Synthesis, biological evaluation, hydration site thermodynamics, and chemical reactivity analysis of α-keto substituted peptidomimetics for the inhibition of Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmcl.2014.01.062

A new series of peptidomimetic pseudo-prolyl-homophenylalanylketones were designed, synthesized and evaluated for inhibition of the Plasmodium falciparum cysteine proteases falcipain-2 (FP-2) and falcipain-3 (FP-3). In addition, the parasite killing activ

Full text of DOI:10.1016/j.bmcl.2014.01.062

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1274–1279
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation, hydration site thermodynamics,
and chemical reactivity analysis of a-keto substituted
peptidomimetics for the inhibition of Plasmodium falciparum
David J. Weldon a,e, Falgun Shah a, Amar G. Chittiboyina a,d, Anjaneyulu Sheri a, Raji Reddy Chada a,
Jiri Gut b, Philip J. Rosenthal b, Develeena Shivakumar c, Woody Sherman c, Prashant Desai a,
Jae-Chul Jung a, Mitchell A. Avery a,d,
⇑
a School of Pharmacy, Department of Medicinal Chemistry, University of Mississippi, University, MS 38677, United States
b Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94143, United States
c Schrodinger, Inc., 120 West 45th Street, 17th Floor, New York, NY 10036, United States
d National Center for Natural Products Research, University of Mississippi, University, MS 38677, United States
e School of Pharmacy, Department of Pharmaceutical Sciences, Loma Linda University, Loma Linda, CA 92350, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of peptidomimetic pseudo-prolyl-homophenylalanylketones were designed, synthesized
and evaluated for inhibition of the Plasmodium falciparum cysteine proteases falcipain-2 (FP-2) and falci-
pain-3 (FP-3). In addition, the parasite killing activity of these compounds in human blood-cultured P. fal-
ciparum was examined. Of twenty-two (22) compounds synthesized, one peptidomimetic comprising a
Received 6 January 2014
Accepted 21 January 2014
Available online 31 January 2014
homophenylalanine-based a-hydroxyketone linked Cbz-protected hydroxyproline (39) showed the most
Keywords:
Malaria
Peptidomimetic
Falcipain
Cruzain
Drug resistance
potency (IC50 80 nM against FP-2 and 60 nM against FP-3). In silico analysis of these peptidomimetic ana-
logs offered important protein–ligand structural insights including the role, by WaterMap, of water mol-
ecules in the active sites of these protease isoforms. The pseudo-dipeptide 39 and related compounds
may serve as a promising direction forward in the design of competitive inhibitors of falcipains for the
effective treatment of malaria.
Ó 2014 Elsevier Ltd. All rights reserved.
Malaria,
a
parasitic disease caused by several plasmodial
Recently, P. falciparum strains have shown decreased respon-
siveness to artemisinin therapy, leading to delayed parasite clear-
ance after therapy, primarily near border areas of Thailand.3 This
finding validates a continued search for effective alternative thera-
peutics targeting key pathways in the life cycle of malaria para-
sites. Among potential malarial drug targets, the cysteine
proteases falcipain-2 (FP-2) and falcipain-3 (FP-3) were identified
as mediators of the degradation of hemoglobin, which is required
by erythrocytic malaria parasites to provide amino acids for
parasite metabolism.7,8 Since the degradation of hemoglobin is
essential for the survival of the parasite, the inhibition of FP-2
and FP-3 is a viable approach for the effective treatment of malar-
ia.9–13 Several studies have validated FP-2/FP-3 as drug targets for
chemotherapy of P. falciparum but both FP-2 and FP-3 must be
inhibited simultaneously to achieve parasite death.14,15,11 Crystal
structures of FP-2 (PDB codes 1YVB, 2GHU and 3BPF) were pub-
lished in 200616,17 and 2009.18 Fortunately, the crystal structure re-
vealed the active sites of FP-2 and FP-3 to be quite similar, with
some narrowing of the S2 subsite in FP-3 compared to FP-2.19,20
Additionally, it was known that the falcipain proteases are closely
related to cruzain proteases (1 is the ligand from cruzain crystal
species, the most virulent of which in humans is Plasmodium
falciparum, is a major health concern in Asia, South America, and
sub-Saharan Africa. With an estimated 219 million cases resulting
in 660,000 deaths worldwide in 2010,1 the need for inexpensive,
easy to use, and well-tolerated treatment options is clear. The first
known treatment of malaria occurred in 1631 with the bark of the
cinchona tree, from which quinine was later purified.2 As quinine is
not well tolerated, new therapeutics were developed, including
chloroquine and related aminoquinolines, antifolates, and meflo-
quine. However, strains of P. falciparum resistant to all of the afore-
mentioned therapeutics have been observed.3–5 Artemisinin, a
natural product from Artemisia annua, and its derivatives have
been identified as rapidly active and highly efficacious antimalari-
als.5 In recent years the standard of care for the treatment of
uncomplicated falciparum malaria has moved to artemisinin-based
combination therapy (ACT), which combines either artesunate, art-
mether, or dihydroartemisinin with a longer acting agent.6
⇑
Corresponding author. Tel.: +1 662 915 5879; fax: +1 662 915 5638.
E-mail addresses: mavery@olemiss.edu, mitchellavery@att.net (M.A. Avery).
http://dx.doi.org/10.1016/j.bmcl.2014.01.062
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

D. J. Weldon et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1274–1279
1275
structure 1F29, PDB code) in structure in the active site.20 This
O
O
O
i
ii
N2
computational information, coupled with the report by Ellman
and others21 of compound 2 as a peptidomimetic inhibitor of cruz-
ain, we proceeded with docking studies of cruzain inhibitor 2 in FP-
2. The studies revealed that inhibitors of cruzain would be a good
starting structural motif for the design of novel inhibitors of falci-
pain cysteine proteases. The overlap in structural features of our
designed motif 3 and the cruzain inhibitor 2 are shown in blue in
Figure 1.
BocHN
BocHN
BocHN
Cl
OH
Ph
Ph
Ph
4
5
6
Scheme 1. Synthesis of
morpholine, isobutyl chloroformate, 1 h, reflux; THF, 0 °C, CH2N2, 2 h, 72%; (ii) HCl–
AcOH (1:1), ether, 0 °C, 1 h, 89%.
a
-chloroketone 6. Reagents and conditions: (i) 4-Methyl-
Of particular note, one of the phenylalanine residues in 2 has
been replaced with a homophenylalanine in 3 and one of the carb-
oxybenzyl-protected (Cbz-protected) phenylalanine amino acids
in 2 has been replaced with a relatively constrained Cbz-protected
proline derivative in 3. The use of unnatural amino acids was delib-
erately made for two reasons: to by-pass peptidase metabolism,
thereby increasing the compound half-life; and to decrease analog
flexibility to improve binding thermodynamics. For the later, utiliza-
tion of cyclic derivatives such as b-amino acids was appropriate. To
probe the available 3D-chemical space for the substrate binding site
of FP-2 and to explore the SAR, we designed and synthesized twenty-
two compounds (21–42) from readily available building blocks.
The syntheses of the twenty-two pseudodipeptidic cysteine
protease inhibitors are dependent upon the construction of a hom-
studies against the W2-strain (chloroquine resistant) of P. falcipa-
rum were also carried out.8,19,22–25 The biological activities of the
derivatives are summarized in Table 1. While several peptidomi-
metics showed moderate activity in one of the three biological
tests, compound 39 was clearly a superior candidate in both iso-
forms of FP-2, FP-3, and W2 inhibition of 80 nM, 60 nM, and
7.70 lM respectively.
In an attempt to understand the SAR of the peptidomimetics,
we first considered the binding affinity in the active site of FP-2
and FP-3. From the experimental SAR, it was evident that hydroxyl
proline in the S2 pocket, an
a-hydroxyketone electrophile in the
S1–S0 pocket, and homophenylalanine in the S1 pocket were suit-
2
able substituents for high binding affinity peptidomimetics. Inter-
estingly, efforts to understand the SAR of this series by means of
steric and electrostatic interactions derived from the docking stud-
ies in FP-2 and FP-3 were not sufficient to explain the variation in
biological data. Van der Waals energy, electrostatics, hydrogen
bonds, or Docking Score from Glide SP26,27 docking calculations
did not provide a statistical correlation with experimental binding
affinity. Furthermore, implicit solvent binding energy estimations
from MM–GBSA (as implemented in the program Prime)28,29 did
not show a significant correlation with experimental binding
affinity.
In light of the above findings, we anticipated the involvement of
water molecules in the binding of these inhibitors, since explicit
water solvation is neglected from all of the above analyses. There-
fore, we generated thermodynamic profiles of water molecules
present in the ligand binding domain (LBD) of FP-2 and FP-3 using
WaterMap (Schrodinger, LLC) in an attempt to understand the ob-
served SAR among the designed pseudopeptidomimetics. Water-
Map computes the location and energetics of water molecules
around a protein using explicit solvent molecular dynamics (MD),
solvent clustering, and statistical thermodynamics.30,31 WaterMap
was chosen to study the protein solvation effects because it has
been effectively applied to a broad range of pharmaceutically rele-
vant targets including PDZ domains,32 kinases,33 G-protein coupled
receptors (GPRCs),34 protein–protein interaction interfaces,35 and
serine proteases.36 Recently, a WaterMap study was reported by
our group to understand binding modes of small molecule inhibi-
tors of falcipain (FP-2 & FP-3) identified by virtual screening.37
ophenylalanine-based scaffold representing the C-terminus: an a-
substituted ketone intended to interact with the catalytic cysteine
thiol moiety; and the N-terminal residue. Peptide-like coupling of
the
N-Cbz proline gives such a pseudodipeptide (3).
Specifically, synthesis of the -chloroketone 6 was accom-
a-chloroketone corresponding to homophenylalanine, 6, with
a
plished by in situ activation of the amino acid 4 as its mixed anhy-
dride followed by quench with diazomethane to yield the
diazoketone 5, best immediately converted to the more stable
a-
chloroketone 6 with HCl in AcOH (Scheme 1). We chose eleven dif-
ferent synthetic amino acids for coupling to 6, N-terminal residues
which occupy the opposing S2/S3 site of falcipain. Hence, the Boc-
group of 6 was removed with 2 M HCl in ether and the resulting
amine-HCl salt 7 was then coupled with the eleven Cbz-protected
unnatural amino acids furnishing the target compounds 9–20
(Scheme 2).
The N-terminal amino acids were all cyclic except for the use of
a homophenylalanine (e.g. 36). Most of these amino acids were
b-aminoacids, normally cis, and include b,b (e.g. 26) or
a,a (27)
1-aminocyclohexyl carboxylic acids. While the amino acids used
for peptide coupling are denoted as R in Scheme 2 for the sake of
simplicity, the exact structural details of the final compounds are
shown in Figure 2. The reactive
a-chloroketone of the derivatives
was easily displaced by O, N, or S nucleophiles resulting in
twenty-two target derivatives, 21–42 (Scheme 3).
The synthesized peptidomimetics were tested against FP-2 and
FP-3 isoforms in vitro. Additionally, in vitro growth inhibition
P3 site P2 site P1 site
P1' site
O
Ph
O
O
H
N
O
R2
O
S
Ph
H
N
Ph
O
N
H
3
S
R3
N
H
R1'
O
Ph
O
R1
1
2
P2 site P1 site
H
P1' site
R1
O
N
N
O
O
O
P3 site
3
Ph
Ph
Figure 1. Structural similarity of vinyl sulfone ligand 1 from the cruzain crystal structure and cruzain inhibitor 2 with our designed inhibitor 3.

1276
D. J. Weldon et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1274–1279
O
O
R
R
H
N
ii
YHN
i
Cl
OH
Cl
+
CbzHN
CbzHN
Ph
Ph
O
O
8
9-20
6 Y = Boc
7 Y= H.HCl
Scheme 2. Synthesis of natural and unnatural dipeptide analogues. Reagents and conditions: (i) 2 M HCl in Et2O, 0 °C to rt, 1 h; (ii) EDC, HOBt, triethylamine, DCM, 12 h.
O
O
O
O
H
N
H
N
H
N
X
X
OH
N
N
O
O
NH
O
O
O
Bn
O
Bn
Bn
O
O
Ph
O
Ph
Ph
26
24
21 X = OH
X = S(CH2)3Ph
X = SC(O)CH3
X = OAc
25 X = OH
22
23
O
O
O
H
N
H
N
H
N
OH
Bn
OH
OH
O
NH
O
O
NH
O
O
NH
O
Bn
Bn
27
28
29
Ph
Ph
Ph
Ph
O
O
O
O
S
O
O
O
O
O
H
N
H
N
H
OH
X
N
X
BnO
N
H
O
Bn
N
H
N
O
O
Bn
O
O
O
33
X = OAc Ph
Ph
32
30
X = OAc
34 X = OH
31 X = OH
HO
O
O
Ph
H
N
O
O
H
N
H
X
X
Bn
N
X
N
O
N
H
O
NH
O
O
O
Bn
O
Bn
O
O
Ph
37 X = OAc
38
Ph
.
35
X = OAc
36 X = OH
Ph
39
X = OH
X = OH
40 X = S(CH2)3Ph
41
X = SAc
42 X = N(CH2CH2)2O
Figure 2. Peptidomimetic inhibitors explored in this work.
O
R
H
N
Cl
CbzHN
i
Ph
v
O
9-20
O
O
O
R
R
O
H
N
H
N
R1
N
iii
iv
CbzHN
CbzHN
Ph
O
Ph
O
R1 = OAc 24,30,33,35,37
42
(
)
ii
(21,25-29,31,32,34,36,38,39 )
R1 = OH
R
O
R
H
N
H
N
SAc
S(CH2)3Ph
CbzHN
CbzHN
Ph
O
23,41
Ph
O
22,40
Scheme 3. Synthesis of target molecules. Reagents and conditions: (i) NaOAc, 18-crown-6, DMF, rt, 12 h, 91%; (ii) LiOH/H2O, THF/H2O, rt, 92%; (iii) 3-phenylpropane-1-thiol,
triethylamine, THF, rt, 12 h, 83% (iv) KSAc, DMF, rt, 12 h, 81%; (v) morpholine, triethylamine, THF, rt, 12 h, 84%.
WaterMap analysis was performed on the published crystal struc-
tures of FP-2 (3BPF, PDB code) and FP-3 (3BWK, PDB code) using
default settings. The WaterMap calculations of the apo protein re-
vealed several highly unstable water molecules in the LBD of FP-2
and FP-3, which could be displaced to yield improved binding
energies.
Typically, water molecules with unfavorable (i.e. positive) free
energy should be displaced by ligand functional groups to gain

D. J. Weldon et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1274–1279
1277
Table 1
184, affording an additional boost in potency. Finally, the hydroxy-
proline moiety displaces two other unfavorable hydration sites
The biological activity of designed peptidomimetic analogues
(W5 and W12 in FP-2; W5 and W6 in FP-3 with
P2.3 kcal molꢀ1 from both enzymes). Compound 39 also displaces
W10 in FP-2 and W2 in FP-3
G = 2.6 kcal molꢀ1
G = 4.2 kcal molꢀ1), with the replacement of a critical hydrogen
bond with Gly83, which is considered important for binding to
the non-prime site by inhibitors of papain family cysteine prote-
ases.39 A portion of the boost in potency of 39 can be attributed
to its proline hydroxyl substituent displacing highly unstable
water sites of the S2 pocket, as the hydration site displacement pat-
tern is the primary difference between 39 and the other
derivatives.
DG
Compds
Falcipain-2
Inhibition
Falcipain-3
Inhibition
IC50 (lM)
Indochina clone
W2 IC50 (lM)
IC50
(lM)
(
D
)
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
>50
48.13
>50
>50
>50
5.52
4.42
>50
>50
>50
>50
>50
36.36
>50
>50
(D
13.77
18.12
>50
>50
>50
47.60
>50
>50
>50
>50
23.29
>50
28.16
>50
37.21
>50
47.21
7.70
19.64
18.64
10.16
>50
0.54
0.91
20.18
23.90
>50
>50
47.23
>50
8.22
>50
11.91
>50
25.44
0.06
0.52
3.56
>50
The Cbz moiety of 39 also displaces several unfavorable water
sites from the S3 pocket of FP-2 and FP-3 (W4, W14, W16, and
W20 in FP-2; W7, W8, W17, and W18 in FP-3) into the bulk of
the solvent, and is a suitable substituent for the S3 pocket. The car-
>50
>50
33.86
3.25
29.67
30.08
>50
33.64
0.08
1.10
>50
bonyl moiety of the
unstable water (W7 in FP-2,
G = 1.4 kcal molꢀ1) from the S1 pocket and replaces its H-bond
a
-hydroxyketone in 39 displaces the proximal
D
G = 3.0 kcal molꢀ1, W16 in FP-3,
D
interaction with the side chain of Gln36/45 and backbone of
Cys42/52 in FP-2 and FP-3, respectively. The hydroxyl group of
the
a-hydroxyketone forms an H-bond with His 174/184 in FP-2
>50
and FP-3. Additionally, the hydroxyketone stabilizes the unfavor-
able water molecule of the S01 pocket (W22 in FP-2 with
D
G = 1.1 kcal molꢀ1; W4 in FP-3 with G = 3.6 kcal molꢀ1) by
D
binding free energy through release of the water molecule into
bulk solvent. Conversely, water molecules with negative free en-
ergy values should be bridged, judiciously replaced with the polar
atoms of ligands to make the same interactions as the water,38 or
forming a water-mediated H-bond with Trp (206/215) in both en-
zymes. To validate the WaterMap analysis, we evaluated deriva-
tives with low and moderate activity to establish their ability to
displace the unfavorable waters. The inactivity of 21, a proline ver-
sion of 39, can be explained by the inability to displace the unfa-
vorable water sites in the S2 pocket (Fig. 4a). The moderate
activity of 26 is a combination of the formation of a strong H-bond
network with Gly83 in addition to the displacement of a few unfa-
avoided altogether. Waters with negative
D
G also have negative
S) is, by def-
enthalpy (
D
H), because the entropy contribution (ꢀT
D
inition, always unfavorable for waters in the binding site relative to
water molecules in bulk solution. Given the above considerations,
the emerging SAR trends of peptidomimetics derivatives can be ex-
plained here in the context of the most active compound 39. The
WaterMap hydration sites in FP-2 and FP-3 binding sites along
with compound 39 are shown in Figure 3.
vorable water sites (
(Fig. 4b).
D
G P2.3 kcal molꢀ1) of the S2 pocket of FP-2
WaterMap was not able to provide insights about the prefer-
ence of
a
-hydroxyketone in the S1–S0 pocket over other electro-
1
According to the hydration site analysis, the homophenylala-
nine in the S1 pocket displaces unfavorable water sites (W19 and
W23 in FP-2; W14 and W19 in FP-3) in both enzymes. In addition,
the proline hydroxyl group of 39 successfully displaces the most
unstable hydration site in the S1 binding pocket of both falcipains
philes with similar hydration site displacement characteristics.
This might be expected, given that WaterMap only accounts for
solvent displacement and not direct interactions made with the
receptor. We anticipate that the reactivity of the carbonyl carbon
in the vicinity of catalytic cysteine [42(FP-2)/52 (FP-3)] with differ-
(W1,
D
G = 4.6 kcal molꢀ1 in FP-2; W1,
D
G = 4.3 kcal molꢀ1 in FP-3).
ent a-substituents might play an important role in the activity of
The hydroxyl also forms an H-bond with the backbone of His174/
these compounds.
W 206
W 215
(a)
(b)
Q 36
Q 45
W23
W22
S1
S1’
W19
W7
S1’
W14
W19
S1
W4
W16
W17
C 52
C 42
W10
W14
W18
H 174
W10
G 93
W2
H 184
N 183
G 83
W20
W12
S3
N 173
S3
W6
W16
W4
W17
W7
S2
W1
W8
W5
W1 S2
W5
Figure 3. The WaterMap profile of 39 in (a) FP-2 and (b) FP-3 is shown. The thermodynamically interesting hydration sites important for SAR are shown in spheres. The
unstable hydration sites ( G. Key hydrogen bonding interactions of
G >1 kcal molꢀ1) are shown in purple. The water sites are labeled based on decreasing value of predicted
39 (shown in yellow) with the falcipain binding (shown in cyan) site residues are displayed.
D
D

1278
D. J. Weldon et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1274–1279
W 206
Q 36
(a)
(b)
W23
W22
S1’
W19
W7
S1
W17
C 42
W14
H 174
N 173
W10
W20
G 83
S3
W12
W16
W4
S2
W1
W5
Figure 4. The predicted binding poses and interesting hydration sites for (a) 21 and (b) 26 are shown FP-2 isoform. Hydration sites labeling and color codes are same as
shown in Figure 3.
explored in this work. For example, WaterMap predicts unstable
Table 2
water (W3 in FP-2 with
D
G = 3.7 kcal molꢀ1; W9 in FP-3 with
Predicted LUMO density of the most electrophilic center (shown by asterisk ⁄) in
D
G = 2.5 kcal molꢀ1) interacting with the backbone carbonyl of
compound 39–42
Ile85/87 lining the S2 pocket of the proteases. W9 is also present
Compds
Functional Group
F_NN_LUMO values
0.52
in the X-ray structure of FP-3.40 Moreover, WaterMap shows a
O
network of stable waters (W24–W26 in FP-2 with
D
G = <ꢀ2.0
kcal molꢀ1; W21 and W22 in FP-3 with
D
G = <ꢀ0.7 kcal molꢀ1) at
39
OH
the bottom of the S2 pocket forming an H-bond with Asp234 in
FP-2 and Glu243 in FP-3. These water sites can be either targeted
(functionally replacing the stable waters) or interacted with
(water-mediated H-bonds) in future work to gain additional bind-
ing affinity.
∗
O
40
41
0.46
0.26
S(CH2)3Ph
∗
The information gained from these computational analyses can
be used to design the next generation of inhibitors of falcipains.
Specifically, we plan to synthesize compounds that are able to dis-
place unstable hydration sites that were not displaced by the com-
pounds presented in this work. This may require developing new
chemical scaffolds, as some of the unstable hydration sites are
not accessible with the R-group positions in the chemotypes pre-
sented in the current manuscript. Further design efforts against
FP-2 and FP-3 will consider the reactivity of electrophiles in addi-
tion to the displacement of unfavorable waters, in particular from
the S2 pocket of FP-2 and FP-3.
O
SC(O)CH3
N(CH2)2O
∗
O
42
0.01
∗
Acknowledgments
To help understand the reactivity of electrophiles with the cat-
alytic cysteine residue and its effect on the inhibitory activity, we
calculated the LUMO variant of Atomic Fukui indices at constant
spin density using the quantum mechanics package Jaguar, as
described previously.19 The compounds 39–42 with different
electrophilic groups were considered for these calculations. The
LUMO variant of Fukui indices was considered for predicting elec-
trophilicity of the carbonyl carbon. The predicted LUMO values
were scaled from zero to one, one being the most reactive, and
are shown in Table 2. The predicted reactivity of electrophiles
We thank the CDC cooperative agreements 1UO1 CI000211-03
and 1UO1 CI000362-01 for financial support. Additionally, Investi-
gation was conducted in a facility constructed with support from
Research Facilities Improvement Program Grant Number C06
RR14503-01 from the National Center for Research Resources,
National Institutes of Health. We are grateful for WaterMap
software support from Schrodinger, LLC.
References and notes
was higher for the compound with an
LUMO = 0.52) than the compound with an
LUMO = 0.46; 41, LUMO = 0.26), which in turn had a higher electro-
philicity than the -amino ketone (42, LUMO = 0.01). The calcu-
a-hydroxyketone (39,
a-thioketone (40,
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a
lated LUMO density values predict correct ordering of the
inhibition of FP-2 and FP-3 for compounds 39–42. These reactivity
calculations complement the hydration site thermodynamic analy-
sis and provide additional insights into the SAR of the compounds
explored in this work.
In addition to elucidating the unfavorable waters that are
displaced by 39, WaterMap also predicts other unfavorable waters
in the ligand-binding domain of FP-2 and FP-3 that were not

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