2017
12 April 2017
Please cite this article as: Sattigeri, J.A., Sethi, S., Davis, J.A., Ahmed, S., Rayasam, G.V., Jadhav, B.G., Chilla,
S.M., Datta, D., Gadhave, A., Tulasi, V.K., Jain, T., Voleti, S., Benjamin, B., Udupa, S., Jain, G., Singh, Y., Srinivas,
K., Bansal, V.S., Ray, A., Bhatnagar, P.K., Cliffe, I.A., Approaches Towards the Development of Chimeric DPP4/
ACE Inhibitors for Treating Metabolic Syndrome, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://
dx.doi.org/10.1016/j.bmcl.2017.04.036
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Approaches Towards the Development of Chimeric DPP4/ACE
Inhibitors for Treating Metabolic Syndrome
Jitendra A. Sattigeri *, Sachin Sethi, Joseph A. Davis, Shahadat Ahmed, Geeta V. Rayasam,
Balasaheb G. Jadhav, Satya M Chilla, Dhrubajyoti Datta, Gadhave, A., Vamshi K. Tulasi,
Tarun Jain, Sreedhara Voleti, Biju Benjamin, Sunitha Udupa, Garima Jain, Yogender Singh,
Kona Srinivas, Vinay S. Bansal, Abhijit Ray, Pradip K. Bhatnagar, Ian A. Cliffe
New Drug Discovery Research, R&D III, Ranbaxy Laboratories Limited, Plot 20, Sector 18,
Udyog Vihar, Gurgaon, Haryana, 122015, India
Corresponding author email address: jitendra.sattigeri@gmail.com Tel: +919810502202
Title running head: DPP4-ACE Dual Inhibitors.
Abstract
Designing drug candidates exhibiting polypharmacology is one of the strategies adopted by medicinal
chemists to address multifactorial diseases. Metabolic disease is one such multifactorial disorder
characterized by hyperglycaemia, hypertension and dyslipidaemia among others. In this paper we report a
new class of molecular framework combining the pharmacophoric features of DPP4 inhibitors with those
of ACE inhibitors to afford potent dual inhibitors of DPP4 and ACE.
KEYWORDS: DPP4-ACE Dual Inhibitor
Diabetes is a growing epidemic in both the developing and developed world. It has been estimated that
there are ~ 420 million diabetics globally with a projected global prevalence of 640 million in 20401.
More than 70% of people suffering from Type 2 diabetes are hypertensive (blood pressure ≥ 140/90
mmHg)2. Additionally, studies have identified the risk of developing type 2 diabetes is much higher in
people with hypertension than in normotensives3. An ideal treatment for this subset of diabetics would not
only address their hyperglycemia but also the hypertensive component of the disease. DPP4 (dipeptidyl

peptidase-4) inhibitors and ACE (angiotensin converting enzyme) inhibitors have been the mainstay for
treating hyperglycaemia and hypertension in these patients. However, patient compliance becomes an
issue when multiple drugs need to be taken on a long-term basis. In addition, there is the risk of enhanced
drug-drug interactions when multiple drugs are prescribed. As a consequence, availability of a single
therapeutic agent which would simultaneously ameliorate the pharmacological processes contributing to
both hypertension and diabetes could be a significant step forward in the treatment of metabolic
syndrome. Here in, we report a designed multiple ligand approach to identify dual inhibitors which
incorporate all the structural elements required for inhibition of DPP4 and ACE.
One of the strategies for designing ligands capable of binding to two phylogenetically diverse biological
targets is to merge the structural elements of two pharmacophores.4 Herein, the identification of molecular
points on both ligands which would allow for merging of the two structures is the most critical step.
Indeed, a detailed analysis of the pharmacophoric features, the size of the enzyme active sites and
knowledge of the tolerability of the respective lead compounds to structural variations is an important
exercise which aids in the design of dual merged ligands.
In our work we critically evaluated ligand-DPP4 and ligand-ACE co-crystal structures5,6 (Fig 1) to design
dual inhibitors. Thus, ACE inhibitors (Fig 1a) need a carboxy group to bind to the S2' site, a zinc
chelating group (COOH or SH), and a hydrophobic group that fits into the S1 pocket. ACE crystal
structure further suggests that the S2' pocket is big enough to accommodate large groups. From the
structures of fosinoprilat (2) and zofenopril (3) (Chart 1)7, it appears that position 4 of the proline ring in
enalaprilat (1) is tolerant to further substitution without loss of activity and that these groups are well
tolerated in S2' pocket. DPP4 inhibitors, on the other hand, require a hydrophobic moiety (e.g.
trifluorophenyl) or an electrophilic group (e.g. CN) to interact with a serine at the S1 site and a basic
amine to form a salt bridge with the glutamic acid diad at the S2 site. A similar analysis of crystal
structure of the DPP4 enzyme (Fig 1b) suggests that the S2 pocket can accommodate large groups as in
sitagliptin (4) and similar compound (5), (Chart 1).8 Thus, based on the structural aspects of the ligands
and enzymes, it appears that enalaprilat and sitagliptin can be merged to form a dual inhibitor by
removing the relatively unessential triazolopiperidine of 4 and forming an amide bond with an amino
group placed at the fourth position of the proline residue of 1. Thus, the proposed dual inhibitor ligands
6a-d incorporate all the essential structural elements required for DPP4 and ACE inhibition (Scheme 1).
Molecular docking simulations were carried out for ligands 6a-d in the ACE crystal structure (1O86)5 and
DPP4 crystal structure (2G5P).6 Comparison of the binding mode of 6a-d (Figure 2) with enalaprilat

(Figure 1a) in ACE shows that the phenethyl group (6a-d, enalaprilat) occupies the S1 pocket and the
adjacent carboxylic acid group (6a-d, enalaprilat) lies in the metal binding region. The R groups of 6a-d
and the methyl group of enalaprilat fit into the S1' pocket and the pyrrolidine group with the carboxylic
acid (6a-d, enalaprilat) positions itself into the S2' pocket.
Comparison of the binding mode of 6a-d (Figure 3) with sitagliptin (Figure 1b) in DPP4 shows that the
2,4,6-triflurophenyl group and the basic amine moiety (6a-d, sitagliptin) binds in the S1 pocket. The S1
pocket is closed and offers tight binding to the docked poses of the ligands, all of which maintain the
same alignments and amino acid interactions, as have been reported for other known DPP4 inhibitors.9
Figure 1b shows that the S2 pocket is wide enough to accommodate bulkier substitutions. Molecular
docking supports this hypothesis and it was observed that the ACE pharmacophore of 6a-d binds into the
S2 pocket of DPP4 (Figure 3).
The ligands 6a-d were synthesized by standard synthetic and peptide coupling methods (Scheme 2).
Amine 710a was coupled with β-amino acid 810b using EDCI to afford 9 from which the Cbz group was
removed to yield the key intermediate 10. The proposed DPP4-ACE dual inhibitors 6a-d were
synthesized from 10 in a simple three-step protocol by coupling the intermediate 10 with 13a-d under
EDCI coupling conditions followed by sequential deprotection of the Boc and ester groups of 11a-d. The
homophenylalanine derivatives 13a-d were prepared according to a literature procedure.11
Merged ligands 6a-d have high MW (>500) and PSAs (162 Å2) (Table 1). These compounds exhibit
gradual increase in cLogPs with increasing alkyl chain length from CH3 to n-Bu (4.23 to 5.78
respectively). Also, the number of rotatable bonds increases from 14 to 17 on going from 6a to 6d
respectively.
Next, compounds 6a-d were evaluated in vitro for their ability to inhibit12,13 ACE and DPP4. For this
purpose, plasma of Wistar rat, ob/ob mouse and human was used as enzyme source for ACE and DPP4.
Table 2 shows the inhibitory activities for compounds 6a-d and the reference standards 1 and 4. As
expected compound 1 inhibited ACE potently (IC50 < 11nM) and did not inhibit DPP4 even at
concentration of 100 µM. Likewise, 4 showed potent inhibition of DPP4 (IC50 < 50 nM) across species
but showed only weak inhibition potential for ACE enzyme. To our delight, compounds 6a-d,
incorporating the features of both ACE and DPP4 inhibitors, exhibited dual inhibition of human ACE and
DPP4. Compounds 6b (ACE IC50 = 8.6 nM; DPP4 IC50 = 57 nM) and 6c (ACE IC50 = 51 nM; DPP4 IC50
= 67 nM) were found to be the most potent dual inhibitors of human ACE and DPP4. Compounds 6a and

6d also displayed dual inhibition of ACE and DPP4 but at relatively higher concentrations (IC50 > 100
nM).
Compound 6a moderately inhibited human ACE. The inter-species shifts in ACE IC50 values for
compound 6a were not significant (within the range of experimental error). Compound 6b exhibited a
very potent inhibition of human ACE (IC50 = 8.9 nM). However, 6b showed a considerable shift (12-fold)
in mouse ACE inhibition (IC50 = 100 nM) and up to 3-fold shift in rat ACE inhibition (IC50 = 24 nM).
Compounds 6c and 6d also exhibited a considerable loss of inhibitory potential against rat ACE
(seventeen- and ten-fold, respectively) and mouse ACE (forty- and eight-fold, respectively). While
compound 6a exhibited similar inhibitory potencies against DPP4 across all three species, a progressive
loss in rat and mouse DPP4 inhibitory potency was observed for compounds 6b-d with the greatest loss
being seen for mouse DPP4 (~2-fold more than that for rat DPP4). The plasma protein binding (PPB) for
6a-d was found to increase in human and mouse plasma with increasing length of R (Table 3) which can
be correlated with increasing clogP (Table 1); the inherent PPB in mouse plasma being higher than in
human plasma. Thus, the upward shift in IC50 of compound 6b-d in mouse ACE and DPP4 (plasma being
used as enzyme source) can be explained to a certain extent based on increased PPB in mouse plasma.
However, the shift in IC50s in rat ACE and DPP4 (plasma being used as enzyme source) cannot be
explained based on PPB. These unusual shifts were not noted for the reference compounds 1 and 4.
Compounds 6a-d were profiled for their inhibitory activity against dipeptidyl peptidase-IV Activity
and/or Structure Homologues (DASH) [eg.DPP2, DPP8 and DPP9] and non-DASH [eg. post-proline
cleaving enzyme (PPCE), neutral endopeptidase (NEP), aminopeptidase P (APP), aminopeptidase N
(APN)] enzymes. Compounds 6b-d exhibited over one thousand-fold selectivity for DPP4 over DPP2,
DPP8 and DPP9 enzymes (Table 4), whilst Compound 6a was approximately one hundred-fold selective.
Compounds 6b-d exhibited high selectivity (> 1000 fold) for non-DASH enzymes PPCE, NEP, APP and
APN. While 6a showed > 1000 fold selectivity for NEP, APP and APN, it was only ~ 100 fold selective
for PPCE.
Compounds 6a-d showed that they had no liability for inhibiting common cytochrome P-450 enzymes
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) up to a concentration of 10 µM. Furthermore, the
compounds were found to be stable to human, mouse, rat and dog liver microsomes. These compounds
had very low Caco-2 permeabilities, which is to be expected from the higher values of calculated
molecular properties.

Despite these compounds showing very low Caco2 permeability, the plasma exposure profiles of
compounds 6a-d were studied at 30 mpk in Wistar rats following oral dosing (Table 5). Compound 6a
exhibited a very low Cmax. and AUC (0.26 µg/mL and 0.61 µg.h/mL, respectively). Compound 6b
exhibited a slightly improved plasma exposure (Cmax = 0.55 µg/mL; AUC = 2.36 µg.h/mL), whilst
compound 6c exhibited good plasma exposure (Cmax = 2.51 µg/mL; AUC = 23.49 µg.h/mL). Compound
6d also exhibited a good Cmax and AUC values (Cmax = 1.33 µg/mL; AUC = 13.94 µg.h/mL); however
these concentrations were lower than those for 6c.
To conclude, rational de novo design and synthesis has been used to identify compounds having a potent
and balanced in vitro potency against both human DPP4 and ACE. The compounds have a favourable in
vitro DMPK profile. While compound 6b exhibited moderate oral plasma exposure, compounds 6c and
6d exhibited high plasma oral exposure in rats. We have thus demonstrated that a merged multiple ligand
approach is a feasible strategy for simultaneously inhibiting DPP4 and ACE enzymes.

Figure 1: Key structural features for DPP4 and ACE inhibition: (a) Enalapril binding in ACE
(PDB ID – 1UZE5) (b) Sitagliptin binding in DPP4 (PDB ID – 1X706)

Figure 2: The complex of ACE with 6a-d. ACE protein is shown as a white color Cα trace. The
active site channel is generated using MOLCAD and is shown as a white transparent surface.
The zinc atom is shown as a white ball in the Metal Binding Region. 6a (yellow), 6b (orange), 6c
(green) and 6d (cyan) are shown as ball and stick model.

Figure 3: The complex of DPP4 with four unique conformations of 6a-d. DPP4 protein is shown
as a white Cα trace. The active site channel is generated using MOLCAD and is shown as a
white transparent surface. 6a (yellow), 6b (orange), 6c (green) and 6d (cyan) are shown as ball
and stick model. The S1 pocket has a well-defined alignment of poses but different poses are
generated in the S2 pocket.

Scheme 1: DPP4-ACE dual inhibitors from the merger of enalaprilat 1 and sitagliptin 4
O
OH
F
F
F
O
N
NH2
O
O
N
F
OH
O
F
F
R
NH2
O
N
N
N
F
NH
R
+
N
N
H
F
HOOC
NH
F
R
HOOC
triazolopiperidine ring
4, sitagliptin
1, enalaprilat
R= Me : 6a
= Et : 6b
=n-Pr : 6c
=n-Bu : 6d
Position 4 of proline ring is tolerant to bulky
substitution (fitting into S2• pocket of ACE) as
seen in zofinopril and fosinoprilat. Hence, it is the
key molecular point where DDP4 ligand can be
merged.
Crystal structure of DPP4 shows that S2
pocket is wide enough to accommodate
bulkier substitutions. Hence, ACE
ligand can be merged at this point.

Scheme 2: Synthesis of 6a-d
(a)EDCI, HOBt, TEA, DMF (dry), 72% ; (b) Pd/C, MeOH, ammonium formate, 62%; (c) EDCI, HOBt,
TEA, 13a-d, DMF (dry), 41-63% (d) TFA, DCM (dry), 59-75%; (e) LiOH, THF, water, 63-97%
O
Ot-Bu
F
F
F
a
NHBoc
O
N
Cbz
N
F
Ot-Bu
Cbz
F
H2N
NHBoc
7
O
N
H
9
F
OH
O
8
b
F
F
F
F
NHBoc
O
N
F
NHBoc
Ot-Bu
O
O
c
Ot-Bu
O
N
H
H
N
F
NH
O
N
H
R
10
O
R= Me; 11a
= Et; 11b
= n-Pr; 11c
= n-Bu;11d
OEt
d
F
F
F
F
F
NH2
O
O
N
NH2
O
e
OH
OH
O
O
F
N
H
N
O
N
H
H
N
H
N
R
R
O
O
OH
OEt
R= Me; 12a
= Et; 12b
= n-Pr; 12c
= n-Bu;12d
R= Me; 6a
= Et; 6b
= n-Pr; 6c
= n-Bu; 6d
O
HO
H
N
R
O
OEt
R = Me; 13a
= Et; 13b
= n-Pr; 13c
= n-Bu; 13d

Chart 1: Representative DPP4 and ACE inhibitors
O
N
O
N
O
OH
O
OH
O
OH
O
N
S
O
NH
P
H3C
R
O
HO
S
HOOC
1, Enalaprilat
2, Fosinoprilat
3, Zofinopril
F
F
CF3
F
F
F
NH2
O
NH2
O
O
N
N
F
N
N
NH
N
F
F
4, Sitagliptin
5
F

Table 1: Calculated molecular properties of 1, 4 and designed merged ligands 6a-d
Compound
Mol Wt
348.39
407.3
cLogP
3.63
2.06
4.23
4.74
5.25
5.76
PSA Å2
107
Rot Bond
8
1
77
5
4
578.58
592.61
606.63
620.66
162
14
15
16
17
6a
6b
6c
6d
162
162
162

Table 2: Inhibition of DPP4 and ACE by 1, 4 and 6a-d
F
F
NH2
O
O
N
OH
O
F
N
H
H
N
R
O
OH
DPP4 Inhibition*
(IC50 nM)
ACE Inhibition*
(IC50 nM)
Compound
R
Rat
Mouse
Rat
Mouse
Human
Human
(Wistar) (ob/ob)
(Wistar) (ob/ob)
-
-
N.D.
33
N.D.
46
> 100 µM
9.6
N.D.
334
11.5
N.D.
58
2.5
1
4
20
11 µM
155
217
Me
Et
255
57
158
8.6
51
6a
6b
6c
6d
(0.61)
(0.85)
488
(2.1)
24
(0.37)
100
319 (5.6)
(8.6)
2470
(36.9)
7170
(70.3)
(2.8)
893
(11.6)
2023
(39.7)
2210
(7.9)
1230
(18.4)
4170
n-Pr
n-Bu
67
(17.5)
2800
(10.0)
102
279
(40.9)
* Plasma from Wistar rat, ob/ob mouse and human was used as source of DPP4 and ACE enzymes.
Data represents average values of at least two independent experiments done in triplicates. Values in
parenthesis indicate fold shift in IC50s on using rat /mouse enzymes over that observed with human
enzymes. N.D. - Not determined

Table 3: Plasma protein binding data (%) for compounds 6a-d, 1 and 4
Plasma
6a
6b
6c
6d
1
4
Human
Mouse
Rat
29.3 36.4 40.3 67.3 65.9 47.0
49
42
87.1 98.5 98.6 57.6 29.3
59.7 43.6 41.7 54.4 41.3

Table 4: Selectivity against DASH and non-DASH enzymes
IC50 (µM)
Compound
DPP8€
DPP9€
DPP2
>100
PPCE NEP APP
>100 >100 >100
APN
>100
>100
27
>100
44
4
>100
> 100
> 100
> 100
31.6
>100 >100
>100
> 100
> 100
> 100
6a
(105)
69
(172)
>100
(>1700)
> 100 > 100 > 100
> 100 > 100 > 100
> 100 > 100 > 100
6b
6c
6d
(1210)
57
>100
(850)
(>1500)
>100
>100
(>1000) (>1000)
€ DASH: DPP4 structure and activity homologues; Numbers in parenthesis represent the fold selectivity
over human DPP4;

Table 5: Plasma exposures of compounds in Wistar rats following oral dosing (30 mpk)
Cmax
AUC 0-24h
AUC 0-∞
Tmax
(h)
(µg/mL)
(µg.h/mL)
(µg.h/mL)
1.50 0.87 0.26 0.09 0.61 0.27
1.67 0.58 0.55 0.25 2.36 0.8
1.17§
6a
6b
6c
6d
2.86 0.67
3.3 1.2
2.0 00
2.5 0.51 21.49 4.11 23. 49 4.75
1.33 0.29 13.94 1.81 16.77 1.31
Values are presented as mean SD, n=3;
§mean values of two rats are reported because the terminal phase of one rat was not well defined

ACKNOWLEDGEMENT
Authors would like to thank Dr. Tridib Chaira for PK studies, Dr.Vanya Shah, for generating the
plasma protein binding data, Mr. M. Bandgar for providing a few intermediates. We wish thank
Mr. Abhishek Gupta, Mr.Varun Jain and Mr. Sreekumar V.B for recording the MS and HPLCs.
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further confirmed by HPLC before submission for biological evaluation.

Graphical Abstract
Approaches towards the development of chimeric
Leave this area blank for abstract info.
DPP4/ACE inhibitors for treating metabolic syndrome
Jitendra A. Sattigeri *, Sachin Sethi, Joseph A. Davis, Shahadat Ahmed, Geeta V. Rayasam, Balasaheb G. Jadhav, Satya M Chilla,
Dhrubajyoti Datta, Gadhave, A., Vamshi K. Tulasi, Tarun Jain, Sreedhara Voleti, Biju Benjamin, Sunitha Udupa, Garima Jain,
Yogender Singh, Kona Srinivas, Vinay S. Bansal, Abhijit Ray, Pradip K. Bhatnagar, Ian A. Cliffe
F
F
NH2
O
O
N
OH
O
IC50 (DPP4) - 57 nM
IC50 (ACE) - 8.6 nM
F
N
H
H
N
6b
O
OH